Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Equine Respiratory Viruses in New Zealand A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Turitea, Palmerston North, New Zealand Magdalena Dunowska 1999 Abstract 111 The outcome of any serious research can only be to make two questions grow where only one grew before. {Veblal T The Place o(Science in Modem Civilization (1919)J Equine respiratory disease is a cause of wastage resulting in financial losses for the equine industry throughout the world. A serological and virological survey was conducted on samples collected from a total of 133 horses from different pm1s in New Zealand. Three groups of foals were sampled on a monthly basis, five outbreaks of respiratory disease were investigated, and samples were collected from 37 yearlings during and following the yearling sales. The only viruses isolated were equine herpesviruses (EHV) types 2, 5, and 4. EHV-2 was isolated from 99% of peripheral blood leucocyte (PBL) samples from foals sampled on a monthly basis and from PBL of 96% of horses from outbreaks and yearlings from the sales. Additionally, EHV-2, EHV -5 or both were isolated from nasal swabs of up to 100% of foals sampled on a monthly basis between March and July. The time of virus excretion from the nasal cavity varied slightly between the three groups. The rate of virus isolation from the nasal swabs was highest at the time when most foals from two of the groups experienced some respiratory signs. Foals from the remaining group, however, were healthy throughout the study period. Of horses from outbreaks and yearlings from the yearling sales, EHV-2, EHV-5, or both were isolated from nasal swabs of 35% of horses showing respiratory signs, 9.5% of healthy horses, and 37.5% of horses for which individual clinical data were not available. EHV -4 was isolated on only one occasion, from PBL of a foal with respiratory disease. There was serological evidence that EHV - I , equll1e adenovirus- I (EAdV -1), and equine rhinoviruses (ERhV) types I and 2 are all present in New Zealand. The average antibody seroprevalence to these viruses was 67%, 61%,78%, and 13%, respectively. All serum samples tested were negative for antibodies to equine arteritis virus, mammalian reovirus-3 and paraintluenza virus-3. Most of the foals sampled showed serological evidence of infection with EHV -1/4 (78%), EAdV-J (61 %), and ERhV-2 (65%) within their first year of life. There was no indication that any of the foals sampled became infected with ERh V - I within the period of study. Samples for virus isolation and two blood samples for serology were collected from 54 of 82 (66%) horses sampled from outbreaks and yearlings from the sales for which individual clinical data were available. These included 35 horses showing signs of respiratory disease around the time of sampling and 19 healthy horses. For the remaining 28 horses, either individual clinical data were not available, or the second blood sample for serology was not collected. Recent viral infection was not associated with development of respiratory signs in yearlings from the sales when all viruses were considered, although this result was not statistically significant (adjusted OR 1.3, p = 0.5). Equine herpesvilUs-2/5 and ERhV-2 IV infections appeared to be associated with development of clinical signs in yearlings from the yearling sales, although these results were significant only for EHV -2/5, and not ERh V -2. However, since none of the foals or horses sampled was examined endoscopically, it is possible that a number of lower airway infections were not recognised. The most common infection among horses with respiratory signs from outbreaks, for which paired serum samples were available, was EHV-2/5 infection (30.4%), followed by ERhV-2 (13.0%), ERhV-J (4.3%), and EHV-1/4 (4.3%) infections. None of the 56 horses for which a full set of data were available showed serological evidence of recent EAdV-1 infection and only two horses showed serological evidence of recent ERh V -I infection. Most horses with signs of respiratory disease that showed serological evidence of recent viral infection also yielded EHV-2 or EHV-5 from their nasal swabs, indicating that EHV -2/5 either predisposes to other infections, or that infection with other viruses re-activates latent EHV -2/5. During the survey, EHV -5 was isolated on 56 occasions. This represented the first isolation of this virus outside Australia. Representative New Zealand isolates were compared to the reference Australian strain by restriction digest of the cloned gLycoprotein B gene. Restriction fragment length polymorphism (RFLP) profiles of all but one New Zealand isolate differed from the RFLP pattern of the prototype strain. With few exceptions, isolates from different horses showed different RFLP profiles. However, isolates from individual horses, collected either at different times, from different sites, or grown on different cells showed identical RFLP patterns. The effect of EHV -2 infection on gene expression in equine leucocytes was investigated by representational difference analysis of cDNA. The results suggested that EHV -2 infection of leucocytes down-regulates the expression of monocyte chemoattractant protein-I. This indicates that EHV-2 has the ability to modulate the chemokine environment of infected cells and may predispose to secondary infections. This work has contributed to the understanding of factors involved in equine respiratory disease in New Zealand. Although infection with none of the viruses was detected only in horses showing respiratory signs, the results suggest that EHV -2/5 and equine rhinoviruses may be more important than previously thought. v Acknowledgements I am grateful to the New Zealand Equine Research Foundation for providing financial support for my research and to Massey University for providing facilities and the doctoral scholarship, which enabled me to financially support myself. I would like to thank my chief supervisor, Or. Joanne Meers, for her friendship, support and enthusiasm throughout this project. I also thank my co-supervisors, Professor Colin Wilks and Dr. Richard Johnson for their time, advice, and encouragement. I wish to acknowledge Or. Brian Goulden, who helped me to establish links with the equine industry. Without him I would not have been able to organise horses for sampling. Thanks are also due to the horse owners who agreed to take part in the project and veterinarians who supplied some of the samples. Thanks are due to people from the Centre for Equine Virology at the University of Melbourne, Australia. Special thanks to Professor M. J. Studdert, for providing me with the reference strains of EHV -2 and EHV -5, antiserum to equine adenovirus-I, and also for hosting me in his laboratory for a period of one month. This made it possible for me to test survey sera for the presence of antibodies to equine rhinoviruses. Many thanks to Nino Ficorilli for his technical assistance in getting through hundreds of serum samples within a short period of time. I also wish to thank Steven Holloway for his input into this work, particularly in designing primers to amplify the gB gene of EHV-5. I am also grateful to all the staff members and fellow students at Massey University who helped me throughout the project. It is not possible to name everybody individually, but I particularly would like to express how much I appreciated the friendly, relaxed and stimulating atmosphere in the Department. I would especially like to thank my Polish friends, Magda and Jacek, for their friendship and support. Special thanks to Matthew for all the times we shared. These acknowledgements wouldn't be complete without mentioning my horse, Travolta. He not only (unwillingly) provided leucocytes for the RDA experiment, but also has been a great companion for the last three years. At last, by no means least, I would like to thank my mother for her love and support, and also for making it possible for me to come and study in New Zealand. VI Table of contents Abstract ...................................................................................................................................................... iii Acknowledgements ..................................................................................................................................... v Table of contents ........................................................................................................................................ vi List of figures ............................................................................................................................................... x List of tables .............................................................................................................................................. xii Abbreviations ........................................................................................................................................... xiii CHAPTER I:LITERATURE REVIEW .................................................................................................. 1 1. 1 EQUINE RESPfRATORY DISEASE ... . . . . . .. .. . . . . . . . . .. . . . . . . .. . . . . . .. . . . . . . . . . .... . . .. . . . . ...... . . . .. . . ........... . . . . .. . . . ...... . I 1.1.1 Respiratory disease as a cause of wastage for the equine industry . . . ....... . ............. . . . . . . .. ...... I 1.1. 2 Equine respiratory disease - definition ..... . . . . . . .. . . . . . . . . . . . . . ......... . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 2 1 .2 INFECTIOUS AGENTS ASSOCIATED WITH EQUINE RESPIRATOR Y DISEASE . .. ..... . . . . . ... . . . . . . . . ... . . . . . . . . . 3 1.2.1 Viruses .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . ... . . . .. .. . . . . .. .. .. . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 3 1.2 .2 Bacteria .. . . . . . . ... . . . . . . .. .. .. . . . . .. . . . . . .. .. .. ... . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . .. . . . ... . .. . . . . . . . . . . . 7 1.2.3 Fungi .. . . . . . . . . . . . . . . . . . . .. . . . . .. .. ... . . . . . . . . . . . . .. .. . . ... ... . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. ... . . .... . . . .... . . . . . . . . . . . . . . . . .. . . . . . . . . . 7 1. 2.4 Other pathogen.s ... . . . . . . . . . . . . . . . . . . ... . .. . . . . . . .. . . . . . ... .. . . .. . . . . . .. . . . . . . . .. . . . . . . . . . . . . .. . . . . . . ... . . . . . . . . . . .... . . . . . .. . . . . . . 7 1.3 EQUINE RESPLRATORY VIRUSES ........ . . . . . . . . . . . ..... ... . .. . . . . . . . . . . . . . ... .... . . . . .. . . . . .. . . . . . . . . . . . .... . . ....... . . . . . . . ..... 8 1.3.1 Equine herpesviruses . . . .. . . . . . . .. ....... . . .. .. . . .. . . .. . . . . . . . .... . . . . . . . . . . . ... . . . . . . .... . . . . .. . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . 8 1.3.2 Equine picorn.aviruses ... . . . .. . . . . . . . . .. . . . . . . . .. .. ... . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . 27 1.3.3 Equine adenovirus . . . . . . . . . . . . . ... . . . .. . . . . . . . . .. . . . . ... . . . . . . . . . . . . . . . ... . . . . . . .. . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . 34 1.3.4 Equine arteritis virus . . . . .. . . . . . . . . . .... . . .... . . . ...... .. .. . . . . . . . . .. . . . . . . . . : . .. .... . . . . . . . . . . . . . ... . . .. . . . . . . .. . . .. . . . . .. .. . . 41 1.3.5 Equine Reo viruses . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . .. . . . . . . . ..... . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . ... . . . . . . . . .... . . . . . . .. . . . .. . 45 1.3.6 Equine parainjluenza virus-3 . . ..... ... . . . .. . . . . . .. . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . .... . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . 49 1.3.7 Equine influenza virus .. .... . .. . . . . . . ... . . . . . . .. . . . . . .. . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . .. .. ... . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. 52 lA AIMS AND SCOPE OF THE THESIS . .. .. ..... . . . . . . . . . . . . . . . . . . . . . ... . . . . . .. . . . . . . ... ... . . . .. . . .. .. ... . . .... . ... . . .. . . .... . . . .. . . 53 CHAPTER 2:DESIGN OF THE SURVEY ........................................................................................... 57 2. 1 INTRODUCTION . . .. .. . . . . .... . . . . . . . . . . . . . . . . . . ....... ... . ..... . . ... . . .... . . . . . . ... . . . . ..... . . . .. . . ... .. . . ... . . . . . . .. . . . . . . . . . . . . . . . . . . 57 2.2 GENERAL MATERIALS AND METHODS . .. . .. . . . . .. ... ... . . ......... . .. . . . . . ..... . . .. . . . . . . ... . . . . . . . . . ... . ... . . . . ....... . .. 58 2.2. 1 Horses .. ... .. ... . . . . . . . . . . .... . . . . .. . . . . . ...... . . . . . . . . . . . . . . . .. . . . .. ... . . . . . . . . . . . . . . . . . . . .. . . . . . . . . ...... . . . . . .. ... . . . . . . . . . . . . . . . . . . 58 2.2.2 Collection of samples . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . .. . . . . . ... . . .. . . . ........ . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . .. .. .. 64 2.2.3 Processing of samples . . . . . . . . . . . . . . . .. . ....... . . .. .. ... . . . . . . .. . . . . . . .... . . . . . . . . .. ........... . . . . . . . . .. .. ... . . . . .. . . . . . . .. .. 65 CHAPTER 3:VIRUS ISOLATION ..................... , ................... ............................................................... 67 3. 1 INTRODUCTION .. .. . . . . . . . ..... . . . ..... . ... . . ... ....... . . ......... ..... . . . . . . .. . . . . ........... . . . . . . . . .. . .......... . . . . . . . . . . . .. . . . . . . . 67 3.2 MATERIALS AND METHODS . . ... . . .. . . ... ......... . . . . ... . . . . ... . . . . ... . . . .. ....... . . . . . . .... . . . . . .. . . . .. . ...... .. . . . . . . . . . . .. . . 68 3.2.1 Cell culture . . ... .. .. .. . . . . . . . . . .. . . . . . . . . . .. ... . . . . . . . . . . ... .. .. . .. . . . . .. . . . . . .. . . . . . . . . . . .. .... . . . . .. . . . . . . . . . . . . . . . . . . ... . . . .. . . . . 68 3.2 .2 Collection of samples . ... . . . . . .. . . . . . . . .. . . . .. . . . . . . . . . .. .. ... . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . .. . . . . . . ... . . . . 70 3.2.3 Processing of samples ... . . . .. . . . . . . ... . . .. .. .. . . . . . .. .. .. . . .. . . . . . . . . . . .. .. .. . . . . . .. .. . . . .. . . . . ......... . . . . . . . . .. . . . .. . . . . . . 70 vu 3.2.4 Virus detection . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . 71 3.2.5 Polymerase Chain Reaction .... . . . . .. . . . .... . . .. . . . . ......... . ..... . .... . . .. .. .... ....... .... . ...... .. . . .. ......... . ..... . 72 3.3 RESULTS ............. . . . . . .. . . .... . .... ..... .. .. . .. ....... .. . ........ . . . . .. .. .. . .. .. . .. ... . . . . . .......... ............... ...... . . .......... 79 3.3.1 Foals . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . .. . . . . . . . . . . . ... . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 79 3.3.2 Outbreaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 82 3.3.3 Yearlings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 3.3.4 Other viruses . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . ... .... . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 83 3.3.5 Isolates negative hy PCR . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . .. . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 3.3.6 Primary isolation . . . . . . . . . . . . . . . .. . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . ... . . . . . .. .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 84 3.3.7 Comparison (�f cell culture and PCR results . . . . . . . . . .. . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 3.3.8 Association with clinical signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 3.4 DISCUSSION ............................................................................................................................. 87 3.4.1 Lack ()f isolation (�f viruses other than herpesviruses . . . . . . . . . .. . . . . ... . . . . . . . .. . . . . . . . ... . ... . . . . . . . . . . . . . . . 87 3.4.2 Unident!fied viral isolates . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . ... . . . . . . .. . . . . . . . . . . . . . .. ... . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 89 3.4.3 Clinical significance ol EHV-2 and EHV-5 infections . . . . . . . . . ... .... . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 90 3.5 SUMMARY .. ..... .. . . .. .. . . . . . ........ ...... . ...... ..... . . . ....... .. . . .... . .. .... ... .. . .. . .. . ... . . ................. . .... .. .. . . .... .. .... . 93 CHAPTER 4 :EHV-1/4 SEROLOGy ..................................................................................................... 95 4.1 INTRODUCTION ........................................................................................................................ 95 4.2 MATERIALS AND METHODS ..................................................................................................... 96 4. 2.1 Serum neutralisation test (SN) . . .. . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . .. . . ... . . . . ... . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 96 4. 2. 2 Blocking ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .... . . . . . .. .. . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . 97 4.2.3 Reproducihility of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 4.2.4 Definition of recent EHV-l and EHV-4 illfectiolls . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . ... . . . . . . . .. . .. . . . . . . . . . . . . . . 98 4.3 RESULTS .................................................................................................................................. 99 4.3.1 Reproducihility (�l results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 4.3. 2 Foals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . ... . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . 99 4.3.3 Yearlings . .. . . . . . . . . . . .. ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . ..... .. . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . 101 4.3.4 Outbreaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 105 4.3.5 Comparison between EL/SA and SN titres . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . 106 4.4 DISCUSSiON ........................................................................................................................... 108 4.4.1 Serology as Cl tool for diagnosis ()f recent EHV-l/4 infection . . . .. . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 4.4.2 Time «fEHV-114 il!fectioll . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . ... .. ...... . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 113 4.4.3 Protection and cross-protection from infectioll . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . 1/5 4.4.4 Correlation between recent EHV-l/4 infection and presence of clinical signs . . . . . . . . . .... . . 116 4.5 SUMMARy ............................................................................................................................. 118 CHAPTER 5 :EQUINE RHINOVIRUS SEROLOGY ....................................................................... 1 19 5.1 INTRODUCTION ...................................................................................................................... 119 5.2 MATERIALS AND METHODS ................................................................................................... 120 5.2.1 Serum neutralisation test .. . . . . . . . . . . . . . ... . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 120 vi i i 5.3 RESULTS ................................................................................................................................ 121 5.3. / Equine Rhinovirus- I . .. . . ....... . . ... . . . .......... . . . . . .. ... . . .. . . .... . . . . . . ....... . .... . .............. ............. .. . . . . . . . 121 5.3.2 Equine rhinoviru-2 . . . . . . . . . . . . ... . . .... . .... . . .... . . . . . . . . . . . . .. . . . . .. . . . . . . . ... . . . ... . . . . . ............... . . ...... . ...... .. .. 123 5.4 DISCUSSION ........................................................................................................................... 126 5.5 SUMMARy .............................................................................................................................. 131 CHAPTER 6:EQUINE ADENOVIRUS SEROLOGY ....................................................................... 1 33 6.1 INTRODUCTION ...................................................................................................................... 133 6.2 MATERIALS AND METHODS ................................................................................................... 134 6.2.1 Haemagglutination Inhibition test (Hl) . . . ........ . . .... . . . .. . . .. . . . . . . .. . . .. . . .. . . .. . . . . . . . . .. . . . . . . . . ...... .. .. . . 134 6.3 RESULTS ................................................................................................................................ 136 6.3.1 Foals ....... . .... ... . . . . . . . . . . . . . . . . .... . . .. . . . ...... . ..... . . .. . . .. .. ... . . . . .. . . .. . . . ...... . .. . . . . . . . . . .. .. . . . .. ... . . . . ..... . .... . . . . . 136 6.3.2 yearlings ..... . . ... . . . . . . ... . . . . . . .. .. . . . .... . . . .. . . . . . ... . ... . . . . . . . . . . ....... . .. .. ... . . . . . . . . .. ..... . . ... ... . . . . . ... . .. . . . . . . . . . . . 138 6.3.3 Outbreaks .. .. ......... ..... .. .. . .... ......... ... . . ... . .. . . ......... . . . ..... ..... . ....... . . ... . . . . ... . .... ..... . . ....... . .......... /39 6.4 DISCUSSION ........................................................................................................................... 140 6.5 SUMMARy .............................................................................................................................. 145 CHAPTER 7 :EQUINE ARTERITIS VIRUS, PARAINFLUENZA VIRUS·3 AND REOVIRUS·3 SEROLOGY .......................................................................................................................................... 147 7.1 INTRODUCTION ...................................................................................................................... 147 7.2 MATERIALS AND METHODS ................................................................................................... 148 7.2.1 Equine arteritis virus . . . . . . . . . . . .. . .. .. ..... . . . .... . . . . . . . . . . . . . . . . ...... . . . . .. . . .. . . . . . . .... . . . . . .. . . .. .... . . .... . . . . . . .. . . . 148 7.2.2 Parainfluenza virus-3 . . . . . . . . . . . . . . .. . . . . . . .......... . . . . . .. . . . . . . . . . . .. . . . . ...... . .. . . ..... . . . . ....... . . ....... . ... . . ...... 148 7.2.3 Mammalian Reovirus-3 . . . . . .. . . . . . . . . .. . . . . .. . . . . . . . .. . . . . . . . . .. . . . .... . . . . . . . . . . . . . . .. . . . . . ........ . . . . . . ..... . .... . . .. .. 148 7.3 RESULTS ................................................................................................................................ 152 7.3.1 Equine arteritis virus .. . . .. . . .. . . . .. . . .. ... . . . . . . ..... . . . .. . . ... . . ... . . . .. . . . . . ... . . . . . . .. . . .. . . . . . ... . . . . . . . . . . .. ..... . .. . . . 152 7.3.2 Paraif�fluenza virus 3 . . . . . . . . ...... . . . . . . . . . . . . . ... . . . . . . .... . . . . . . ... .. .. . .. . . . . . . . . . ..... . . . . . . . . . . . . . . ..... . .. . . .... . . ... 152 7.3.3 Mammalian Reovirus 3 .. ... . . . . . . . .... . . . . . ... . . ... . . . . . .... . . .. . . .. . ... . . . . . .... . . . . . . . . . . . ...... . . . ...... ... . .. .. . . . . . .. . 152 7.4 DISCUSSION ........................................................................................................................... 153 CHAPTER 8 :THE SURVEY - GENERAL DISCUSSION ............................................................... 155 8.1 INTRODUCTION ...................................................................................................................... 155 8.2 VIRUSES CIRCULATING IN NEW ZEALAND HORSES AND ASSOCIATION WITH CLlNICAL SIGNS 156 8.2.1 Outbreaks ()f respiratory disease . .. . . . .. ..... . .. . . .. . . . . . . .. . . . . . . .. . . . . .. . . . .... .. .. ... . . . . . . . . . . . ... . . . . . ... . . ... . . . 156 8.2.2 Yearlings from the yearling sales . . . . . . . . . .. . . . . .. . ... . . . . . . . . . . . . . . .. . . . . . .. . . . ....... .. . .. . .... . . . . . . . . . . . . . . . ...... 159 8.2.3 Foalsfallowed on Cl monthly basis . .... . . . . .. . . . . . . . . . .. . . .. . . . . . . . . . . ...... . . . . . .... .. . . . . . . . . . . . . . . . . . ... . . .. . . . . . . 160 8.3 TIME OF VIRAL INFECTIONS IN FOALS ..................................................................................... 1 61 8.4 SUMMARy .............................................................................................................................. 163 CHAPTER 9:GENOMIC COMPARISON OF EHV-5 ISOLATES ................................................. 169 9.1 INTRODUCTION ...................................................................................................................... 169 9.2 MATERIALS AND METHODS ................................................................................................... 170 IX 9.2.1 Viruses ................................... .............. .... . ............... . . ..... .. . . , .... ,., .. , .. '" .. , .".'"." ......... '." , .. 170 9.2.2 I)NA extraction ... " .. . ............. .. "" ..... . . . " ........... .. . . .................. . . . . . .. .......... , ...... "' ... "",, .. .... 171 9.2.3 Polymerase Chain Reaction ...... . . . ....................... .......... .. .. " .............................. " .. """" ... 171 9.2.4 Cloning PCR products .. " ............. . .. ..... ... ........ . . . " ...... " ... ......... . ... " .......... . . .......... " .... .. .. . . 171 9.2.5 Colony screening . . ........... . . ....... . . . ....................... . . ... ........ . . " ... . . . ... , . . . . .................. . . ....... , .. 173 9. 2.6 RFLP of glycoprotein B gene ......... ... . . .. ................. . .................. . . .. .. .. " ........ . . ........ "" . . . .... 173 9.2.7 Sequencing and sequence comparison ..... . . . . .. " ................ . . .. . . ............... ... . ...... .... ............. 174 9.3 RESULTS . . . .............. .......... . .. .. .. . . ................ ...... ........ .. . ........ ...... .. . . . . . . . . . . . .. . ................. ...... ...... 174 9.3.1 EHV-5 isolatedfrom different horses had different RFLP profiles . . . ..... " . .... ..... " ... " ... . . . 175 9.3.2 RFLP {Jr()tlles of EHV-5from the same horses were identical ............... ... . ..................... 175 9.3.3 Sequence comparison ........... . . ......................... ..... . . .... . ....... . ...... . ... .. .. .. .. , .......... ' . .. ........... , 177 9.4 DISCUSSION . . . ........... . .. ........ . .. ............ ............. . . ....... ....... . ....... ..... . . . . ........... ........ ..... ............. 177 9.5 SUMMARY ............ .... . . ........... . . .................................. .. .. . . ............ .. . . .. . . .. . .... . .......... , .......... , .. ,. 183 CHAPTER lO:REPRESENTATIONAL DIFFERENCE ANALYSIS OF EHV-2 INFECTED EQUINE LEIJCOCYTES ..................................................................................................................... 1 85 10.1 INTRODUCTION .... " .. . . . . . ,,, .. ... . . . . .. . . . . , , . .. . . . ' .. . . . . . . . .. . . . . . ,, . . . . , , . . , . , , . ... . . . . ..... . . . . . . . . . . ... . . . . . .. . .. . . .. . . . .. . " 1 85 10.1.1 Approaches to investigatioll (�f differentially expressed geneL . . .. . . . . . . . . ....................... . ... 186 10.2 MATERIALS AND METHODS .. . . ... . . . . . " ................................................. , .......................... " ....... 189 10.2.1 Equine herpesvirus-2 ........ . ...... . . . .. . . .................... . . . . . ................ , . . .............................. " . . . . , 189 10. 2.2 RNA extraction .. " .... ...... . . .... ......... " ....... .. ............... . . .................... . .. ........................... . ..... 190 10.2.3 cDNA synthesis .................... . ....... .. .... . ...... . . .. . . . .................. , ..... . .. . . ..... .. .. .... . . . " ........ . . ....... 190 10.2.4 Preparation «{driver and tester ................ " ........ ... . . ....... . . ...... . . . ..... . . ........... .. ......... " ...... 191 10.2.5 First round (�f ampl(fication generation ()f the first difference product (DP 1) . .... .. .. .... 194 10.2.6 Ligatiofl (�ffesters to J adaptors .......... . . . . .. . . . . . ... . . . . ... .. ........... . . ..... ...................... ........ ..... 194 10.2.7 Second round of amplification - generation ()f the second difference product (DP2 ) ..... 195 /0.2.8 Third round of ampl(ficatioJl - generatioll (if the third difference product (DP3 ) ........... 196 10.2.9 Cloning of the DP3 amplicolls ......... . ................ . . .... .... .. .. .. ............. ......... .... . . ............ . . ..... 196 10.2.10 Dot blots .... . . .. . ......... . . ... . . . .... ............. . . . ..... . . .. . . . . ................. . . . ........ .. .. .. ......................... 197 10.2.11 Preparation ofprohes ... ..... . .... . ............ . . ....... . . ............. ............................................... 197 10.2.12 Hyhridisation ...... . . ............................................ . . .... . . ........ . . . . . .. .. .. .. . . ............ .. ....... . ..... 198 10. 2./3 Selection of clones for further analysis .... .. . . .............. . . . . ...... . . . ....................... . . ......... . . 198 JO.2.14 Sequencing and molecular analysis .. .. ........ . . ........ .. . . . ....... . . . . . ......... . ........................... 199 10.3 RESULTS . . . .... . .... ......... . . .. .. . . .... . . .... . ...... ...... .. . . ............ ... . ............... .. .. . . . . ....... . . . ....................... 199 10.4 DiSCUSSiON . . .... . . ..... .. ...... . . . .. . . . . . . ......... .. ..... . .... . . .... . . . . ... . . . ... . . ... . ........ . . . . ..... . . . ....................... .. 203 1 0.5 SUMMARY . . . ..... . . . .... . . ......... ..... .............. . . ...... . . .............. . . . . ........... . . .... . . .. .. . ...... . . .. ... .... ......... . . 209 CHAPTER 1 1 :CONCLUDING REMARKS ....................................................................................... 2 1 1 APPENDiCES .......................................................................................................................................... 215 BIBLIOGRAPHy .................................................................................................................................... 237 x List of figures Figure 3.1 : Results of EHV- l /4 PCR visual ised on a I .S�. EtBr stained gel and a corresponding dot blot probed with either EHV - I or EHV -4 probe as indicated . . . ... . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 80 Figure 3.2: Viruses i solated from foals fol lowed on a monthly basis: group A (A), group B (B), and group C (C) . . . . . . . .. . . .. . . . .. . ... . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . .. . . . . . . . . .. . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . 8 1 Figure 3.3: An example of ampl i fication products from a PCR with EHV -2 (upper part of a gel samples I' to 1 6') and EHV -5 ( lower part of a gel - samples I to 1 6) primers, visuali sed on 1 .5% ErBr stained gel (a) and a corresponding dot blot probed with either EHV-2 probe (b) or EHV-5 probe (c) . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . .. . .. . . . . . .. . . . .. . . . . . . . .. . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. 85 Figure 4.1 : Foals group A: EHV- 1 /4 SN titres (A) and EHV- I ELISA titres (B) ..... . . . . . . .. .. . . . .. . . . . . . . . . . . . .. . 1 00 Figure 4.2: Foals group B : EHV- 1 I4 SN titres (A) and EHV -I ELISA titres (B) . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . .. . .. 1 0 1 Figure 4.3: Foals group C: EHV- 1 /4 SN titres (A) and EHV- I ELISA titres (B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 02 Figure 4.4: Yearli ngs: EHV- 1 /4 serology: SN titres (A) and blocking ELISA titres (B) . . . . . . . . . . . . . . . . . . . . . . . 1 04 Figure 4.5 : Outbreaks: EHV- I SN ti trcs (A) and ELISA t itres (B) . .. . .. . . . .. . . . . . . . ... . . . .. .. ... . . . .. . . . . .. . . . . . . . . . . . . . . . 1 06 Figure 4.6: Comparison between EHV - I block ing values and SN titres of sera from foals (A), yearl ings from the yearl ing sales (B) , and horses from outbreaks of respiratory d isease (C) . . . .. . . .. 1 07 Figure 5.1 : Foals group A: ERhV - I serology . . . . . .. . . . . . . .. . .. . . . . . . . . . . . . . .. . . ... . . . . . . . . . .. . . . . .. . . . . . . . . . . . .. . . . . . . . . . . ... . . . . . .. . 1 22 Figure 5.2: Foals group A: ERh V -2 serology .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . .. .. . . . . . . . . . . . . . . . .. .. . . . . . ... . . . . .. . . . . . . .. . . . 1 24 Figure 5.3: Foals group B : ERhV-2 serology . . . . .. .. . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . 1 25 Figure 5.4: Foals group C : ERhV-2 sero\ogy . . . . .. . . . . . . . . . .. . . . . . . . . . . . .. . . . . .. . . . . . . . . . . .. . . . . . . . . .. . . . . . .. . . . . . . . . . .. . . . . . .. . . . . . . . 1 25 Figure 5.5: Outbreaks: ERhV-2 serology . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ... . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . 1 26 Figure 5.6: Yearli ngs: ERhV-2 serology . . .. . . .. .. .. . . . .. . . .. . ...... . . .. . . . . ... . . .. . . . . . . . . . .. . ...... . . . . . . . . . .. . . . . . . . . . . . . . .. . . .. . . . . . 1 26 Figure 6.1 : Foals group A: EAdV - \ serology . . . . . .. . . . . . . .. . . ..... . . . . . . ...... . . . . .. . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . ...... . . . . . .. .. . 1 36 Figure 6.2: Foals group B : EAdV- 1 serology . . .. . . . .. . . . . . .. . . . . . . . . . . . .. . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . .. ... . . . . . . . . . . . . . . . . . . . . . . . . . 1 37 Figure 6.3: Foals group C : EAdV - I serology . ... . .. . .. . . . . . .. . . . ... .. . . . ......... .. . . .. . . ....... .. . . . . . . . . .. . .... . . . . . . . . . . . . . . ... . .. J 38 Figure 6.4: Yearl ings: EAdV - I serology . ... . . . . . . . . . . . . . .. . . . .. . . . . .. . . . . . . . . . . .. . .. . . . . . . . .. . .... . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . 1 39 Figure 6.5 : Outbreaks: EAdV- \ serology . . . . . . . . . . .. . . . . . . . . . . . . ... . . . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . .. . . . . . . .. . . . . . .. . . 1 39 Figure 8.1 : The activity of equine respiratory viruses i n horses and foals from outbreaks of respiratory disease . .. .. . .. . . ... . . . . ... . . . .... . . . . . .. . . . . . . . . .. . . . . . . . . . . . . ......... ... .... .. . . . . . . . ... .. . . . ...... ..... . . . . . . . .... .. . . ... . . . . J 57 Figure 8.2: Association between the presence of c l inical sings and recent viral i n fections in yearl i ngs from the yearl ing sales . . . . . . . . . . . .. . .. . . . . . . . . .. . . . . . . .. . . . . . . .. .. . . . . . . . . . . . .. . . . . . . . . . . . . .. .. . . . . . . . . . .. . . . . . . . . . . . . . . . . I S9 Figure 8.3: Foals group A (A), B (B) and C (C) : mean l i tres to EHV- 1 /4 ( i ) , EAdV-l ( i i ), ERhV-2 ( i i i ) , ERhV- 1 ( i v ), and virus isolation from nasal swabs (v) . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 65 Figure 9.1 : Amplification products of PCR with primers ( rp + fp) specific for EHV -5 gB . . . .. . . . . ... . . .. . .. 1 74 Figure 9.2: Amplification products of PCR colony screen i ng with EHV -5 rp and T7 primer. . . .. . . . . . . . . . . 1 7 4 Figure 9.3: The position o f predicted Bfa I sites i n a cloned EHV - 5 rp-T7 primer ampli fication product (yellow) and in a corresponding fragment of EHV-2 sequence (white) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 75 Figure 9.4: Bfa 1 d igest ofEHV-5 rp-T7 primer amplification products .. . . . .. .. . . . . . . . . . . . . . . . . . . . . . .. ... .. ... .. .. . . . . 1 76 Figure 9.5: Comparison of the nucleotide sequences of the EHV -5 inserts from the clones l isted on the left . . ..... . . . . . . . . . . . . . . . .. . .. . . ... .. .......... . . .. .... . .... . . . . . . ... . .. . . ..... . .. . . . ......... .............. .. . . . . . .. .. . . .. ....... . . ... .... 1 78 Figure 9.6: Comparison of the predicted amino acid sequences from the N term inus of gB . . . . . . .. . . . .. .. . . 1 79 Figure 10. 1 : Schematic diagram of cDNA RDA (adapted from Frazer et al. 1 997) . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . 1 9 1 Figure 10.2: Approximately O.S - I !lg of R representations (A) , DP 1 (B) , DP2 (C), and DP3 (D) run on 1 % agarose gels and stained wi th gel star nucleic acid stain (FMC) . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . .. 200 Figure 10.3: Colony dot blots of DP3( -) or DP3( +) products, probed with either DP3( +) or DP3(-) probes. as indicated . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . ... . . .. . . . . . . . .. .. . . . . . . . .. . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . 20 1 Xl Figure 10.4: Re-amplification products of peR using selected DP3( -) amplicons (top) and DP3( +) amplicons (bottom) as target DNA. . .. ....... . . . . . . . . . . . . . . . . .. . .. . . . . . . . .. . . . .. . ..... . . . . .. . .. . . . . . . . . . . .. .. . .... . . . . . . . . . . . . . 202 Figure 10.5: Approximately 500 ng of R representations, DP I, DP2, and DP3 products, obtained after subtraction of either (+) Of (-) samplcs, probed with DIG-labelled probes, as i ndicated . .. . .................... .. . . . .. . ................ . ............... . . ....... . . . . . ............. . . . .. .... . . .. . . .............. 203 XlI List of tables Table 2. 1 : Foals sampled on a monthly basis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Table 2.2: Yearl i ngs from the yearl i ng sales 1 997 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 Table 2.3: Horses from outbreak TA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Table 3. 1 : PCR primers and programs used in the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Table 3.2: EHV -5 isolation from foals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Table 3.3: Viruses isolated from horses from outbreaks of respiratory d isease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Table 3.4: Viruses isolated from yearlings from the sales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Table 3.5: Isolates negati ve by PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Table 3.6: Herpesvirus (EHV -2, EHV -5, or both) isolation from horses from outbreaks of respi ratory disease and yearlings from the yearl ing sales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Table 5. 1 : Outbreaks of respiratory d isease - horses positi ve for ERh V - I ant ibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 23 Table 7. 1 : Haemolysis standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1 Table 7.2: Haemolysis allowances for complement controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1 Table 9. 1 : Details of the source of the EHV-5 isolates used i n the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 70 Table 9.2: Comparison of RFLP patterns obtained for d ifferent EHV -5 isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 76 Table 10. 1 : GenBank sequences with signi ficant homology to the listed clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 Abbreviations AGID AIDS ATP ATV bp BLAST BSA CF Con A COPD CPE CTL CV DD ddNTP DIG dNTP DP EAdV EAV EBV EDTA EFK EHV ELH ELISA EM ERhV F FBS FM DV fp gB GM agar gel immunodiffusion acquired immunodeficiency syndrome adenosine-5' -triphosphate Antibiotic / trypsin I verse ne base pair basic local alignment search tool bovine serum albumin complement fixation concanavalin A chronic obstructive pulmonary disease cytopathic effect cytotoxic T Iymphocyte coefficient of variation differential display dideoxynucleoside-5' -triphospate digoxigenin deoxynucleoside-5' -triphospate difference product equine adenovirus equine arteritis virus Epstein-Barr virus ethylenediamine tetra-acetic acid equine foetal kidney equine herpesvirus Earles lactoalbumin hydrolisate enzyme linked immunosorbent assay electron microscopy equine rhinovirus fusion (glycoprotein) foetal bovine serum food-and-mouth disease virus forward primer glycoprotein B growth medium X III XIV GPCR HA H&E HCMV HI HIV HN HSV HVS IAA IL IPTG kbp LAT LB MAb MC MCP MEM+n MHC MIP MM NK O.D, OPD ORF PBL PBS PCR PHA PI-3 PNK PSK RANTES G-protein-coupled receptor haemagglutination haematoxilin and eosin human cytomegalovirus haemagglutination inhibition human immunodeficiency virus haemagglutinin-neuraminidase herpes simplex virus herpesvirus saimiri isoamyl alkohol immunoglobulin interleukin isopropylthio-p-D-galactoside kilobase pairs latency associated transcript Luria-Bertolini broth monoclonal antibody mononuclear cells monocyte chemoattractant protein minimal essential medium + non-essential amino acids major histocompatibility complex macrophage inflammatory protein maintenance medium natural killer optical density m'tho-phenylenediamine dihydrochloride open reading frame(s) peripheral blood leucocyte(s) phosphate buffered saline, pH 7.0 polymerase chain reaction phytohaemagglutinin parainfluenza virus-3 polynucleotide kinase penicillin I streptomycin I kanamycin regulated on activation of normal T cell expressed and secreted RBC RDA Reo RFLP RK-13 rp RT RT-PCR SDS SN SPF SV40 TAE TBE TCIDso TE TGF Thl TK TNF SN Vero red blood cell(s) representational difference analysis mammalian reovirus restriction fragment length polymorphism rabbit kidney-l 3 reverse primer room temperature reverse transcriptase-PCR sodium dodecyJ sulphate serum neutralisation specific pathogen free simian virus 40 tris / acetate / EDTA tris / borate I EDTA tissue culture infective dose 50% tris I EDTA transforming growth factor CD4+ T helper type I Iymphocyte thymidine kinase tumor necrosis factor semm neutralisation African green monkey (cells) xv XVI CHAPTER 1: LITERATURE REVIEW 1 . 1 EQUINE RESPIRATORY DISEASE 1.1.1 Respiratory disease as a cause of wastage for the equine industry Respiratory disease is common among horses worldwide. While usually not fatal, it has been regarded as an important cause of wastage for the equine industry. The term 'wastage' refers to financial losses due to any causes that prevent a hor e from successful competition. In most reports, wastage was defined a any injury or disease that results in lost training days, prolonged spell , or early retirement from racing (More 1 999; Bai ley & Rose 1 997; Linder & Dingerkus 1 993; Ros dale et aL. 1 985) . Respiratory problems were considered the second most important cause of wastage among British Thoroughbreds in training (Rossdale et aL. 1 985; Jeffcott et al. 1 982) . In England, approximately 20% of training day in a 2-year study period were lost due to equine respiratory disease (RossdaJe et al. 1 985 ) and respiratory problems prevented 1 2% of horses from racing at al l during one flat racing season (Jeffcott et al. 1 982) . In Germany, 1 2% of 348 recorded cases of training failure were due to respiratory infections (Linder & Dingerkus 1 993) . Coughs, colds and viral respiratory problems were considered the most important causes of wastage by Austral ian trainer (Bailey & Rose 1 997). Respiratory problem were also ranked as the third most important group of health problem among the equine population in Michigan ( Kaneene et al. 1 997b; Kaneene et al. 1 997a). They were ranked a the second most important by 1 1 49 veterinarians from different parts in the USA (Traub-Dargatz et al. 1 99 1 ). Factors interfering with successful breeding are also important causes of wastage (Jeffcott et aL. 1 982) . According to the results of leffcott et al. ( 1 982), wastage among breeding stock was predominantly caused by failure to conceive or carry the foal to term. However, pneumonia and respiratory tract disease were the most common causes of disease and death of foals 32 to 1 80 days old (Co hen 1 994). Thus, respiratory disease constitutes an important problem for the equine industry. Chapter I 2 1 . 1 .2 Equine respiratory disease - definition One of the problems associated with investigations of respiratory disease in horses is definition of the condition. The disease is commonly referred to as 'colds' or 'the virus' by trainers and owners. However, cl inical signs vary considerably. Some horses present with typical respiratory signs, but others look apparently healthy and the only 'sign' of disease is their 'poor performance' as subjectively assessed by their trainers. Based on observation and cl inical experience, Mumford & Rossdale ( 1 980) defined cases of respiratory disease as those presenting with one or more of the fol lowing signs: • Unexpectedly poor performance, during training gallops or in racing. • Excessive fatigue or respiratory distress after cantering or faster work. • Varying degrees of inappetance. • Rectal temperature more than 38.3 qc. • A 'staring' coat, with excessive detritus, hair loss or itchiness. • A serous to seromucous nasal discharge or conjunctivitis. • Enlargement of submandibular lymph nodes. • Intermittent or persistent coughing. • Small , dry or moist, foul smell ing faeces. • 'Stiffness' or 'setfast-l ike' signs before or after exercise. This i l lustrates well the difficulties in defining equme respiratory disease. Strict adherence to these guidelines would probably result in overestimation of the prevalence of the condition. On the other hand, more recent investigations revealed that many racehorses had subclinical lower airway inflammation detectable only by endoscopic examination (Burrell et at. 1 996; Sweeney et at. 1 992) . Indeed, horses involved in one study spent 33% of their t ime in training with a degree of lower airway disease, evidenced by an increased amount of tracheal mucus, that was l ikely to affect their abi l ity to perform (Burrel l et at. 1 996). One impl ication of these findings is that the role of some pathogens may have been underestimated, considering that most of the studies into the causes of equine respiratory disease and the relative importance of different pathogens involved were based on observation of overt c l inical signs. Equine respiratory disease is more frequently observed in 2-year-olds, in comparison with older horses (Linder & Dingerkus 1 993 ; Rossdale et at. 1 985) . It often occurs in a Literature Review 3 seasonal manner on both racetracks and stud farms (Cohen 1 994; Linder & Dingerkus 1 993 ; Sugiura et al. 1 987; Sherman et al. 1 979). This could indicate the influence of the weather on the activity of the infectious agents involved, but could also be related to the time of foaling (Cohen 1 994) or the seasonal racing pattern (Linder & Dingerkus 1 993) . 1 .2 I NFECTIOUS AGENTS ASSOCIATED WITH EQUINE RESPIRATORY DISEASE Pathogens that have been associated with equine respiratory disease i nclude viruses, bacteria, fungi and mycoplasmas. 1 .2.1 Viruses Several viruses have been associated with respiratory disease in horses (Mai r 1 996; PoweIJ 1 99 1 ; Jolly et al. 1 986; Mumford & Rossdale 1 980). A viral aetiology is often difficult to establish, as many outbreaks are investigated some time after development of acute c l inical signs. At that stage the i solation of respiratory viruses, if attempted, i s often unsuccessful . The results of some of the virological and serological investigations conducted in different countries are presented below. Germany According to a serological survey conducted in Germany in the years 1 986-87, between 40 and 80% of unvaccinated horses sampled had been exposed to i nfluenza virus, equine herpesvirus (EHV)- 1I4, mammalian reoviruses (Reo) or rhinovirus- l (ERh V- 1 ) and nearly 100% to equine adenovirus- l (EAdV - J ) . The prevalence of antibodies to equine arteritis virus (EA V) was less than 10%. Prevalence of antibodies to EA V and EAdV - 1 were found to be similar in c l inically normal horses, diseased horses and horses with chronic respiratory disease. The antibody prevalence to equine influenza viruses, reoviruses, ERh V - 1 , and EHV - 114 was found to be higher in chronicall y diseased horses than i n horses with acute disease o r c linicall y normal ones. Among horses with acute disease, the most common infection was EHV - 114 ( 2 1 .2 %) , fol lowed by influenza A equi 2 ( l L7 %) , ERhV- l ( 10.2 %) , EAdV- l (4%) and Reo- I /3 ( 1 %) . None of the horses with acute disease showed serological evidence of recent Reo-2 or EA V infection (Herbst et al. 1 992) . Chapter 1 4 United Kingdom Respiratory disease has been recognised as a problem in Britain and, fol lowing recommendations made In a report to the Joint Racing Board in 1 97 1 , several investigat ions of the problem were conducted. Equine herpesvirus- 1 I4 (Powell et al. 1 978; Powel l et al. 1 974), equine influenza viruses (Powell et al. 1 978; Rose et al. 1 974), EHV-2 (Rose et al. 1 974), ERhV- l and EAdV (Powell et aL. 1 978; Powell et aL. 1 974) were all found to be associated with outbreaks of respiratory disease. However, infections with each of these viruses were also de cribed in healthy horses. Also, many outbreaks remained undiagnosed (Powell et aL. 1 978) . Switzerland The viruses found to circulate among horses in Switzerland included EHV - 1 14, influenza A equi 1 and 2, ERh V - 1 and 2, EAdV, and EA V (Gerber et al. 1 978; Hofer et aL. 1 978; Hofer et aL. 1 973) . Equine herpesviruses I and 4 were often isolated from animals showing respiratory igns (Hofer et al. 1 978; Hofer et al. 1 973) . However, EHV - 1 14 infection was diagnosed serological ly in a number of hor es without overt disease (Hofer et aL. 1 978). S imi larly, equine rhinovirus (ERhV) infection were identified in healthy animals as wel l as those showing respiratory signs (Hofer et al. 1 978; Hofer et al. 1 973) . Influenza infections were associated with respiratory disease in one study (Hofer et al. 1 973), but no evidence of equine influenza infections could be found during another survey among horses submitted to one equine clinic (Hofer et al. 1 978). From the serological results it was also apparent that EA V was present in Switzerland, and respiratory disease due to EA V infection was described by Gerber et aL. ( 1 978). The Netherlands According to a serologicaJ survey conducted in The Netherlands on equIne sera col lected from foal and horses of various breeds and locations, 76% of 288 had antibody to EHV- 1 I4, 39% of 264 to EAdV, 65% and 59% of over 500 sera tested were positive for neutral ising antibodies to ERhV- l and ERhV-2, respectively, 1 0% and 33% of 600 sera were positive for haemagglutionation inhibition (HI) antibodie to Reo- l and Reo-2, respectively, and 3 .6% of 39 1 sera for H I antibodies to Reo-3 . Although 33 .5% of adult racehorses were positive for EA V antibodies, all 1 47 sera tested from Literature Review 5 horses born after 1 968 were negative. Generally, the highest prevalence of antibody to al l viruses was observed in racehorses over 1 year of age. Sera from studs with overt respiratory disease were excluded from this survey (de Boer et al. 1 978) . Poland In Poland, a survey conducted on 722 healthy horses from three breeding farms revealed that 48.5% of horses sampled had antibodies to equine i nfluenza, 33 .5% to parainfluenza virus-3 (PI-3) , 23 .8% to Reo- I , 0.55% to EAdV and 1 2.9% to EHV- 1 /4 (Zmudzinski et al. 1 980). The low prevalence to EAdV was most probably a result of testing methods, as EAdV antibody was detected by complement fixation (CF) with adeno-3 antigen. No further information on the source of this antigen was provided. Canada Viruses that have been identified among Canadian horses include equine infl uenza A equi 1 and 2, EHV- 1 I4, EHV-2, ERhV- I and ERhV-2. Equine influenza i nfections (Sherman et al. 1 979; Ingram e t al. 1 978) or ERh V -2 infections (Carman et al. 1 997) were most often associated with development of c l inical signs. Influenza vims usual ly affected horses younger than 5 years of age. Equine herpesvirus- 1 I4 was i solated on several occasions from horses showing respiratory s igns, but i t was associated with sporadic cases rather than with any of the outbreaks (Sherman e t al. 1 979; Ingram et al. 1 978) . However, seroconversion to equine influenza and EHV - 114 viruses was also demonstrated in apparently healthy horses. Equine herpesvirus-2 was frequently isolated from nasal swabs, but the significance of these isolations was not estab l ished (Sherman et al. 1 979). United States of America The viruses most commonly associated with outbreaks of respiratory d isease among horses in the USA included EHV - 114 and equine influenza v iruses (Mumford e t ai. 1 998; Powel1 1 99 1 ) . During a recent survey conducted among horses with upper respiratory disease in Colorado, influenza virus infection was i dentified in 43 of 1 12 horses sampled, EHV - 1 14 infection i n 1 8, and mixed EHV - 1 14 and influenza virus infections in four horses. The study did not involve sampling of healthy control horses, and thus the causative role of the viral infections in the horses sampled could not be estab l ished (Mumford et al. 1 998) . In another study, approximately 3 1 % of 523 horses Chapter 1 6 sampled i n different states of the USA, usual ly as part of routine health examinations, had antibodies to EHV- 1 /4, 97% to EHV-2 , and 1 1 % to EAV. The antibodies to all three viruses were most prevalent in Standardbred horses, although this may have been influenced by the relatively small number of S tandardbred horses sampled (McGuire et al. 1 974). Additionally, several c linical outbreaks of respiratory disease due to EAV infection have been reported (Timoney & McCollum 1 990). Japan In Japan, a large-scale epizootic of influenza at the end of 1 97 1 caused races to be cancelled for several months (Kono et al. 1 972) . In a serological survey conducted i n this country at two training centres from 1 980 to 1 986, seroconversion to a variety of infectious agents was observed in 23 .5% of the horses with pyrexia (Sugiura e t al. 1 987). Of the horses showing seroconversion, about 74% seroconverted to EHV - 1 14, 1 4% to ERhV- l , 9 .3% to rotavi rus, 3 .2% to EAdV and one horse to reovirus. None of the 1 05 horses tested showed serological evidence of recent EHV-2 infection, although all were positive for CF antibodies to EHV -2, and none of the horses tested had H I antibodies against PI-3 . Thus, EHV- 1 I4 seemed to be the most common cause of pyrexia among horses sampled. However, in 76.5% of cases the cause of pyrexia was not determined. Also, the serum samples collected were not checked for antibodies to equine influenza viruses, probably because they were regarded as protected from influenza infection by vaccination. However, vaccination to equine influenza not always protects against i nfection (Mumford et al. 1 998) . Thus, some of the horses in which the cause of pyrexia was unrecognised may have been infected with equine i nfluenza virus. New Zealand Several v iruses which cause equine respiratory disease overseas, have also been found to circulate in New Zealand horses (Jolly et al. 1 986). Approximately 66% of sera, collected from 68 5- to 1 1 -month-old foals w ith histories of respiratory disease, were positive for EHV - 1 14 antibodies, nearly 1 00% for EHV -2 antibodies, 1 2.3% for antibodies against ERhV- l , 41.2% for antibodies against ERhV-2 and 3% for EAV antibodies. Of 55 sera collected from 1 - to 9-year-old Thoroughbred racing horses, 37.7% had antibody to ERhV- l , 84.9% to ERhV-2, and only one horse had antibody against EA V (Jol ly et al. 1 986). Adenoviruses were isolated from cl inical ly sick horses in New Zealand and the seroprevalence of infection in the northern part of North Island - - - - - - ---- -� -- - - - - - -- - - - - - - Literature Review 7 was found to be 39% as measured by an agar gel immunodiffusion (AGID) test (Homer & Hunter 1 982) . There has been no reported evidence of influenza v irus, reovirus, coronavirus or PI-3 infection in New Zealand horses (Homer & Ledgard 1 988; Jolly et al. 1 986). 1 .2.2 Bacteria Several bacterial species have been isolated from horses with respiratory disease (Nordengrahn e t al. 1 996; Burrel l e t a!. 1 996; Wood et al. 1 993; Hoffman e t al. 1 993; Traub-Dargatz et al. 1 99 1 ; MacKintosh e t al. 1 988) . Streptococcus equi subspecies zooepidemicus, S. pneumoniae or Pasteurella/Actinobacillus-like species were most commonly associated with lower airway disease (Ward et al. 1 998; Burrel l et al. 1 996; Burrel l et al. 1 994; Hoffman et al. 1 993 ; Wood e t al. 1 993) . In some cases, bacterial infections were preceded by viral infections, which may indicate a predisposing role of viruses for secondary bacterial infections. However, such an association was not found using the statistical methods employed (Burrel l e t al. 1 996; Burrel l e t al. 1 994) . The distal trachea of normal horses is not sterile, but has flora that reflects the environment of the horse (Whitwell & Greet 1 984). Thus, results of some studies that used bacteriological culture of nasal or nasopharyngeal swabs (Carman et al. 1 997; Burrel l et al. 1 986) to assess the importance of different bacterial species i n equine respiratory disease should be interpreted with caution (Sherman et al. 1 979). 1 .2.3 Fungi Fungal infections have been associated with the development of chronic pulmonary disease in horses (McPherson et al. 1 979). Fungal i nfections are uncommon in New Zealand horses. Many fungal species that are capable of causing respiratory s igns i n horses are exotic to New Zealand. A spergillus species are present i n New Zealand and i nfection w ith them can cause guttural pouch mycosis with respiratory s igns (Julian 1 992). 1 .2.4 Other pathogens Chlamydia psittaci has been associated with a variety of c l inical syndromes in horses including pneumonia, polyarthritis, hepatitis, keratoconjunctivitis, rhinit is and abortion. However, there is no clear association between the isolation of C. psittaci and disease Chapter I 8 and it has been isolated from the respiratory tract of both healthy and diseased animals (Mair & Wills 1 992; Burrell et al. 1 986) . Mycoplasma felis and M . equirhinis have been isolated from both healthy horses and those showing signs of respiratory disease (Antal et al. 1 989; Poland & Lemcke 1 978; Moorthy et al. 1 976; AlIam & Lemcke 1 975; Windsor 1 973) . Mycoplasma felis has been isolated from horse with pleuritis (Hoffman et al. 1 992 ) and has been hown to cause pleuritis after experimental infection (Morley e t al. 1 996; Ogilvie et al. 1 983) . Recently, Wood e t al. ( 1 997) reported an outbreak of respiratory disease among British racehorses in training associated with M. felis infection . Also, both M. felis and M. equirhinis were isolated from horses with acute re piratory disease during a recent study in Canada, with M. equirhinis isolated from more than 50% of hor es examined (Carman et al. 1 997) . 1 .3 EQUINE RESPIRATORY VIRUSES 1.3.1 Equine herpesviruses The family Herpesviridae Family Subfamily H erpesviridae A lphaherpesvirinae � Simplexvirus Varicellovirus Equ ine herpesviru - \ (EH V -I) Genus Genus Subfami ly Genus Genus Genus Subfamily Genus Genus (Murphy e t al. 1 995) Betaherpesvirinae Cytomegalovirus Muromegalovirus Roseolovirus ---l Gammaherpes virinae I Lymphocryptovirus Rhadinovirus Classification and general characteristics A typical herpesvirus consists of: Equine herpesviru -4 (EHV-4) Equine herpesv irus-2 (EHV-2) Equine herpesvirus-5 (EHV-5) • a core containing a l inear double stranded DNA 1 24-235 kbp in size ��-�--------- - -- - - Literature Review • an i socahedral capsid containing 1 2 pentameric and 1 50 hexameric capsomers • an amorphous tegument located between the capsid and the envelope 9 • an envelope derived from altered nuclear membrane of an infected cell containing glycoprotein spikes The size of virions ranges from 1 20 to 300 nm in d iameter. They are unstable i n detergents or other lipid solvents and less stable in low than neutral pH values (Murphy e t al. 1 995) . Herpesviruses have been divided into three subfamilies: A lphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae based on biological properties, and i nto six groups designated A through F based on the genome arrangement (Roizman 1 996) . Due to the existence of varied number of repeats in herpes viral genomes, the size of the individual genomes may vary by more than 1 0 kbp (Roizman 1 996). All herpesviruses code for several enzymes involved in nucleic acid metabolism and DNA synthesis . They are also able to establish latent i nfections in their hosts. Biological properties of herpesviruses differ between different members of the famil y. Alphaherpesviruses are characterised by a variable host range, relatively short reproductive cycle, rapid spread i n culture, efficient destruction of infected cell s and the abil ity to establish latent infection in sensory ganglia (Roizman 1 996). Betaherpesviruses are characterised by a restricted host range, long reproductive cycle and slow spread of infection from cell to cell in culture. Infected cells frequently become enlarged and carrier cultures are readily establ ished. The v imses can establish latency in secretory glands, lymphoreticular cel ls , kidneys and other tissues (Roizman 1 996) . Gammaherpesviruses have usual ly a l imited host range. In vitro, they replicate i n lymphoblastoid cells . Some can I yticly infect epithelial and fibroblastic cel l s . Gammaherpesvimses are usually specific for either T or B lymphocytes. In the lymphocyte, infection is usually either at the latent or pre-Iytic stage, but without production of infectious v irus . Latency is established in l ymphoid tissue (Roizman 1 996; Murphy et al. 1 995) . Al l herpesviruses attach to one or more cellular receptors and enter the cell through the fusion of the plasma membrane with the viral envelope. The capsid is transpOlled to the nuclear pore and v iral DNA enters the nucleus, where it is circularised. During l ytic Chapter J 1 0 infection, transcription of early genes i s induced, mRNAs are transported to the cytoplasm and translated. Early proteins are transpOIted back to the nucleus and are involved in production of beta mRNAs, which are necessary for replication of viral DNA by rol l ing circ le mechanism and transcription of the late mRNAs, which code mostly for structural proteins. Unit lengths of viral DNA are packaged into empty capsids. Virus replication and the formation of infectious particles occur in the nucleus. The envelope is acquired in the process of passing through the nuclear membrane into the cytoplasm of infected cells. The final processing of glycoproteins occurs in the Golgi apparatus and finally virions are released into the extracellular space. This i s always accompanied by the destruction of an infected cell (Roizman 1 996; Murphy e t al. 1 995). Latency Latent infection is defined as non-productive infection in which the v iral genome i s present, but gene express ion i s l imited and infectious virus i s not produced . Latency i s a typical feature of herpesviruses and serves as a sophisticated strategy for escape from surveil lance by the immune system. Al l herpesviruses establish l atency, although molecular mechanisms and sites of latency are different for different viruses. Latency of herpes s implex virus (HSV) has been the most extensively studied. During latency, the HSV genome is harboured in neural cel l s and only a part of the genome ( latency associated tra�script or LA T) is expressed. There is no known LA T protein product, but even if there were one it could not be appropriately presented to T cells, s ince neurones express only low levels of major histocompatibi l i ty complex (MHC) proteins, necessary for presentation of a foreign antigen to T cell s . Human cytomegalovirus (HCMV) establ ishes latency in lymphocytes and macrophages and its gene expression i s l imited to immediate early and early genes. It has not been determined whether any protein products are produced in latently infected cells (Banks & Rouse 1 992; Mocarski e t al. 1 990; R ice et al. 1 984). By contrast, at least nine viral proteins have been identified in B lymphocytes latently infected with Epstein-Barr virus (EBV) (y-herpesvirus) (Rickinson & Kieff 1 996). Some of these protei ns can be recognised by the immune system. Despite this, latently infected B lymphocytes expressing these foreign antigens on their surface survive and are not eliminated by the immune system. Additionally, cell s expressing all latent proteins are protected from Literature Review 1 1 programmed cell death - apoptosis (Banks & Rouse 1 992) . The exact mechani sms behind these phenomena are not ful ly understood (Gregory et at. 1 99 1 ) . Equine herpesvirus-2 Equine herpesvirus-2 was first isolated in 1 963 (Plummer & Waterson 1 963) . It was provisional ly included among p-herpesviruses (Roizmann et al. 1 992), but was recently reclassified as a y-herpesvirus (Telford et al. 1 993) . The exact role of the virus in producing disease in horses i s not known, although i t has been associated with a variety of clinical signs (see later this section) . B iology i n vitro The biology of EHV -2 in vitro has been reviewed by Browning & Studdert ( 1 988) . The virus has a relatively broad host range and can productively infect cell s of equine origin as well as rabbit, guinea pig, fel ine and ovine cell cultures. Some i solates also grow in bovine, monkey and hamster kidney cells, bovine endothel ial and African green monkey (Vero) cell s . Cytopathic effect (CPE) consists of foci of small , rounded, refract i le cell s that eventuall y detach. Some isolates produce bal looning of cells with syncytia formation, which is most apparent in rabbit kidney (RK- 1 3 ) cells . Stained cell preparations show swollen nuclei and large, distinct, intranuclear inclusion bodies (Wharton et al. 1 98 1 ; Horner et al. 1 976; Wilks & S tuddert 1 974; Kemeny & Pearson 1 970; Plummer & Waterson 1 963) . Viral structures are first observed in the nuclei of infected equine cells 48 hours post infection, and by 72 hours post infection enveloped virions are readi ly detectable in the cytoplasm (Wharton et al. 1 98 1 ). Equine herpesvirus-2 has a typical herpesvirus structure, with a diameter of approximately 1 40 nm (Wharton et al. 1 98 1 ) . Genome structure The EHV-2 genome i s a l inear, double stranded molecule with a buoyant density value of 1 .7 1 65 glee, which corresponds to G+C content of 57 .7% (Wharton et al. 1 98 1 ) . The genome of EHV-2 strain 86/67 was pmtially c loned and mapped by digestion with restriction endonucleases (Browning & Studdert 1 988) . A totall y d ifferent structure was proposed by Calacino et al. ( 1 989), but i t was revised by Raengsakulrach & Staczek ( 1 992) to agree with the one described by Browning and Studdelt. In 1 995, the complete DNA sequence of EHV-2 strain 86/67 was published (Telford e t al. 1 995) . Chapter I 1 2 The sequence consists of 1 84,427 bp, comprising a unique region of 1 49,32 1 bp flanked by 1 7 ,553 bp terminal direct repeats (class A) . In contrast to other known y­ herpesviruses, the G+C distribution is uniform in the entire genome. Similarly to EBV and herpes virus saimiri (HVS) , the EHV-2 genome is deficient in CG dinucleotide, which is probabl y a consequence of methylation of a latent virus (Honess et al. 1 989). Seventy-nine open reading frames (ORF) have been identified, which are predicted to code for 77 distinct proteins. The genome analysis confirmed that EHV -2 belongs to the subfamily Gammaherpesvirinae and is marginally c loser to HVS than to EBV (Telford et al. 1 995; Telford et al. 1 993) . There are, however, marked differences between the genomes of EHV -2 and other y-herpesviruses, i ncluding G+C distribution and possession of interspersed, non-coding regions. This may suggest that EHV -2 comprises another, distinct subgroup of y-herpesvimses (Telford et al. 1 995) . Genomic and antigenic heterogeneity Different isolates of EHV -2 were shown to be antigenical ly related in serum neutralisation (SN) tests (Kemeny 1 97 1 ; Plummer et al. 1 969). Other reports demonstrated considerable antigenic heterogeneity in simi lar tests (Steck et al. 1 978 ; Harden et al. 1 974; Plummer et al. 1 973) . These authors, however, did not distinguish between EHV-2 and the newly discovered EHV-5 (see below). Accidental i nclusion of EHV-5 isolates among the EHV -2 isolates tested may have lead to the greater heterogeneity observed, as i t has been shown that EHV -2 and EHV -5 share many common epitopes, but also possess type speci fic antigens (Agius & Studdert 1 994) . Although rabbit antisemm raised against EHV - 2 cross-reacted with EHV - 5 and vice versa in ELISA tests, calculations of antibody t itres showed that the titre of homologous semm was almost ten-fold higher than the titre of heterologous semm in both cases (Agius et al. 1 994) . Common antigens among EHV-2 isolates could be demonstrated by CF and indirect immunofluorescence (Steck e t al. 1 978), although other authors showed that these techniques detect common herpes viral antigens (Watson et al. 1 967), so they may not distinguish between EHV-2/5 and other equine herpesvimses. Browning & Studdert ( 1 989; 1 987b) showed considerable heterogeneity among EHV-2 isolates using restriction enzyme analysis. Of 75 sites mapped, 40 were variant among 1 5 isolates tested, with 3 to 2 1 sites variant when any two strains were compared. Literature Review --�-�- - - - - 1 3 Polymorphism independent of c leavage s ite variation was also observed (Browning & S tuddert 1 989) . Structural proteins and glycoproteins S tructural proteins and glycoproteins of EHV -2 were examined by Caughman et al. ( 1 984) and Agius et al. ( 1 994) . At least 37 proteins have been identi fied in purified EHV -2 preparations, including nine major nucleocapsid proteins. The most immunodominant protein seemed to be glycoprotein B (gB) , but at least 1 1 proteins from infected cel l lysates were immunoprecipitated by rabbit antiserum raised against purified EHV-2. The genome structure and protein profile of EHV-2 and EHV-5 are colinear with those of other y-herpesviruses (Telford et a!. 1 995; Agius & S tuddert 1 994; Tel ford et al. 1 993) , although both v iruses also code for type specific proteins not specified by other herpesviral genomes (Telford et a!. 1 995 ; Agius & Studdert 1 994). Interaction with other viruses The possibi l i ty of in vitro interference between EHV -2 and EAdV was suggested by Gleeson & Studdert ( 1 977) . In another study, recovery of latent EHV- 1 and EHV-4 was always accompanied by isolation of EHV -2 from the same samples (Welch et al. 1 992) . The authors suggested that EHV -2 might play a role in trans-activation and reactivation of latent EHV - 1 and EHV -4. This hypothesis was supported by the findings of Purewal et al. ( 1 993) , who showed that EHV -2 can trans-activate immediate early genes of EHV - 1 and HSV - 1 in vitro. The same authors also reported the existence of homology between immediate early genes of EHV -2, EHV - 1 and HSV - 1 in cross-hybridisation studies. The similarity of immediate early genes of EHV -2 and EHV - 1 could faci l i tate trans-activation, but it could also lead to competition between these two viruses. Dutta et al. ( 1 986) demonstrated the interference between EHV -2 and EHV - I in vitro. Equine herpesvirus- I expression in dually infected lymphocyte cultures was markedly reduced in the early stages of virus multiplication when compared to cultures infected with EHV - 1 alone. However, this effect was transient and after ini tial interference, EHV-2 infection seemed to have a stimu lating effect on EHV - 1 expression i n lymphocyte cultures . In dually infected equine dermis cel ls , the i nterfering effect was more prolonged with no subsequent stimulation observed, but these cultures were studied for a shorter time after infection than were lymphocytes. Whether these reactions occur in vivo and how important they are has not yet been established. Chapter I 1 4 Disease The exact role of EHV-2 in producing disease has not been estab l ished. However, there is increasing evidence that the importance of this virus may have been underestimated due to its low pathogenicity and widespread distribution. Equine herpesvirus-2 infection has been l inked to a variety of c lin ical signs including upper respiratory d isease (Fu et a!. 1 986; Palfi e t al. 1 978; Kemeny & Pearson 1 970), chronic pharyngitis (Blakeslee et al. 1 975), keratoconjunctivitis (Collinson et al. 1 994), lower respiratory d isease (Murray et al. 1 996; Nordengrahn e t al. 1 996; Belak et al. 1 980; Palfi e t al. 1 978; Studdert 1 974), or general malaise and poor performance (Jensen-Waern e t al. 1 998; Rose e t a l . 1 974). Nordengrahn et af. ( 1 996) provided indirect proof of EHV-2 involvement in respiratory disease in young foals . These authors prevented the occurrence of Rhodococcus equi pneumonia in foals by vaccinating them with an EHV- 2 subunit vaccine. Murray e t al. ( 1 996) showed a greater rate of EHV-2 i solation from tracheal aspirates from foals with c lin ical signs of lower respiratory d isease than from cl inically unaffected foals. Also, in another study (Borchers et al. 1 997a), a h igher prevalence of EHV -2 infection was found i n horses with respiratory problems and ataxia (70-7 1 %), than in c linicall y normal horses (42%) . Al l of these observations, although suggestive, do not provide definite proof of the involvement of EHV -2 in equine respiratory d isease. Experimental infection with EHV- 2 produced either no overt disease or onl y mild respiratory signs (Borchers et al. 1 998 ; Gleeson & Studdert 1 977; Blakeslee et al. 1 975) . Epidemiology Equine herpesvirus-2 i s widespread among horse populations. The prevalence of infection in healthy, adult horses aproaches 90% (Roeder & Scott 1 975; Kemeny & Pearson 1 970) . Foals are born EHV -2 negative and become infected soon after birth often in the presence of maternal antibodies (Murray e t al. 1 996; Fu et al. 1 986; Wi lks & Studdert 1 974). The route of infection is via the respiratory tract. The level of colostral EHV-2 antibodies is usual ly low and there is a steady increase in antibody level during the first few months of l ife, which indicates an immune response to persistent or repeated infections (Murray et al. 1 996; Bel 1 00) protected horses from infection, suggesting that humoral immunity may also play some protect ive role. However, the levels of cellular immunity were not reported in this study. Therefore, the protection observed in horses with high antibody levels may have been conferred by cellular immunity rather than humoral responses, although i n none of the studies (Edens et al. 1 996; Pachciarz & Bryans 1 978; Wilks & Coggins 1 976) was any correlation between cellular immunity and neutralising antibody levels to EHV - 1 observed. Subsequently, further studies were conducted in order to determine the rel ative importance of different mechanisms of cell mediated immune responses i n protection from infection. Although the results of different investigations varied sl ightly, all indicated the presence of MHC restricted T-ceI l mediated cytotoxicity in l ymphocytes stimulated with either EHV - 1 or EHV -4 antigens. The presence of cross-reactive epitopes was also evident (Ell is et al. 1 997; Edens et al. 1 996; Al len et al. 1 995; Edington et al. 1 987) . Antibody dependent cel lular cytotoxicity responses (ADCC) and complement dependent lysis have also been shown to be induced by EHV - 1 vaccination (Stokes & Wardley 1 988). They did not, however, prevent horses from being infected with EHV - 1 in challenge experiments, which indicates that ADCC or complement dependent l ysis are not l ikely to be the main immune mechanisms in protection from EHV - 1 infection. Most probably, it is co-operation between several different defence mechan isms, including systemic cell mediated and humoral responses, but also local musocal immunity, that confer protection from EHV - 1 infection and abortion (Allen e t al. 1 999), Chapter 1 24 Cross-reactivity and cross-protection between EHV- J and EHV-4 The level of cross-reactivity and cros-protection between EHV- l and EHV-4 has been of interest with respect to vaccination. The data regarding this issue, however, are mostly conflicting (Stokes et al. 1 99 1 ; Edington & Bridges 1 990). This is well i l lustrated by the fact that the immunization against both viruses u ing commercially avai lable vaccines has proved to be very unpredictable and often not protective (Burki et al. 1 99 1 ; Burki et al. 1 990; Burrows et al. 1 984; Dutta & Shipley 1 975) . The main difficulty hindering any investigation is the necessity to use either SPF/gnotobiotic foals or conventional horses with a known EHV - 1 14 status. The production and use of SPF or gnotobiotic animals is expensive and it has been reported that they may react differently to conventional animals, most probably due to different bacterial flora (Fitzpatrick & Studdert 1 984). Defining the EHV-free status of conventional horses is even more problematic, as negative serology results do not necessari ly mean that the given animal has had no contact with EHV in the past. Envelope glycoproteins have been identified as major targets for the immune response (Crabb et al. 1 99 1 ). Extensive cross-reactivity between epitopes recognised by EHV- l . and EHV -4 specific sera has been demonstrated by Western blot analysis (Crabb & Studdert 1 990). However, the existence of type-specific antigens was also evident, and this has been utili ed in the development of type-specific serological tests (Van de Moer et al. 1 993 ; Crabb & Studdert 1 993) . Fitzpatrick & Studdert ( 1 984) reported that primary neutral ising EHV- I re ponses were type-specific, whereas primary EHV -4 responses were cross-reactive. Secondary responses to both viruses were cross-reactive. On the contrary, Tewari et al. ( 1 993 ) found minimal cro s-reaction in SN test , and only partial cross-reaction in CF test after both primary and secondary EHV - 1 infect ion . Primary EHV -4 infection induced only poor serological responses, but subsequent EHV - 1 challenge induced respon es of a secondary nature to both EHV - 1 and EHV -4 antigens. The authors suggested that this cross-reactivity was due to reactivation of latent EHV-4 rather than true cross-reaction between EHV - 1 and EHV -4 antigens, since immunological responses to secondary EHV - 1 infection after primary EHV - 1 infection were sti ll type-specific. In another study, primary SN responses to both EHV - 1 and EHV -4 antigens were type-specific, while primary CF responses were cros -reactive after EHV - I chal lenge and type- - - - - - - - - - - - - - - ------------ - - - - - Literature Review 25 speci fic following EHV -4 challenge (Edington & Bridges 1 990). Secondary infection with EHV -4 evoked responses that were cross-reactive with EHV - 1 , while secondary EHV - \ challenge evoked neutralising responses that were sti l l type-specific . However, after the inoculation with heterologous v irus, ponies infected with EHV - 1 developed similar responses to both EHV - 1 and EHV -4 antigens, while ponies infected with EHV- 4 developed higher neutralis ing t itres to heterologous EHV - 1 than to EHV -4. Thus, cross-neutrali sation data from in vitro studies showed considerable variabi l i ty , indicating that the extent and the nature of these interactions sti l l need to be e lucidated. Immunosuppression Immunosuppression IS one of the mechani sms employed by herpesviruses to successful ly estab li sh persistent infection in their host species (Brodsky 1 999; Confer e t al. 1 990; Koff et al. 1 987; Plaeger-Marshall et al. 1 983) . Lymphocyte responses have been shown to be suppressed fol lowing EHV - 1 infection (Charan et al. 1 997; Hannant et al. 1 99 1 ; Dutta et al. 1 980; Wilks & Coggins 1 976) . The immunosuppressive effect of sera of ponies exposed to e ither l ive or DV­ inactivated EHV - I on T cell proliferation in response to both specific (EHV - 1 ) as well as non specific (phytohaemagglutinin, PHA) antigens could be demonstrated for up to 2 1 days after exposure. It was associated with induction of production of transforming growth factor-beta (TGF-P) (Charan et al. 1 997) . The i nduction of TGF-p was also associated with immunosuppression in cultures of EBV (Bauer e t al. 1 99 1 ) and HCMV (Michelson et al. 1 994). Hannant et al. ( 199 1 ) described depressed polyclona1 T cell act ivation for at least 40 days after EHV - 1 infection in response to PHA only. The EHV - I speci fic T cell responses were not affected in this study. Dutta e t al. ( 1 980) reported stimulation of lymphocyte transformation in response to both mitogens and EHV - 1 antigen . This initial stimulation was fol lowed by suppression of l ymphocyte transformation i n response to PHA only. The lowest levels of PHA-induced responses coincided with the highest levels of antigen-induced responses. In contrast, Wi lks & Coggins ( 1 976) showed the inhibitory effect of sera from EHV - 1 infected horses on lymphocyte responsiveness to EHV- J anti gen only, and not PHA. Thus, although all the investigators reported suppression of l ymphocyte responses fol lowing EHV- l infection, the results differed in rel ation to the level and specifici ty of this inhibitory effect. These d iscrepancies could reflect the differences in experimental set-up, such as the source Chapter I 26 and amount of ant igens u ed, or the existence of differences in immune responses to EHV - 1 between individual horse . In contrast to EHV - L , there was no period of suppression observed in proliferative responses of lymphocytes col lected from foals experimental ly infected with EHV -4 in response either to EHV -4 or Concanavalin A (Con A) (Bridges & Edington 1 987) . Latency The typical site of latency for a-herpes viruses is neural tissue, in particular sensory ganglia (Roizman 1 996) . The reactivation of latent EHV - I from horses has not always been successful and many investigators fai led to demonstrate EHV - I latency using tissue explantation or corticosteroid treatment (All en & Bryans 1 986). In one study, reactivation of latent EHV - I from experimental ly infected horses resulted mainly in viraemia with only occasional nasal secretion (Edington et aL. 1 985) . Others (Slater et al. 1 994) reported successful detection of EHV - I by cocultivation and PCR in trigeminal ganglion, thus providing evidence for the neurotropism of EHV - 1 . Equine herpesvirus-4 has been shown to establ ish latent infection in trigeminal gangl ia (Borchers et aL. 1 999; Borchers et aL. 1 997b; Alien & Bryans 1 986). In contrast, Browning e t al. ( 1 988) did not recover EHV -4 from trigeminal ganglia of corticosteroid-treated horses, although reactivation of latent EHV -4 was evident by nasal shedding of the virus. The inconsistency in ability to recover latent EHV - 1 14 from neural tissue may be explained in different ways. One po ibility is that this is not the predominant site of latent infection in the horse. Equine herpesviru - 1 can be readily isolated by co­ cult ivat ion of lymphocytes (Gleeson & Coggin 1 980; Bryans 1 969) and it has been suggested that lymphoid tissue may be the site of latent EHV - 1 infection (Scott et al. 1 983) . In agreement with this, EHV - 1 was detected in cells of the lymphoreticular system of experimental ly infected horses using PCR and co-cultivation (Welch et aL. 1 992) . The other possibil ity is that the number of infected neurones is extremely low, resulting in inconsistent results in co-cultivation studies. Literature Review 27 The molecular basis of EHV - l /4 latency has not been extensively studied. Chesters et al. ( 1 997) reported detection of LA T of EHV - 1 in equine lymphocytes, but not in trigeminal ganglia. On the contrary, Baxi e t al. ( 1 995) reported the existence of EHV- 1 LAT in ganglionic neurons and Marshal l & Field ( 1 997) demonstrated EHV - 1 latency in olfactory bulbs of mice experimentally infected with EHV- l . Additionally, Chesters and Baxi's groups identified different parts of the genome as potential LAT. One explanation may be that EHV- I is capable of establ ishing latency in different t issues with different part of the genome expressed. Whether this is the case remains to be elucidated. 1 .3.2 Equine picornaviruses The family Picornaviridae Family Picornaviridae Genus Genus Enterovirus Rhinovirus Genus Hepatovirus Genus Cardiovirus Genus Aphthovirus (Murphy et al. 1 995) Unassigned members of the family: Equine rhinovims- J ( ERhV- \ ) Equine rhinovims-2 ( ERhV-2 ) Equine rhinovims-3 (ERhV-3 ) Picornavimses are non-enveloped RNA viruses, 25 to 30 nm in size, of roughly spherical symmetry. The genome consists of positive sense ssRNA, 7 .5 to 8 .5 kb in size. The capsid consists of four major polypeptides. Additionally, a protein VPg is covalently l inked to the 5' end of the RNA. Virions repl icate in the cytoplasm of infected cells. The release of vims is accompanied by the destruction of an infected cel l . Enteroviruses, hepatoviruses and cardioviruses are stable over a wide pH range, while aphthoviruses and rhinoviruses are very sensitive to low pH and are efficiently inactivated at pH lower than 5 (Murphy et al. 1 995) . Picornaviruse have been isolated from healthy humans and animals, but also from those with different cl inical manifestations including enteric, respiratory and reproductive ystem failure (enterovimse ), common cold and upper respiratory infections ( rhinoviruses), hepatitis and gastroenteri tis (hepatovimses), or encephal itis and myocarditis ( cardioviruses) (Studdert 1 996c; Wutz et al. 1 996). The genus Aphthovirus includes only foot-and­ mouth disease virus (FMDV). It causes a general ised disease in cloven-hoofed animals . Chapter I 28 The malO cl inical feature inc lude fever, depression, anorexia, vesicular lesions on epithelial surfaces, drop in milk production in lactating animals and myocarditis in calves (Mann & Sellers 1 990). Classification of equine picornaviruses Equine picornavirus was first described by Plummer & Kerry ( 1 962) in the United Kingdom. Subsequently, picornaviruses were isolated from horses in many other countries (Studdert 1 996b) . Most isolates were found to be identical or similar to Plummer and Kerry's isolate . One of these isolates, NM l l , has been chosen as the prototype ERh V - 1 (Burrows 1 970). A serological ly different Canadian isolate, E26, was proposed to be assigned as ERh V -2 (Ditchfield & Macpherson 1 965) . However, this isolate was not available for further study and the Swiss isolate, P 1 43617 1 , has been accepted as the prototype ERhV-2 (Steck et al. 1 978) . Another Swiss isolate, P3 1 3175, has become the prototype ERhV-3 (Steck et a L . 1 978) . Additonal ly, Mumford & Thomson ( 1 978) described the isolation and characteristics of acid-stable equine . . pIcorna vIruses. Based on physico-chemical properties and the cl in ical manifestat ions of infection, equine picornaviruses were originally regarded as rhinoviruses (with the exception of the acid-stable picornaviruses) and named accordingly. However, analysi of the genomes of both ERhV- l and ERhV-2 showed a greater similarity to FMDV than to rhinoviruses (Li et al. 1 996; Wutz et al. 1 996) . At a protein level, ERhV- l was most similar to FMDV (genus Aphthovirus) , whereas ERh V -2 showed greater simi larity to encephalomyocarditis virus (genus Cardiovirus) (Wutz et aL. 1 996) . At present, all three serotypes of equine rhinoviruses, as wel l a acid-stable picornaviruse of horses remain unassigned in the family Picornaviridae (Murphy et al. 1 995) . Equine rhinovirus-} Virus structure and properties The physical and chemical characteristics of ERh V - 1 have been reported by Newman et al. ( 1 977), Plummer ( 1 963), Ditchfield & Macpherson ( 1 965), Studdert & Gleeson ( 1 977) and Burrows ( 1 970). The virions are 25-30 nm in size and contain an RNA genome. Equine rhinovirus- l is resistant to chloroform treatment, but readily - - - - - - - - - - - - ------ ----- --- - - - - - - Literature Review 29 i nactivated at pH lower than 5. The virus, diluted in water, i s not stable when exposed to 50 QC and the addition of 1M MgCh does not stablise against i nactivation (Studdert & Gleeson 1 977; Burrows 1 970; Ditchfield & Macpherson 1 965) . By contrast, virus preparations in maintenance medium were found to be resistant to the same heat treatment (Studdert & Gleeson 1 977; Burrows 1 970). The density in CsCI was reported to be 1 .40- 1 .456 g/cm:l (Studdert & Gleeson 1 977; Burrows 1 970) . Equi ne rhi novirus- l does not haemagglutinate human 0, guinea pig, equine or chicken red blood cells (Studdert & Gleeson 1 977) . Cultivation i n cell culture Unlike human rhinoviruses, ERh V - 1 does not have requirements for special culture conditions, such as low carbonate, low temperature and rol l ing. It replicates i n cells derived from several species at 37 QC (Burrows 1 970). The CPE consists of rounding of cells that become refractile, dense, and eventually detach from the surface (Ditchfield & Macpherson 1 965) . The study of Li et af. ( 1 997) showed that ERh V - 1 does not always produce an obvious CPE in cell culture. These authors infected Vero cells with nasopharyngeal swab filtrates collected from horses from outbreaks of respiratory disease. Cytopathic effect was not observed in any of the cultures, yet replicating ERh V - 1 was detected i n 1 0 of 1 5 samples by immunofluorescence (Li et al. 1 997). These findings may explain why ERhV- J was rel atively rarely isolated in cell culture by other authors, even from horses that showed serological evidence of recent ERh V- I infection (Carman et al. 1 997; Powell et al. 1 978; PowelI et a l . 1 974; Hofer e t al. 1 973) . Clinical signs i n infected animals Experimental infection of horses with ERh V - 1 was described by Plummer & Kerry ( 1 962) . The incubation period was 3 to 7 days. After that t ime, infected horses developed v iraemia that lasted for 4 to 5 days. The termination of the viraemic phase coincided with the development of neutralising antibodies. Cl in ical signs i ncluded fever, nasal discharge of varying severity and mucopurulent reaction in the pharynx . Based on virus i solation from faeces, the authors postulated that ERh V - 1 persisted i n the pharygneal area for at least a month after infection, at which stage the horses were euthanased. Chapter J 30 Experimental i nfection of animals other than horses did not result i n any c l inical signs, although rabbits, guinea pigs, monkeys and man became i nfected as judged by v irus isolation and serological response. Other laboratory animals including mice, hamsters, chickens or embryonated eggs did not support infection (Plummer & Kerry 1 962) . Clinical signs in natural i nfections vary from sub-cl inical to overt c l inical disease with signs including fever, anorexia, nasal d ischarge, pharyngitis, i ncreased respiratory and heart rates, cough and lymphadenitis (Li et al. 1 997; Hofer et al. 1 978 ; Burrows & Goodridge 1 978; Rose et a!. 1 974; Hofer et a!. 1 973 ; Burrows 1 970; Ditchfield & Macpherson 1 965) . Epidemiology Serological diagnosis of infection with equi ne picornaviruses could be difficult due to the rapid development of antibody responses in infected animals. Samples collected after the development of c l inical s igns may already possess high levels of antibody and therefore a significant rise i n titre between acute and convalescent samples may not be demonstrated (Burrows & Goodddge 1 978 ; Mumford & Thomson 1 978; Plummer & Kerry 1 962). Serological results indicate that ERh V- I infection is widely spread among horses worldwide. The prevalence of antibodies to ERh V - 1 was reported to be 77% i n the United States (Holmes et al. 1 978), 65% i n The Netherlands (Morail lon & Morail lon 1 978), 64 to 80% in the UK (Powell et al. 1 978 ; Rose et al. 1 974), 8 1 to 88% i n Germany (Herbst et al. 1 992), 47.9% in Australia (Studdert & Gleeson 1 977) and 37.7% in New Zealand (Jolly e t al. 1 986) . The relatively lower prevalence of ERhV- l antibodies i n Australia and New Zealand i n comparison with figures from America and Europe may renect different husbandry conditions in these countries. In these two countries, the stabling of horses is less i ntensive than in the Northern Hemisphere and animals tend to be paddocked for a greater proportion of the year. Newborn foals are negative for antibodies to ERh V- I . Soon after birth they acquire high levels of maternally derived antibodies, which decl ine steadily . Foals older than 5 to 7 months of age are mostly negative (Holmes et al. 1 978 ; Hofer et al. 1 973) . The prevalence of ERh V - 1 infection was found to be higher among horses 4 years and older Literature Review 3 1 in comparison with younger ones (Holmes et al. 1 978) . Young horses are l ikely to acquire ERh V - 1 infection when brought to points of concentration. In one study (Hofer e t af. 1 973), 50% of horses arriving at a Swiss remount depot station were positive for antibodies to ERh V - 1 . Within 3 months of arrival, 98% of remounts were posit ive, and all cavalry horses tested that had been in the army depot for more than a year, were positive for ERhV- l antibodies. Simil arly, in the United States and The Netherlands, the prevalence of ERh V - 1 antibodies was found to be higher in race horses or riding school horses than i n pleasure or solitary horses (Holmes et al. 1 978; Morail lon & Morail lon 1 978) . Also, most serological evidence of infection with ERh V - 1 occurred during the first winter in training of young Thoroughbred horses in the UK (Powel l et al. 1 978) . In that study, in some stables, c lose to 1 00% of horses showed serological evidence of ERh V - 1 infection, whereas i n others only a few horses became infected during the same period of time. These findings indicate that other factors, apart from housing and concentration of l arge number of horses at one place, are also important in the spread of infection. High t itres of neutrali sing antibodies to ERh V - 1 can be detected in the blood of horses as early as 1 to 2 weeks after experimental i nfection with the v irus (P1ummer & Kerry 1 962) . Neutral is ing antibodies remain at high levels for years (Burrows & Goodridge 1 978) . While this could reflect the estab lishment of persistent infection, none of the susceptible horses kept together for over 7 years with horses showing high ERh V- 1 antibody titres showed serological evidence of infection (Burrows & Goodridge 1 978) . In a study in the USA, 1 7% of post racing urine samples contained ERhV- l (Mc Collum & Timoney 1 992) . The same authors demonstrated that shedding of the virus in urine persists for up to 146 days in individual horses. Also, Plummer & Kerry ( 1 962) detected small amounts of ERh V - 1 in faeces. The possibil ity of ERh V - 1 transmission via urine and faeces, together with the reasonable stabil i ty of this virus in the environment may explain the usuaI ly rapid spread of infection at points of concentration, such as quarantine stations, race tracks or riding schools. Equine rhinovirus-2 The structure and physico-chemical properties of ERh V -2 are similar to those of ERh V­ I (Steck et af. 1 978) . Similar to ERh V - 1 , ERh V -2 infection can be detected in both Chapter I 32 healthy horses and horses showing cl inical signs of re piratory disease (Carman et aL. 1 997; Steck et aL. 1 978; Pow ell et at. 1 978; BUlTOW 1 978; Hofer et at. 1 978; Rose et aL. 1 974). The growth characteristics in cell culture and the CPE produced by ERhV-2 are similar to that described for ERh V- \ . However, in contrast to ERh V - I , which was reported to produce CPE in a variety of cell l ine , ERh V -2 produced clearly visible plaques only when grown on actively dividing RK- 1 3 cells (Holmes et al. 1 978; Steck et al. 1 978) . Despite these similarities, ERh V - 1 and ERh V -2 are clearly two distinct viruses. They do not show any cross-reaction in cross-neutral isation studies (Steck et al. 1 978) . Also, although both ERhV- l and ERhV-2 are similarly widely spread in horse populations worldwide, the epidemiological picture of infection with these two viruses differs. The prevalence of ERhV-2 antibodies is simi lar to that reported for ERhV- l , with figures of 83% reported for the United States (Holmes et aL. 1 978), 59% for The Netherlands (Morai l !on & Morai l lon 1 978) , 73% for Switzerland (Steck et at. 1 978) and 84.9% for New Zealand (Jolly et at. 1 986). In the UK, the prevalence of ERh V-2 antibodies among Thoroughbred racehorses wa reported to be 20% in one study (Rose et al. 1 974), but 56.4% in another (Powel ! et al. 1 978) . The latter figure i probably more representative, as it was calculated using a larger number of horses. In contrast to ERhV- l , the prevalence of antibodie to ERhV-2 does not show a specific age distribution. In a study by Holmes et al. ( 1 978) , foals acquired low levels of ERh V- 2 antibodies soon after birth. The titres remained low thereafter, with occasional increases at sporadic times, until the study ended when the horses were 2 years of age. In agreement with these findings, 47% of 7- to 1 2-month-old Swiss bred foals and 4 l .2% of 5- to l l -month-old New Zealand foals, had antibodies to ERhV-2 (Jolly et al. 1 986; Steck et at. 1 978) . Arrival at points of concentrat ion, although facil itating spread of infect ion, does not seem to be as important an epidemiological factor as for ERh V- I infection. Most of the hor es sampled acquired ant ibody before arrival at the racetrack (Holmes et al. 1 978) . S imi larly, about 40% of English Thoroughbred horses showed serological evidence of ERh V -2 infection before entering racing stables (powell et at. 1 978) . In Switzerland, 68% of horses arriving at an army depot already had antibodies to ERhV-2. However, 98% of cavalry horses staying at the depot for longer than a year - - - - - � ---------------- - - - - - - Literature Review 33 were positive (Steck e t al. 1 978) . Also, the prevalence of ERh V -2 antibodies i n Danish leisure horses (32%) was sti l l considerably lower than that observed in Danish race horses (67%) (Morail lon & Morail lon 1 978) . I n a study among Thoroughbred racmg horses in England, approximately 20% of seronegative horses seroconverted to ERh V -2 during the racing season, while 1 3% of the seropositive horses reverted to seronegative status (Powel l et at. 1 978) . Thus, unlike antibodies to ERhV- l , antibodies to ERhV-2 do not seem to persist at high titres for years. Also, the range of neutralis ing antibody t itres against ERhV-2 is generally 1 0 to l OO-fold lower than that reported for ERh V - ] (Steck et al. 1 978) . Burrows & Goodridge ( 1 978) observed the spread of ERhV-2 infection among a group of Pirbright experimental ponies. The i nfection was i ntroduced to this group by two horses, which had been infected with ERhV-2 7 weeks or 1 0 months previously. W ithin the following 2 years, ERh V -2 infection became endemic in the herd. In general, the mean antibody titres of the herd increased during periods of housing and decreased when ponies were kept at pasture. I n individual horses, a significant four-fold i ncrease in the ERhV-2 antibody titre could not be demonstrated between samples taken 1 month apart, although it was observed between samples taken 2 to 3 months apart. This may have reflected either seasonal activity of the virus in persistently infected animal or repeated infections of pm1ial ly protected animals, as experimental infection of seronegative colts resulted in a four-fold i ncrease in neutralising t itres within 2 weeks after experimental infection. Nearly 2 years elapsed between the first and last ERhV-2 infection in this herd. This, together with the fact that ERhV-2 was i solated from pharyngeal samples of two experimentally infected horses 2 years after ERh V-2 infection, further suggests the abi l ity of the virus to either establ ish persitent infection or to frequently re-infect horses (Burrows 1 978; Burrows & Goodridge 1 978) . I n contrast to ERh V - 1 , neither viraemia nor faecal or urine shedding of ERh V -2 has been demonstrated in any of the studies. Chapter I 34 Other equine picornaviruses Equine rhinovirus-3 The existence of the third serotype (P3 1 3175) of equine rhinovirus was proposed by Steck et al. ( 1 978) . A serological survey among ponies in the Pirbright herd showed that about 50% of ponies sampled had antibodies to thi or a similar virus (Burrows 1 978) . No other information on ERh V -3 is avai lable. A cid-stable picornaviruses Mumford & Thomson ( 1 978) reported the isolation of acid-stable picornaviruses from nasopharyngeal or faecal samples of horses. These i olate were found to be stable at lower pH and were stabi l i ed against inactivation at 50 QC by addition of 1 M MgCh. The buoyant density in CsCI of one of these isolates was 1 .40- 1 .43 g/cm3. Although acid-stable picornaviruse were clearly distingui hed from ERhV- l and ERhV-2, there was some cross-reaction with ERh V -2 antigen in SN and CF tests. Sera col lected from horses from three outbreaks of respiratory di ease were posit ive for antibodies to the representative acid stable picornavirus ( i solate 4442). Although the majority of horses showed high titres, a four-fold rise between acute and convalescent ample could not be demonstrated. A significant increase in CF antibody was demonstrated in another outbreak of respiratory di ea e among routinely ampled horses. The outbreak was reported in Apri l . Only two horses showed a significant rise in titre between April and May, and four other horses had decreasing antibody levels at the same t ime. However, in 8 of 1 2 arumal a significant increase in ti tre was demonstrated retrospectively between samples col lected in March and Apri l , ugge ting that equine picornaviruses were involved in thi outbreak of respiratory disease. 1 .3.3 Equine adenovirus The family Adenoviridae Family Adenoviridae Genus Genus (Murphy et al. 1 995) Mastadenovirus A viadenovirus Equine adenoviru - I (EAdV -I) Equine adenovi ru -2 (EAdV -2 ) - - - - - - - - - - --- -------------- Literature Review 35 Group characteristics Adenoviruses are non-enveloped icosahedral v iruses 80 to 1 10 nm in d iameter with a double stranded DNA genome. The capsid consists of subunits arranged i nto 240 hexamers and 1 2 pentamers with penton fibres projecting from apices (Murphy et af. 1 995) . The penton bases are composed from group-specific antigens that are common to most members of the family. Antibodies raised against these proteins possess only low levels of neutralizing activ ity. The hexons and fibres are major antigenic sites. The fibre protei n i s respons ible for attachment of the viru s to RBC in a haemagglutination reaction. This reaction is type-specific with some subgroup speci ficity. Hexon proteins el icit a heterologous population of antibodies, some of which are family cross-reactive in a CF test and others show marked type specificity in SN tests (Horwitz 1 996; Murphy et (If. 1 995) . Adenoviruses are usual ly confined to one host speCIes or c losely related speCIes (Horwitz 1 996). S imilarly, they usuall y replicate only in cell cultures derived from their natural host. The CPE consists of bunches of rounded, refractile cells with characteristic basophilic intranuclear inclusion bodies (Hsiung 1 982) . Human adenoviruses were i solated from healthy people for 24 months after i ni tial infection, suggesting the abil i ty of the virus to establish persi stent infection in its host (Horwitz 1 996). Also, i t has been suggested that adenoviruses can establ ish latent infection in lymphocytes and lymphoid tissue. The exact mechanisms of adenovirus latency, however, have not been established (Horwitz 1 996). It has been shown that three adenovirus genes encode products that have the potential to interact with the host' s immune response, and therefore could faci litate persistence or latency of the virus. Cellular responses to a and � i nterferons are inhibited by E t A proteins and VA RNAs, while E3 protein protects infected cel l s from l ysis mediated by cytotoxic T lymphocytes (CTL) and tumor necrosi s factor a (TNFa) (Mahr & Gooding 1 999; Shenk 1 996) . Although there i s no evidence of association between adenoviruses and any human neoplasm, several human serotypes can produce tumours in rodents (Shenk 1 996). Tumour formation is associated with integration of adenovirus DNA into the host cel l ' s chromosome with no production of mature viruses (Horwitz 1 996) . Chapter 1 36 Adenoviruses have been isolated from many species. They are reasonably stable in the environment. Human adenoviruses usual ly produce asymptomatic infections, but have also been associated with c l inical i nfections affecting a variety of organs i nc luding respiratory, ocular, urinary and gastrointestinal systems (Horwitz 1 996; Murphy et al. 1 995) . Typical ly, adenoviral disease i s short-lived, self-l imi ted and with no serious consequences in immunocompetent persons. However, adenoviruses can cause more serious respiratory disease in young or immunocompromised hosts. Additionally, i n such patients, they can disseminate to other organs causing acute systemic i nfection (Horwitz 1 996). Equine adenovirus Virus properties The biological and physiochemical properties of equine adenov irus have been described by Wilks & Studdert ( 1 973) , England et al. ( 1 973) , Fatemie-Nainie & Marusyk ( 1 979), Ardans e t al. ( 1 973) , Moorthy & Spradbrow ( 1 978a) , Konishi et al. ( 1 977), Dutta ( 1 975) , and Harden ( 1 974) . Equine adenovirus is not sensitive to chloroform or ether and is stable within the pH range 4 to 7, but s l ightly i nactivated at pH 3 (Konishi et al. 1 977) . However, there are some differences between isolates, as equine adenoviruses described by Ardans et al. ( 1 973) and Dutta ( 1 975) were stable at pH 3, while the i solate described by Harden ( 1 974) was i nactivated at pH 3. Additional ly, other characteristics reported for d ifferent isolates differed slightly. The cell l ines used to propagate the virus were usual ly primary cell s of equine origin , as these were the only cel l s that supported the growth of all the i solates characterised. Other cells supported the growth of some isolates, but not others (Konishi e t al. 1 977; Harden 1 974; England et al. 1 973) . Different isolates also differed in their haemagglutination characteristics. Most of the equine adenoviruses described haemagglutinated human type 0 and equine RBC. However, Moorthy and Spradbrow's i solate was not tested against human 0 RBC and Dutta's i solate haemagglutinated only human 0 RBC, and not equine ones. All i solates that were tested for haemagglutination with rat RBC gave positive reactions and the t itres were much higher than those obtained with RBC from other species (Moorthy & Spradbrow 1 978a; Harden 1 974) . The virion s ize reported by different authors ranged from 70 to 82 nm, and the buoyant density in CsCI ranged from 1 .27 glcc to 1 . 34 glcc Literature Review 37 (Konishi et al. 1 977; Kamada et al. 1 977; Dutta 1 975 ; Harden 1 974; Ardans et al. 1 973 ; England e t al. 1 973 ; Wilks & Studdert 1 973) . A ntigenic serotypes Adenovirus serotypes are identified on the basis of SN and HI assays (Murphy et al. 1 995) . Two viruses are defined as separate serotypes if they show either no cross­ reaction or an homologous/heterologous titre ratio greater than 1 6 in both directions i n the SN test. For heterologous/homologous titre ratios of 8 and 1 6, a serotype assignment is made on the basis of lack of cross-reaction in HI tests and/or the existence of major biophysical or biochemical differences (Murphy et al. 1 995) . Most of the equine adenoviruses compared were closely related and constituted a single serotype designated as EAdV - 1 (Studdert 1 978 ; Thompson et al. 1 976), although there were s l ight anti genic d ifferences between i solates (Fatemie-Nainie & Marusyk 1 979; Studdert et a l . 1 974) . In 1 982, the i solation of an adenovirus antigenical ly different to EAdV - 1 was reported (Studdert & Blackney 1 982) . The i solate was obtained from diarrhoeic foal faeces and was found to be totally unrelated to EAdV- l by the SN test. Furthermore, i t did not haemagglutinate any RBC tested including human, monkey, equine, porcine, guinea pig and chicken. A new serotype was designated EAdV-2 (Homer & Hunter 1 982; Studdert & B lackney 1 982) . Both EAdV - 1 and EAdV -2 were i solated i n New Zealand from young Thoroughbred horses with c l inical signs of i l l ­ thrift, upper respiratory disease or d iarrhoea (Homer & Hunter 1 982) . Viral DNA DNA restriction analysis of the genome of EAdV - 1 has been publi shed (Sheppard e t al. 1 992; H igashi & Harasawa 1 989; Ishiyama et al. 1 986). The comparison of restriction maps of three different i solates of EAdV -1 confirmed the homogeneity among different i solates of EAdV - 1 , despite some polymorphism that could be detected with appropriate restriction enzymes (Higashi & Harasawa 1 989). Several genes from EAdV- l and EAdV-2 have been cloned and sequenced (Reubel & Studdert 1 997b; Reubel & Studdert 1 997a). Sequence data confirmed that EAdV- l and EAdV-2 are two separate viruses . Phylogenetic analysis indicated that both EAdV - 1 and EAdV -2 evolved separately from each other and from other adenoviruses (Reubel & Studdert 1 997b; Reubel & Studdert 1 997a) . Chapter J 38 Disease Equine adenovirus usually produces subclinical or mild respiratory tract i nfection i n immunocompetent horses (Studdert 1 996a) . Adenoviruses were i solated from cases of fatal respiratory disease in Arab foals (Thompson et al. 1 976; Ardans et al. 1 973; McChesney et al. 1 970; Todd 1 969), as well as from Arab and non-Arab foals and older horses with milder, non-fatal respiratory disease (Moorthy & Spradbrow 1 978a; Kamada et al. 1 977; Dutta 1 975; England et at. 1 973) and healthy animals (Wilks & S tuddert 1 972; Harden et al. 1 972 ; Petzoldt & Schmidt 1 97 1 ). Adenovirus was also i solated from the bronchus and lung of a 3-month­ old Thoroughbred colt with fatal mucopurulent pneumonia, although Corynebacterium SI'. was also i solated from pneumonic lung tissue of the foal and was probably the cause of death (Konishi et al. 1 977). Equine adenovirus was also isolated from two of three cases of cauda equina neuritis . The adenovirus infection in neural t issue was considered to be latent as determined by the fai lure to demonstrate virus in tissue sections by immunofluorescence and the need to co-cultivate andlor passage the virus several times before the occurrence of any visible CPE in cell culture. None of the three animals had antibody to EAdV - I as determined by immunofluorescence, HI and SN (Edington et al. 1 984). Fatal adenovirus pneumonia III Arab foals occurs in foals with pnmary, severe combined immunodefic iency disease (PSCID) . As maternal ant ibody wanes, these foals become susceptible to infection with many pathogens among which adenovirus seems to p lay a predominant role (Studdert 1 996a). In these foals , EAdV can be i solated from many different organs. The outcome of i nfection is usual ly fatal (Studdert 1 996a). The disease signs and pathology of adenoviral infection in PSCID foals has been reviewed (Studdert 1 996a) and wi l l not be discussed in detail . C linical signs observed i n non-fatal respiratory d isease caused by equine adenovirus are variable and include rhinitis, serous to mucopurulent nasal d ischarge, cough, enlarged lymph nodes or stunted growth (Moorthy & Spradbrow 1 9780.; Kamada et al. 1 977; Dutta 1 975) . It i s possible that adenoviral infection at the time of trans ient immunosuppression caused by factors such as stress, cold or other infections, can lead Literature Review 39 to more severe cl inical signs than infection in immunocompetent hosts. For example, the adenovirus i solated by Dutta ( 1 975 ) came from a young foal with severe respiratory tract disease. Equine herpesvirus- 1 I4 was i solated from other foals with milder respiratory disease on the same farm. As EHV - 1 infection causes transient immunosuppression in the horse (Charan e t al. 1 997; H annant et al. 1 99 1 ; Dutta et al. 1 980), adenovirus i nfection at the time of this transient immunossuppression may have lead to more severe disease in this foal . Experimental infection The pathogenicity of equine adenovi rus has been studied in experimentally infected animals (Glee son et al. 1 978; Pascoe et af. 1 974; McChesney et al. 1 974). Cl in ical signs i n both conventional and SPF foals experimentall y infected with adenovirus varied from subclinical infection to severe respiratory disease. In a large-scale experimental infection of 35 foals, the cl inical signs observed i ncluded intermittent fever, nasal and ocular discharges, polypnea and cough (McChesney et al. 1 974). Colostrum-deprived foal s showed more apparent c l inical s igns and more extensive post-mortem lesions than the colostrum fed foals . Cl in ical signs disappeared spontaneously by 1 0 days post infection. Only two colostrum-deprived foals died, and both of them had concurrent bacterial infections . Foals that were not ki l led for necropsy grew and developed normall y. Experimental infection of foetuses resulted in death and abortion. Pathological changes on post-mortem examination consisted of pulmonic lesions involving the cardiac region of the lungs, characterised as atelectasis , suppurative bronchopneumonia and i nterstitial pneumonia. H i stologicall y, hyperplasia, swell ing, necrosi s and i ntranuclear i nclusions of epithelial cel l s were observed. Two-month-old and 4-month-old foals exposed to equine adenovirus developed only mild respiratory disease with few or no cl inical signs. Similarly, post-mortem examination revealed no gross lesions and much milder histological changes in epithelial cel ls , as compared with younger foals (McChesney et a/. 1 974) . Epizootiology Several serological surveys conducted throughout the world have indicated that adenoviral infection is common among horses and often seroconversion occurs without any cl inical s igns ( Herbst e t al. 1 992; Homer & Hunter 1 982; S tuddert & Blackney 1 982; Farina et al. 1978 ; Harasawa et al. 1 977 ; Harden et al. 1 974). Chapter I 40 Serological evidence of adenoviral infection in hor e was first presented by Darbyshire & Pereira ( 1 964) , who reported that 24. 1 % of 1 78 horse sera tested in the United Kingdom had precipitat ing antibody to group-specific adenoviral antigens. The results of similar surveys in Iran (Afshar 1 969), Ireland (Timoney 1 97 1 ) , New Zealand (Fu et al. 1 986), Nigeria (Obi & Taylor 1 984) and Italy (Farina et al. 1 978) were 1 0.9% of 73 horses, 1 0.6% of 227 horses, 39% of 1 83 , 4 .5% of 44 and 1 8 .5% of 694 horses, respectively. Also, 88.6% of hor es imported to Japan from different countries had neutral ising antibodies to EAdV - 1 (Harasawa et al. 1 977) and 39% of horse sera tested in The Netherlands had CF antibodies to EAdV (Morail lon & Morai llon 1 978) . Of 63 1 equine serum tested in another study (Studdert et al . 1 974) , 73 . 1 % had H I antibody to EAdV - 1 . Sera included in this survey originated from horses from different countries, different breeds and age groups. There wa no significant difference observed in antibody prevalence between horses from different countries or different breeds. The prevalence was sl ightly higher in horses 1 year of age and older than in younger foals. A similar age distribution was reported by others (Homer & Hunter 1 982; Harden et al. 1 974) . I t has been demonstrated, that the AGID test is relatively less sensitive than SN and H I tests for detection o f antibodies to EAdV ( Kamada 1 978; Harden e t al. 1 974; Pascoe et al. 1 974) . In one study, 77% of 433 erum samples tested had neutral ising antibodies to equine adenovirus, whereas only 1 4% had precipitating antibodies detectable by AGID and 53% of the same sera were positive by CF (Harden et al. 1 974) . Complement fixation titres were two to three dilutions lower than neutrali ing ones. However, CF had the advantage of detecting common group antigen and on 1 6 occasions it detected anti-adenoviral antibody in the absence of type specific neutral ising antibodies to EAdV - 1 , indicating for the first time the existence of the econd type of EAdV 10 Austral ia (Harden et al. 1 974) . A more recent survey conducted in Austral ia distinguished the prevalence of infection with EAdV - 1 and EAdV -2. About 80% of 339 horse sera tested were positive for SN antibodies to EAdV-2 and 86% for antibodies to EAdV- l . Most positive horses were positive for antibodies to both viral serotypes. There were, however, horses positive for antibodies to only one of the viruses (Studdert & Blackney 1 982) . Literature Review 4 1 1 .3.4 Equine arteritis virus The family Arteriviridae Equine arteri tis virus has been recently reclas ified and placed in a newly created order - Nidovirales (Cavanagh 1 997) . ORDER Family Genu Family Genus Genus NIDO VlRA LES Arteriviridae A rteri virus Coronaviridae Corona virus Torovirus -----i�� Equine arteri ti virus ( EA V) (Cavanagh 1 997; Murphy et al. 1 995 ) General characteristics The molecular characteristics of the genome structure and the biology of members of the genus A rterivirus have been recently reviewed by Snijder & Meulenberg ( 1 998) . Arteriviruses contain a positive sense RNA genome, 1 2 .7 to 1 5 .7 kbp in size, surrounded by a nucleocapsid and a lipid envelope. The envelope possesses characteristic, ring-like surface structures. The virions are about 40 to 60 nm in diameter and contain at least four structural proteins. Arteriviruses exhibit a very restricted host range in vitro, and can be grown in primary macrophage cell cultures. The buoyant density in ucrose is 1 . 1 3 to 1 . 1 7 g/cm3• They are unstable at pH outside the pH 6-7 .5 range, and stabil ity decrea es with increase in the temperature (Snijder & Meulenberg 1 998; Murphy et al. 1 995) . Equine arteritis virus The biology of EA V and the economic ignificance of infect ion have been recently reviewed (Glaser et al. 1 996; Holyoak et al. 1 993) Equine arteritis virus differs from other arterivi ruses in its abil ity to repl icate in a variety of cells, includjng primary cultures of macro phages and kjdney cells, and continuous cell l ines l ike BHK-2 1 , RK- 1 3 or Vero (Snijder & MeuJenberg 1 998; GJaser et al. 1 996). Chapter 1 42 Genomic and antigenic variability The neutrali sation epitopes of EA V have been mapped to specific sites i n the envelope glycoprotein GL (Bal asuriya e t al. 1 997 ; Glaser e t al. 1 995; Chirnside et al. 1 995; Balasuriya e t al. 1 995; Balasuriya e t al. 1 993) . Analysis of genomic diversity between different EA V i solates revealed considerable variabi l i ty mapped to the regions associated with neutral i sation domains, i ndicating the existence of strains with different antigenic properties (S tadejek et al. 1 999; Stadejek et al. 1 999). However, only one serotype of EA V has been recognised (Glaser et al. 1 996) . Clinical signs Clinical signs of EA V infection range from inapparent infection to severe disease. The i ncubation period is usually 3 to 1 4 days. Clinical signs in horses with severe disease i nc lude pyrexia, depression and anorexia, profuse serous nasal discharge, which may become mucopurulent, conjunctivitis and rhinitis, leucopenia, stiffness, periorbital or supraorbi tal oedema, mid ventral oedema involving prepuce and scrotum of the stall ion and mammary glands of the mare, urticarial-type rash most commonly on both sides of the neck, and abortion in the mare. Less frequently observed c li nical sings include respiratory distress, coughing, d iarrhoea, ataxia, stomatitis, icterus, mild anaemia, photophobi a and colic (Del Piero et al. 1 997; Glaser e t al. 1 996; E ichhorn e t al. 1 995; Monrea1 et a l . 1 995; Wood et al. 1 995; Timoney & McCollum 1 993 ; Evans 1 99 1 ; Huntington e t al. 1 990a; Gerber et al. 1 978; McCollum & Bryans 1 973) . Experimental infection Several experimental infections have been repOIted (Evans 1 99 1 ; McCollum et al. 1 988; Neu et al. 1 988; McCollum et al. 1 97 1 ; Doll et al. 1 957) . Clinical s igns in experimentally infected animals varied from typical signs of natural arterivirus infection to subcl in ical infections with no signs at al l . Young horses usually showed more pronounced cl inical signs than did older ones. Fol lowing i ntranasal inoculation, the ini ti al replication of the VlruS occurs m lung macrophages. By the third day post i nfection all body fluids except for the cerebrospinal fluid contain virus. Development of the characteristic vascular lesions, a vasculit is with focal or segmental necrosis and l ymphocytic infi l tration of tunica media and adventitia, - - - - -- - - - - --------- Literature Review --- -- - -- - - 43 i s caused by virus replication in endothelium and tunica media of blood vessels . Virus replication also takes place in the mesothelium and epithelium of some organs such as adrenals . Vascular lesions give rise to oedema and haemorrhage in many organs and tissues and produce characteristic c l inical signs of infection (Mumford 1 994; Timoney & McCollum 1 993) . Usuall y, EA V can be easi ly recovered from experimentally infected animals . However, it is often difficult to i solate the virus from field cases even during the acute phase of i nfection (Wood et al. 1 995 ; Monreal et al. 1 995) . Epidemiology Antibodies to EA V were found in horses, donkeys and mules (Paweska et al. 1 997) . The results of various serological surveys have i ndicated that EA V infection i s present in Europe, America, Africa, New Zealand and Australia (Glaser e t al. 1 996; Eichhorn et al. 1 995; Huntington et al. 1 990b; McKenzie 1 989; Morail lon & Morai l lon 1 978; McCollum & Bryans 1 973) . The seroprevalence varied between different populations and breeds (Glaser e t al. 1 996). Particularly, EA V seroprevalence was found to be significantly higher in Standardbred horses in comparison with Thoroughbreds (Hunt ington et al. 1 990b; Timoney & McCollum 1 990; McCollum & Bryans 1 973) . However, since the first EA V outbreak involving 38 Thoroughbred breeding farms, several other cl inical outbreaks of EA V were recorded among Thoroughbreds, mostly in America (Timoney & McCollum 1 990) . Although an i ncrease i n the number of outbreaks of disease associated with EA V infection has been recently noticed, serological surveys i ndicate that the majority of infections occur without any cl inical signs (Timoney e t al. 1 992; Timoney & McCollum 1 990). Transmission The virus is transmitted either by respiratory secretions or by semen. Transmission via the reproductive system constitutes a much bigger problem because of the existence of carrier stallions. These animals shed virus in the semen continuously without any cl inical signs and the virus can not be demonstrated in any other body t1uids. Artifici al insemination makes it possible to disseminate the virus from one infected stallion over a wide geographic area. Virus shedding i n persistently infected stall ions was shown to be testosterone-dependent. Thus, chronic virus shedding was not observed in geldings or - -- - -- - - - - ---- -��������--- -- -- - Chapter I 44 after castration of persistently infected stallions . However, castrated stalions treated with testosterone continued to shed the virus (Little et al. 1 992) . In agreement with these findings, EA V was not isolated from the reproductive tract of mares l ater than a month after infection and there is no evidence to suggest that mares are ever persistently infected (Glaser et a l. 1 996). In infected mares, EA V is shed in respiratory secretions, vaginal secretions, urine and faeces for 2 to 3 weeks after primary infection (McCollum e t al. 1 97 1 ). The virus i s reasonably fragile i n the outside environment and lateral spread by the respiratory route can be effectively halted by physical separation of infected from seronegative animals (Monreal et al. 1 995 ; Wood et al. 1 995) . Transmission via fomites does not seem to play an important role (Wood e t al. 1 995; Timoney & McCollum 1 993) . Soon after birth, foals from seropositive mares are protected by maternall y derived antibodies to EA V that persist for 2 to 8 months (Hul l inger e t al. 1 998). Neutralising antibodies fol lowing natural infection persist for many years (Glaser e t al. 1 996; Gerber et al. 1 978) . However, the exact immune mechanisms in protection from infection have not been determined. Occasional induction of disease in horses with neutral ising antibodies i ndicates that other mechanisms, apart from humoral immunity might also be important in protection (Glaser e t al. 1 996). Alternatively, this could be a reflection of the existence of EA V strains with s l ightly different antigenic properties (see above). Sign�ficance and control The main economic significance of EA V infection l ies in the abi lity of the virus to induce abortions and the potential for rapid dissemination of the virus among large numbers of horses by persistently infected stallions. Live attenuated and killed vaccines are commercially available. However, vaccination prevents occurrence of disease, but does not prevent infection (Glaser et al. 1 996). Programmes designed to control EA V infection have been introduced in several countries. They combine serological survei l lance and restricting the mating of persistently infected stalions to seropositive mares with vaccination and isolation of infected animals (Glaser et al. 1 996). Literature Review 1 .3.5 Equine Reoviruses The family Reoviridae Reoviridae mammalian reovirus- l (Reo- I ) Family Genus Genus Geneus Genus Genus Genus Genus Genus Genus Orthoreo virus � mammal ian reovirus-2 (Reo-2 ) (Murphy et al. 1 995) Orbivirus Rota virus Coltivirus Aquareovirus Cypovirus Fiji virus Phytoreovirus Oryzavirus Group characteristics mammal ian reoviru -3 (Reo-3) 45 Reoviruses are non-enveloped, isosahedral viruses 60 to 80 nm in diameter. The capsid consists of an inner core surrounded by several protein layers. The buoyant density in CsCI is 1 .36 to 1 . 39 g/cm3. The viruses are moderately resistant to heat, organic solvents and to non-ionic detergents, while pH stabi l ity varies among genera (Murphy e t al. 1 995) . Viral RNA is double stranded and consists of 10 to 1 2 segments of total size 1 6 to 27 kbp (Fenner 1 993) . Genetic reassortment occurs within each genus. Members of Reoviridae replicate in the cytoplasm of infected cel ls . Virions are released from the cytoplasm fol lowing lysis of cells (Nibert et at. 1 996; Murphy et al. 1 995; Fenner 1 993) . Some of the members of the family repl icate only in vertebrate hosts, some additional ly in arthropod vectors and there are also plant and insect viruses in the family (Murphy e t al. 1 995) . Genus Orthoreovirus The distribution of orthoreoviruses i s ubiquitous worldwide. They infect only vertebrate hosts. The route of transmission is either respiratory or oral-faecal . Antibodies to mammal ian reovi ruses, of which three serotypes have been recognised, have been found in a wide range of species, but the disease associated with infection is usual ly mild or - - - - - - - ------- - � - - - Chapter 1 46 inapparent. Sometimes they cause respiratory disease or diarrhoea. Orthoreovirus infection in mice causes hepatoencephalomyelitis, characterised by jaundice, ataxia, oily hair and growth retardation and some avian reoviruses are pathogenic for poultry (Tyler & Fields 1 996; Murphy et al. 1 995; Fenner 1 993) . Orthoreoviruses possess a double capsid shell and the diameter of an i ntact particle i s 80 nm. They are stable over a wide pH range (Murphy e t al. 1 995) . Virions have to be degraded to infectious subviral particles or core particles in order to initiate the i nfection (Nibert et al. 1 996) . Thus, proteolytic enzymes i ncrease the infectivity of orthoreoviruses, as they help to loosen their outside coat. The S I segment of the viral genome codes for a cell attachment protein , which has haemagglutinin activity. It is type specific and reacts also with neutralising antibodies (Tyler & Fields 1 996; Nibert e t al. 1 996; Murphy et at. 1 995) . All mammalian orthoreoviruses share a common CF antigen (Tyler & Fields 1 996) . Equine reo vi ruses There are relatively few publications about equine reoviruses and their significance in equine respiratory disease is unknown. The host specificity of equine reoviruses has not been determined. In one study, horses were successfully infected with human isolates. During the experiment, a stable worker developed respiratory disease due to Reo-3 i nfection, as judged by serological results (Thein & Mayr 1 974) . Thus, it is possible that horses are one of the many host species for mammalian reoviruses, without the existence of equine-speci fic serotypes. Several serological surveys have indicated that reovirus infection is widespread and common among horses in Europe and America(Conner et al. 1 984; Reinhardt et al. 1 983 ; Sturm et al. 1 980), although differences were observed between different countries and different breeds of horses (Herbst et al. 1 992; de Boer et al. 1 978) . In Germany, Reo-3 infection was found to be most common, with a seroprevalence of 48 to 76% reported in different surveys. The seroprevalence of Reo- l and Reo-2 varied between 22 to 50% and 8 to 43%, respectively (Herbst et al. 1 992 ; Thein & Mayr 1 974) . Reovirus-3 infection was also more prevalent than both Reo- l and Reo-2 infections in England, Belgium, and Chile with seroprevalence values 1 5 to 25 .6% for Literature Review 47 Reo- I , 5 to 1 2 .6% for Reo-2 and 5 1 to 63.9% for Reo-3 (Reinhardt e t al. 1 983 ; Thein & Mayr 1 974) . In contrast, Reo-3 (3 .6- 1 4.6%) infection was not significantly more common than either Reo- l (8 .8-24.5%) or Reo-2 (9.8-42.2%) infections among Dutch and American horses (Conner et al. 1 984; de Boer e t al. 1 978; Thein & Mayr 1 974). The results of examination of 2596 semm samples from Thoroughbred and S tandardbred racehorses in Ontario showed marked differences between these two breeds of horses (Sturm et at. 1 980) . Thoroughbred sera were positive mostly to Reo- I , fewer to Reo-2, with Reo-3 being the least frequently implicated infection. Infection with one type of reovims gives rise to a homologous antibody response as well as a lower heterotypic reaction (Thein & Mayr 1 974). Therefore, Thoroughbred horses i n this survey were most probably infected exclusively with Reo- I . The prevalence of positive sera was markedl y lower in young Thoroughbreds than older ones, with a sudden i ncrease between the groups of 5 - and 6-year-olds. A similar pattern was not observed among Standardbred horses. They seemed to become infected early in l i fe with all three serotypes of the vims. The reason for the differences in the distribution of reovirus antibodies among these two populations of horses is not known. A simi lar s ituation was not observed in other parts of the world. It was particularly surpris ing that the prevalence was low in Thoroughbreds up to 5 years of age, and increased in older animals, at the time when they usuall y retire from racing. It might be that the older Thoroughbred horses sampled in this survey were mostly horses that stayed at the racing track for longer than a few years. This, together with the fact that the numbers of older Thoroughbreds included in the survey were much smaller than that of 2- and 3- year-olds, may have influenced the results obtained. If so, the higher prevalence in older horses would reflect the differences between the different age groups in terms of number of horses and the period of time they spent in racing rather than i ndicate the predilection of the vims for older animals . The fact that a similar situation was not observed in Standardbred horses, where the representative groups of horses of different ages were more similar to each other might support the former. However, the latter can not be excluded. The c linical significance of reovims infections in horses has not been ful ly establi shed. Thein & Mayr ( 1 974) reported an outbreak of respiratory disease in an Arab stud, which was believed to be due to Reo- l and Reo-3 i nfections based on serological evidence. Chapter I 48 The outbreak fol lowed an introduction of two new mares into the stud. Cli nical signs, including intermittent cough, nasal and ocu lar discharges and loss of condition, were observed among horses in this stud for several months after introduction of infection. Three weeks after the first signs in horses, a dog kept on the premises developed respiratory disease with cough, possibly indicating spread of i nfection from the horses . However, in another study, recent reovims infection was identified only i n 1 of 252 German horses with respiratory disease (Herbst e t af. 1 992) . This suggests that reovimses were not a significant cause of respiratory problems in the horses sampled. Alternativel y. the l ack of detection of recent reovims i nfection in horses sampled may have reflected the rapid development of antibodies. A significant rise in the HI t itre fol lowing experimental infection of horses with human reovims isolates was observed as early as 3 days post infection with Reo-3 , and 7 to 8 days after i nfection with Reo- l (Thein & Mayr 1 974) , Maximum titres were reached 8 to 10 days after infection with both serotypes. The t itres decl ined 16 to 36 days after infection with Reo- I , while remaining high up to the end of the observation period ( 106 days) after i nfection with Reo-3. The immune response was not always serotype-specific, although heterotypic responses were usual ly lower and decl ined faster. Conner et al. ( 1 984) isolated Reo-3 from a foal with diarrhoea. This i solate differed from respiratory ones in that i t haemagglutinated pig RBC, and not human 0 or bovine RBC, as did the respiratory isolates (Erasmus et al. 1 978; Thein & Mayr 1 974). Horses experimentally infected with Reo- l (two horses) or Reo-3 (one horse) developed relative ly mild respiratory disease. The disease signs were exacerbated after work on a lunge, with all three infected horses showing marked dyspnoea, rise in temperature and massive watery nasal discharge. The infection was confirmed by virus isolation and serology (Thein & Mayr 1 974) . In another study, two 3-year-old horses infected with either Reo-2 or Reo-3 showed no clinical signs apart from induced cough in the horse infected with Reo-3, although both horses became infected as judged by serological responses and vims isolation (Erasmus et a!. 1 978) . Literature Review 1 .3.6 Equine parainfluenza virus-3 The family Paramyxoviridae Family Subfami ly Genus Genus Genus Paramyxoviridae Paramyxovirinae Paramyxo virus -----. Morbilli virus Rubulavirus (Murphy et al. 1 995) Group characteristics Properties 49 Equine parainfluenza viru -3 Paramyxoviruses are pleomorphjc, enveloped RNA viruses. The particle size ranges from 1 50 to 300 nm. Within the virion, the nucleocapsid is wound around itself, but when released following disruption of the virus it has a rod l ike structure about I IJ1ll in length. The viral RNA is a single stranded, non-segmented molecule of negat ive sense, covered with nucleocapsid proteins. It codes for six structural and one, or po sibly two, non-structural proteins (Lamb & Kolakofsky 1 996). The haemagglutinin-neuraminjdase (HN) protein is the major antigenic deterrr unant of paramyxoviruse that elicits neutral ising and neurarrunidase-inhjbiting antibodies. It is responsible for adsorption of the virus to host cel l receptors that contain sialic acid. It also possesses neuraminidase activity necessary to cause enzymatic cleavage of ial ic acid (neuraminic acid) residues on the virus in order to prevent self-aggregation of released virus particles (Lamb & Kolakofsky 1 996). Viral repl ication takes place in the cytopla m of infected cel ls . Maturation of the VIruS occurs by budding through the cel l membrane. During tills process the virus acquires an envelope, which had been previously modified by insertion of viral HN and fusion (F) glycoprotein pike . Virions are easily inactivated by heat, lipid solvents, non-ionic detergents, formaldehyde and oxidizing agents (Murphy et al. 1 995) . Chapter 1 50 Classification and disease signs Parainfluenza viruses belong to the family Param}'xoviridae. Four serotypes of parainfluenza viru ses have been described (CoBins e t al. 1 996) . Parainfluenza virus- l (PI- I ) produces subcl inical infections i n humans, monkeys, guinea pigs and rabbits and can cause severe respiratory disease with high mortali ty in breeding colonies of rats and mice (Fermer 1 993) . Parainfluenza virus-2 (PI-2) i nfection occurs in a variety of species and is usually subcl inical. In dogs, however, it plays a role in the "kennel cough" syndrome. Antibodies to PI-3 have been detected in a number of species including man, cattle, sheep, goats, pigs, cats, dogs, rats, monkey and others (Fenner 1 993) . The infection i s usually subcl inical . However, under certain conditions such as stress, transport, poor hygiene, crowding or harsh weather conditions, PI-3 together with other respiratory viruses predisposes animals to secondary bacterial infections. In particular, PI-3 i s a major predi sposing factor for severe bronchopneumonia caused by Pasteurella species i n sheep and cattle (Fenner 1 993) . Parainfluenza virus-4 has been l inked to mild respiratory tract disease in humans (Collins et af. 1 996) . Respiratory disease caused by paramyxoviruses can range from an inapparent infection to l ife threatening lower respiratory tract disease. Parainfluenza virus-3 is the second most important cause of pneumonia in children less than 6 months of age. It often causes i l lness in the presence of c irculating maternal antibodies. Transmission of parainfluenza viruses occurs by person to person contact or by large droplet aerosols (Coll ins et al. 1 996) . Equine parainfluenza virus-3 Information about PI-3 i nfection in horses is rather sparse. Equine parainfluenza virus-3 was first i solated in 1 96 1 from 1 2 of 48 Thoroughbred yearlings with acute upper respiratory disease (Ditchfield 1 969; Ditchfield e t af. 1 963) . All 48 yearlings showed a four-fold or greater rise in HI antibody t i tres to the equine i solate of PI-3, whereas none of 259 cl ini call y normal yearli ngs and adult horses on the same farm showed a similar rise in titre. However, all healthy adult horses and most healthy yearlings examined were positive for antibodies to PI-3, which indicated that the virus was widespread in this particular group of horses . Parainfluenza v irus-3 was also i solated from cl inical ly sick horses i n I l l inois (Sibinovic e t a!. 1 965) . The equine strain of PI-3 Literature Review 5 1 haemagglutinated guinea pig and human 0 RBC and was found to be more closely related to human PI-3 than to the bovine one in one study (Ditchfield 1 969) , but i ndisti nguishable from the bovine strain in antoher (Sibinovic et al. 1 965) . This indicates the existence of antigenic differences between isolates . The reported prevalence of PI-3 antibodies in selected equine populations varied from 0 to about 90% (Todd 1 969; Ditchfield 1 969; Ditchfield et al. 1 963) . These differences may indicate geographical differences in distribution of the virus. Alternatively, they may reflect differences between diagnostic tests used or the existence of different antigenic types, as Todd ( 1 969) could not detect neutralis ing antibodies in sera previously reported to be positive for CF antibodies to equine PI-3 by another l aboratory. The pre-existing levels of PI-3 antibodies in the horses from the outbreak of respiratory disease described by Ditchfield et al. ( 1 963) were not reported. This information would be usefu l in determining the level of neutralising antibodies that could be regarded as protective . Although the levels of antibodies in acute serum samples was not reported either, all the adult healthy horses on the farm had neutralising antibodies to parainfluenza viruses 1 , 2 , 3 and Sendai v irus (murine PI- I ) at a titre of 1 6 or more. Complement fixing antibodies in horses from which PI-3 was isolated disappeared by the fourth month after infection, whereas HI antibodies persisted for at l east a year (Ditchfield 1 969). Cl inical sIgns associated with PI-3 infection in horses include fever, anorexia, dyspnoea, enlargement of submandibular lymph nodes, and seropurulent nasal discharge. Most horses recover spontaneously by 7 to 9 days. However, in some animals, mucopurulent nasal discharge and enlargement of lymph nodes persist for several weeks (Ditchfield et at. 1 963) . The cl inical signs observed in horses in I l l inois were more severe, probably because the PI-3 infection was complicated by infection with S. equi (Sibinovic et al. 1 965) . Persistent c linical s igns observed in some horses infected with PI-3 (Ditchfield e t al. 1 963) could be due to secondary bacterial infections, as it is known that PI-3 infection in sheep and cattle predisposes animals to bacterial invasion (Fenner 1 993) . This Chapter 1 52 probably results from the impairment of the function of alveolar macrophages in which PI-3 is capable of growing (Brown & Ananaba 1 988; Davies e t al. 1 986; Toth & Hesse 1 983) . Also, the outcome of PI-3 infection may depend on the level of pre-exi sting protective antibody at the time of infection and the load of infectious vims. Huberman et al. ( 1 995) showed that the degree of cytopathology i n cell monolayers infected with human PI-3 depended on the multipl icity of infection. Low multiplicity of infection caused cell to cell vims spread and syncytia formation, but at high multiplicity of infection, sufficient sialic acid receptors were removed by v iral neuraminidase to prevent cell to cell fusion, while stil l allowing viral i nfection and spread, leading to persistent infection. Whether the same is tme in vivo has not been established. The few papers mentioned above are the only reports on PI-3 infection in horses. Generally it is not considered to be a common or important pathogen of horses . Parainfluenza vims-3 infection has not been reported in New Zealand horses, although PI-3 i nfection is common in sheep and cattle in this country (Oliver e t al. 1 976; Carter & Hunter 1 970). The factors influencing host specificity of PI-3 v imses have not been ful ly evaluated. I t has been suggested that the efficacy of the F protein cleavage, which depends on both the vims strain and the host cel l , may be an important factor in determining the vimlence and tissue tropism of Sendai vims and Newcastle disease vims (Coll ins et al. 1 996) . Thus, s imi lar mechanisms may be important for the host range restriction of PI-3 vimses. From that perspective, molecular comparison of equine, human and bovine strains of PI-3 would be interesting. 1 .3.7 Equine influenza virus Equine influenza vims is exotic to New Zealand (Horner & Ledgard 1 988; Jolly et al. 1 986). The introduction of this vims poses a major threat for the New Zealand equine industry. However, equine influenza does not contribute to equine respiratory d isease in this country at present. Therefore, equine influenza infection wil l not be d iscussed i n this l i terature review. Several reviews on the subject have been publ ished (Timoney 1 996; Hannant & Mumford 1 996; Wil son 1 993) . Literature Review 1 .4 AIMS AND SCOPE OF THE THESIS 53 The long-term aim of the studies reported i n this thesis is to minimise wastage due to equine respiratory disease in performance horses. In order to control, or prevent, the occurrence of equine respiratory disease, knowledge of which pathogens are involved i n causing disease i s essential . The New Zealand environment is peculiar in that some equine respiratory viruses, most importantly equine int1uenza, are exotic to this country. Thus, the in itial stage of the project was to establish which viruses circulate among New Zealand horses, at what age foals become infected and which v iruses are most commonly associated with development of cl in ical signs. The design and results of a virological and serological survey performed are presented in Part I of the thesis. The des ign of the survey i s described in Chapter 2 . Results of virus i solation are reported i n Chapter 3 . Serological testing for the presence of antibodies against EHV- I , EHV-4, ERhV- l and ERhV-2, EAdV- 1 , PI-3 , EA V and equine reoviruses are reported in Chapters 4 to 7 . The results of the survey are summarised and discussed in Chapter 8 , Part 2 of the thesis reports investigations of selected aspects of the biology of EHV-2 and EHV -5. During the survey, several isolates of EHV -5 were obtained. This represented the first i solation of this virus outside Australia. Genomic comparison of representative New Zealand i solates with the original Australian strain is described i n Chapter 9 . During the survey, the most commonly i solated virus was EHV-2. I t was i solated both from healthy animals and those showing cl inical signs of respiratory disease. Several features of the genome of EHV -2 suggest that it may be involved in causing respiratory disease i ndirectl y, by modulating the immune responses of infected horses. In order to gain more knowledge about the role this virus plays in equine respiratory disease, gene expression in equ ine leucocytes infected with EHV-2 or exposed to inactivated EHV-2 was compared. The results of this investigation are presented in Chapter 1 0. Some problems associated with establ ishing a causative l ink between any infectious agent and disease, as well as possible future directions in investigation of causes of equine respiratory disease are discussed in Chapter 1 1 . Part 1 : The Survey 2.1 INTRODUCTION CHAPTER 2: DESIGN OF THE SURVEY The horse racing industry i well developed in New Zealand. In the 1 998 season, more than 500 race meetings were held, and the betting turnover for Thoroughbred and Standardbred racing exceeded 700 mill ion New Zealand dol lars (Anon.b) . S imi larly to the situation overseas, respiratory disease constitutes a recognised economic problem for both breeding farms and racing stables (Anon . 1 999). The infectious agents capable of causing respiratory problems in horses include viruses, bacteria, fungi and mycoplasmas. The relative importance of these pathogens, however, has not been clearly defined. Outbreaks of respiratory disease usually occur among young animals at times of stress, for example at weaning, yearl ing sales, or when 2- year-olds are brought in for training. Most of the animals at any particular place are affected and they rarely re pond to antibiotic treatment. The e feature are consistent with a viral aetiology. However, many outbreaks of respiratory disea e remain undiagnosed, and many are never investigated. If they are investigated, it is u ual ly some time after the development of clinical signs, when the trainer or owner decides that the disease has become a problem. At this stage, a viral aetiology, even if correct, is often difficult to prove for several reasons. Firstly, viruses can be i olated most readily only before, or at the onset of cl inical signs. Secondly, serological diagnosis without an "acute " serum sample is usual ly inconclusive. Thirdly, primary viral infection are often complicated by secondary bacterial infections. Equine respi ratory viruses include equme herpesviruses, equme influenza viruses, equine adenoviru e , equine rhinoviruse , equine arteritis virus, reoviru e , coronavirus and parainfluenza virus-3. Herpesviruses and influenza viruses have been most often associated with outbreaks of respiratory disease in horses overseas (Sugiura et at. 1 987; Alien & Bryans 1 986; Ingram et al. 1 978; Rose e t a t . 1 974). New Zealand is fortunate in the fact that equine influenza is exotic to this country (Jolly et at. 1 986) . However, there has not been a comprehensive study conducted on the importance of other Chapter 2 58 respiratory vIruses 10 causll1g viral respiratory disease i n New Zealand. A l imi ted serological survey conducted in 1 98 1 indicated that equine herpes v iruses, equine rhinoviruses, equine adenoviruses and equine atierit is virus were all present in New Zealand (Jolly e t al. 1 986). There has been no reported evidence of reovlruses, coronavi rus or parainfluenza virus-3 infections in New Zealand horses. The aim of the present survey was to identify which respiratory virus infections are most common in the New Zealand equine population, at which stage of l ife horses become infected and which of the v iruses are most commonly associated with development of cl inical signs. In order to answer these questions, three groups of foals were fol lowed on a monthly basis, samples were collected from horses from outbreaks of respiratory disease and also from yearlings at the yearling sales. The yearli ng sales were chosen as one of the foci of the survey, as they provide a good environment for development of respiratory disease. Within a few days, hundreds of young animals are shifted to and from the crowded location. This, together with stress connected with travell ing and a new environment, makes an ideal situation for viral respiratory infections to occur. The "acute" samples of nasal swabs and blood were collected at the sales or just after. It was hoped that if any of the sampled horses developed respiratory d isease due to v iral infection during the sales, the t iming of the first sampling would increase the chance of the i solation of the virus. Also, the availabil i ty of "acute" and "convalescent" serum samples would allow for serological d iagnosis in infected animals . 2.2 GENERAL MATERIALS AND METHODS 2.2.1 Horses Foals Three groups of foals were sampled on a monthly basis . The dates of samplings and number of foals sampled at each time are shown in Table 2 . 1 . The Survey ------- - - - -- - - - - - - Table 2.1 : Foals sampled on a monthly basis Group A Group B <:lli ng T I fi l ly healthy T2 fi l ly healthy T3 fil ly healthy T4 fi l ly had a 'cold' few days after the sales TS colt healthy T6 colt healthy T7 colt healthy TS colt healthy 1 horses sampled only at the first sampling t ime Group SA J sf sampling 6 1 2nd sampling All the colts from group SA (SA l to SAS) were paddocked i nd iv idually, whereas fil lies (SA9 to SA 1 5 ) were paddocked in groups. Horses SA I l , SA 1 2, and SA l 3 were paddocked together. Fil l y SA 1 2 showed cl inical signs, as well as one of her paddock­ mates. The stud workers, however, did not remember whether it was fil ly SA I l or SA l3, and hence these two horses were regarded as horses for which there were no Chapter 2 62 cl inical data available. Horses SA9, SA 1 0, SA 14 and SA 1 5 formed another group. Two of these yearl ings, SA9 and SA 1 4, showed clinical signs of respiratory disease about 3 weeks after the sales. All fi l l ies were treated with antibiotics. Although colts SA3 and SA5 showed sl ight nasal discharge, they did not develop cl inical signs that warranted treatment. All horses in this group were vaccinated with influenza vaccine 3 to 4 weeks after the sales. Group W Horses from group W, at times, were boxed individuaJ ly during the month after the sales. When paddocked, however, these horses were kept in groups. Some of the yearlings developed respiratory s igns 1 to 2 weeks after the sales. Similarly to SA horses, the general impression of the stud stuff members was that it was mostly fi l l ies that presented with cl inical respiratory s igns. None of the horses in this group was treated with antibiotics. Group T Group T consisted of eight hor e brought from the sales to a racing stable for training. The yearl ings were boxed separately in two different locations : horses T l , T2, T4, and T5 were kept at the racing stable, whereas hor e T3, T6, T7, and T8 were stabled at the yard at a different location. The only horse that was reported to have respiratory problems a few days after the sales was horse T4. All other horses remained healthy, as assessed by their keepers. Outbreaks Samples from 45 foals and hor es from five outbreaks of respiratory disease were col lected. Outbreak TA Samples from 1 0 horses from the Te Awamatu region were sent to Massey University by the local veterinarian. These horses developed respiratory disease after the yearl ings were brought back home from the yearl ing sales. Other hor es, apart from yearl ings, were also affected. The horses were sampled for the first time on 1 6 February 1 996, and the second blood sample were collected 3 weeks later. The horses were first sampled 1 -- --- - - - - - The Survey 63 to 2 weeks after the development of c lin ical signs, so they may not have been in the acute state of i nfection. The horses and the c lin ical signs reported are l isted i n Table 2 .3 . Table 2.3: Horses from outbreak TA Horse numher Description TAl yearl ing fi l l y from sales TA2 yearl ing fi l ly from sales TA3 2-year-old TA4 2-year-old TA5 3-year-old TA6 4-year-old TA7 4-year-old TA8 yearl ing from sales TA9 5-year-old TA 1 0 4-year-old Outbreak H Clinical signs s l ight nasal d ischarge s l ight nasal discharge cough in contact wi th TA3 history of nasal d ischarge cough chronic nasal discharge enlarged l ymph nodes sudden onset of i nappetance, watery nasal discharge i n contact wi th T A9 Group H consisted of seven horses from a small racing stable in the central North Island. The horses were 2- to 3-year-old colts (horses H I , H2, H4, H5, H6 and H7) and one 2-year-old fil ly (horse H3) . The respiratory problem in these horses started i n December 1 995 and was characterised mainly by poor performance and weakness of all the horses from the stable. The horses were examined and treated by a local veterinarian. However, the situation did not improve and the horses were put out of training at the end of March 1 996. The horses seemed to be weak, did not want to work and had a watery, milky nasal discharge after work. Two of the horses, H i and H7, were brought to Massey University equine clinic for examination. No obvious cause for the condition was found. The first survey samples, nasal swabs and blood, were collected from six of these horses on 20 April 1 996 (all except H6). The second samples were taken from all seven horses on 1 3 June 1 996. These samples included nasopharyngeal swabs and blood for serology (horses H I to H6) or only blood sample for serology (H7). Chapter 2 64 Group B T Group BT consisted of I S Thoroughbred foals that were 2- to 3-months of age. The foals were first sampled on 3 December 1 996, and the second t ime on 23 January 1 997. All foals sampled, apart from BT 1 0 and BT 1 1 , showed s igns of respiratory d isease at the time of first sampling and for up to 3 weeks previously. Foals BT l O and BT 1 ! did not show any respiratory signs and were i ncluded as healthy foals. S ingle acute serum samples were collected from foals BT l , BT3, and BT6; single convalescent serum samples were collected from foals BT8 to BT I 5 ; and paired serum samples were collected from foals BT2, BT4, BT5 and BT7. Group SS Group SS comprised eight yearl ings, which were being prepared for the yearling sales at a small training centre in the Manawatu region. Clinical s igns in this group of horses started with serous nasal discharge, which later became mucopumlent and some animals also had enlarged lymph nodes and coughed. Horses SS 1 and SS2 were healthy horses in contact with sick animals. Horse SS3 was most severely affected. At the t ime of first sampling (6 March 1 996), horses SS5 and SS6 had been s ick for 4 days, whereas horses SS3, SS4, SS7 and SS8 had been sick for 9 days. The second serum sample was collected a month later (6 April 1 996) from only two horses (SS6 and SS I ) that were available for sampling. Group F Group F consisted of four 3- to 4-month-old Thoroughbred foals and one dam from a small stud in the Manawatu region. All foals presented with a cough, enlarged l ymph nodes and nasal discharge. First samples were collected on 26 Febmary 1 996, approximately 2 weeks after the development of c l in ical signs, and the second blood samples were taken 6 weeks later ( 1 2 March 1 996) . 2.2.2 Collection of samples Samples collected from each horse on the first sampling consisted of a nasal swab (Virocult, General Wire and Equipment), one tube of b lood for serology and one tube of blood on heparin. At the second sampling, usual ly only blood for serology was collected. However, on a few occasions, nasal or nasopharyngeal swabs and blood on The Survey 65 heparin were also collected, if the horse sampled showed signs of respiratory disease at the t ime of the second sampling. 2.2.3 Processing of samples Samples were shifted to the laboratory as soon as possible, and processed. Sera were allowed to c lot at 4 QC overnight. On the fol lowing day, the blood clots were separated by centrifugation at 2000 g for 20 min, sera collected, and stored in aliquots at -70 QC until further use. Sera collected from the eight foals from group B that were sampled only once as well as sera from foals SA 1 0 and SA 1 5 were not included in the majority of serological tests conducted. All serological tests were performed at the end of the sampling period, after col lection of all the samples. During each serological test, paired serum samples from horses from outbreaks of respiratory disease and yearli ngs from the yearling sales were processed preferentiall y on the same plate, or at least in the same run of the test. For virus isolation, samples from all 1 14 foals and horses included in the survey were processed as described in section 3 .2 . 2.2.4 Statistical analysis For statistical analysis, only horses for which ful l data sets were available (clinical data, nasal swab and two blood samples for serology) were included. Probabi lities (p) were calculated using Fisher 's exact test. If any of the value used for the calculation of the odds ratio (OR) was 0, the value of 0.25 was added to each of the values used for that calculation and the result presented as corrected OR. The association between recent viral infection and presence of c l inical signs of respiratory disase in yearlings from the sales was tested by Mantel-Haenszel method and results presented as adjusted OR. Results were regarded as statistically significant if p s; 0.05 . All calculations were performed using the NSCC 2000 statistical package. The outbreaks were investigated to determine which VIruses were most commonly present i n horses with respiratory disease. As such, results o f these investigations were presented as percentage data only. CHAPTER 3: VIRUS ISOLATION 3.1 INTRODUCTION The successful isolation of a virus from a cl inical specimen depends on several factors. Apart from the use of suitable laboratory techniques, the time of sample collection, and the conditions of transport of the specimen, are the most important. Traditional methods for viru detection involve cultivation of a laboratory specimen in a su itable system. Cell culture systems are used most frequently, with embryonated eggs, tissue transplants or animals being used less commonly, and only for viruses that are difficult to grow in cells . The growth of a virus can be detected by the presence of the CPE in cultured cells . With viruses that do not produce a vis ible CPE in cel l culture, several other methods can be appl ied to detect the presence of a virus. The most commonly used include electron micro copy (EM), haemagglutination (HA), haemadsorpt ion, ELISA, hi tological staining, immunostaining, and detection of viral nucleic acids using either PCR or DNA probes. The use of these methods is not exclusive and often combinations of several are used. Also, most of them can be used for direct detection of a virus in a cl inical specimen, without attempting virus isolation fir t, or for further identification of a cytopathic viral isolate from cell culture. The main advantage of using methods to detect a virus directly is peed and specificity of diagnosis. Also, viral components are detected even in sample that were not kept under appropriate condit ions during transportat ion. Thus, these methods may be preferred for detection of sensitive viruses that are difficult to grow in cell culture, or in ca es when speed of diagnosis might influence the treatment and outcome of disease. The advantages of isolating a virus in ceI J cul ture are definitive proof of the pre ence of a viable virus in the animal from which the sample was col lected, and avai lability of unlimited material for further characterisation and study. In the present chapter, the attempted isolation of viruses from nasal swabs and PBL of the horses included in the survey is presented. All equine re piratory viruse , except for Chapter 3 68 equine influenza virus, grow well in a cell culture sy tern. The cel ls most commonly reported to be used for isolation of equine respiratory viruses include primary cells of equine origin, RK- 1 3 and Vero cel ls . Therefore, these cells were used for primary inoculation of col lected specimens. Several methods including PCR, haemagglutination, electron microscopy and hi stological staining were used for characterisation or detection of viruses in the inoculated cell cultures. 3.2 MATERIALS AND METHODS 3.2. 1 Cell culture The three cell culture sy terns routinely used in the study were commercially available continuous cells l ines of RK- 1 3 and Vero cel ls , and primary equine foetal kidney cells (EFK). Throughout the study, standard laboratory procedures were used for propagation and maintenance of cell cultures ( Freshney 1 994), and for isolat ion and propagation of viruses ( Lennette et aL. 1 988). Media The growth medium (GM) consisted of minimal essential medium with non-essential aminoacids (MEM+n, S igma) supplemented with 1 0% v/v foetal bovine serum ( FBS) and 1 % v/v antibiotic olution ( PSK) (Appendix E). Maintenance medium (MM) was the same as GM, except that it contained 2% v/v FBS. For preparation of primary cel ls, the GM was supplemented with 1 0% v/v lactalbumin hydrolysate (ELH, S igma). All media were prepared from powder according to the manufacturer' s instruct ions. Subculturing of cells The cel ls were routinely subcultured twice weekly. The monolayers were washed twice with warm phosphate buffered saline, pH 7.0 (PBS ) and cells were disassociated with antibiotic-trypsin-versene solution (ATV) at 37 cc. After cells detached from the flask, they were resuspended in an appropriate volume of GM. Usually, a split ratio of 1 : 2 was used for EFK cel ls, and a split ratio of 1 :4 to 1 : 8 for RK- 1 3 and Vero cells . For seeding 24-well plates, 2 ml of freshly diluted cell suspension was added to every wel l . Cell cultures were maintained at 37 cC in 5% CO2 atmosphere. For viru i olation, RK- 1 3 and Vero cel ls at passage 30-200, and EFK cel ls at a maximum passage five were used. EHV- J/4 Serology 69 Cryopreservation of cells For freezing, cel l s were trypsinized, resuspended in 1 0 ml of GM, counted, pelleted by centrifugation at 300 g for 1 0 minutes, and resuspended in freezing medium (FM) at a concentration of 2 x 1 07 cells per m!. Freezing medium consisted of MEM+n with 20% v/v FBS and 1 0% v/v diemethyl sulfoxide (DMSO, S igma). The cell suspension was dispensed into 1 .7 ml cryo-vials (Nunc) , and cooled slowly at 1 QC per minute in a 1 cC freezing container (Nalgene) at -70 QC, before v ials were placed in l iquid nitrogen. In order to reconstitute the cells , one vial was thawed and transferred to a 1 75-cm3 or 80- cm' flask. An appropriate volume of warm GM was added slowly and cell s incubated at 37 °C in 5% CO2 atmosphere. Preparation of primary equine kidney cells The preparation of primary cell cultures were based on the methods described by Freshney ( 1 994) . Enzymatic disaggregation in either cold or warm trypsin was used. The kidneys were removed aseptical ly from an equine foetus. The cortex was c leaned from unwanted tissue, chopped to fine pieces and washed three times in PBS . After the last wash the cortex pieces were transferred to a trypsinization flask and approximately 1 00 ml of 0.25% trypsin per 10 g of t issue was added. Tissue suspended in warm trypsin was stirred at 200 rpm at 37 °C for 2 hours. Every 1 5 minutes the pieces were allowed to settle, supernatant was collected and fresh trypsin added to the flask. The first supernatant was discarded, and all remaining supernatants were centrifuged at 500 g for 5 minutes. The cell pel let was resuspended in 1 0 m] of GM, and stored on ice. The chi l led cell suspensions were pooled and seeded into 1 75-cm2 flasks at a concentration of 2 x 1 06 cells/ml . The tissue suspended in cold trypsin was left undisturbed at 4 QC overnight. Then, the supernatant was removed, and the tissue was incubated in residual trypsin at 37 QC for approximately 20 to 30 minutes, after which time cells were resuspended in approximately double the original trypsin volume of warm GM by gently pipetting up and down and seeded into flasks as described above. Irrespective of the method used, the cells were passaged once before they were frozen in liquid nitrogen. Chapter 3 70 3.2.2 Collection of samples Nasal swabs Nasal swabs were collected using the Virocult transport system (Virocult, Medical Wire & Equipment Co Australasia Pty Ltd), consi sting of a J 5-cm long swab and a transport tube with a foam pad saturated with 1 .2 ml of transport medium. After swabbing the nasal cavity of a horse, the swab was placed i n the transport tube and transported to the laboratory as quickly as possible. For samples collected from places more than 30 to 60 minutes drive away from the laboratory, the swabs were transported on ice. For samples collected from closer locations, the swabs were usually transported at ambient temperatures. The manufacturer of the Virocult system assures survival of viruses at ambient temperatures for long periods of time: adenoviruses - 9 days, parainfluenza viruses - 3 days, herpesviruses - up to 1 2 days. B lood samples B lood for virus i solation was collected by venipuncture using 1 0-ml heparinised vacutainer tubes (green caps) . One tube of blood for virus i solation from PBL was collected from every horse. 3.2.3 Processing of samples Nasal swabs Nasal swabs were processed directly on arrival at the laboratory according to the manufacturer' s i nstructions. Approximately 2 ml of MEM+n was added to the transport tube with the swab in situ. The tube was vortexed briefly and squeezed several t imes to mix the contents. The liquid medium was withdrawn from the tube, passed through 0.22 f.lm filter, d ispensed i nto 1 .7 ml Eppendorf tubes, and e ither used directly for i noculation of cell cultures, or frozen at -70 °C . B lood Peripheral blood leucocytes were separated from heparin ised blood as previously described (Gleeson & Coggins 1 985) . The blood was allowed to settle for 1 0 to 20 minutes at room temperature (RT). After the RBC separated, the buffy coat rich plasma - - - - - - -- -- -- ---------- - - EHV- J/4 Serology 7 1 was collected, mixed with an equal volume of RBC lysing buffer (0.85% NH4CI , 0.0 1 7 M Tris, pH 7 .4) and incubated at RT for 5- 1 0 minutes. The cells were pelleted by centrifugation at 250 g for 1 0 minutes, washed once in PBS, pelleted again , and finally resuspended in 4 ml of warm PBS . Primary inoculation and subculturing For virus i solation, 200 /-11 of either PBL suspensIon or nasal swab filtrate was inoculated onto each of three different cell cultures, grown in 24-wel l plates. Thus, each sample was inoculated into three wells containing three different cell cultures (EFK, RK- 1 3 and Vero). Three one-week passages were performed. Each time, cell cultures were freeze-thawed and 200 !-1l of the cell lysate was transferred to a corresponding well in a new plate. The well s were inoculated either at the same time as the cel l s were seeded into plates, or on the foIJowing day, when monolayers were approximately 80% confluent. 3.2.4 Virus detection The cell cultures were observed for CPE at least twice weekly. Additionall y, HA tests were performed as described below. Occasional ly, electron microscopy and histologic staining of fixed cells were also used. Isolated herpesviruses were typed using PCR (3 .2 .5) . The samples were considered negative if no CPE was observed after three passages and, when applicable, the HA and PCR results were negative. Haemagglutination Haemagglutination tests were performed in order to detect v iruses that do not produce an obvious CPE in t issue culture, including reoviruses and PI-3 v irus. The freeze­ thawed cell lysates from the third passage on RK- 1 3 and Vero cell s were used in HA tests. Red blood cell s were prepared and stored as described in section 6.2. 1 . Two-fold dilutions i n duplicates (50-!-1l volume) in PBS of lysates from inoculated cell cultures were made in a microtitre plate (V -bottom, Nunc) from 1 : 1 0 through to 1 :40. Then, 50 /-11 of RBC suspension was added, the mixtures left undisturbed unti l haemagglutination was observed in control wells (approximately an hour) and the results read. Human 0 RBC (0.75%) or guinea pig RBC (0.5%) were used as an i ndicator system for Vero and RK- 1 3 cell cultures, respectively. The tests were performed at RT (RK- 1 3 cell s ) or 4 DC Chapter 3 72 (Vero cel ls ) . The posit ive controls consisted of bovine Reo-3 (Vero cel ls ) and bovine PI-3 (RK- 1 3 cel ls ) . The control v ilUses were grown in the respective cell l ines and treated in the same way as survey samples. Negative controls, consisting of freeze­ thawed cultures of non-inoculated Vero and RK- 1 3 cells were also i ncluded with every lUn of the test. Al l samples showing a c lear pellet of sedimented RBC were considered negative. Electron microscopy After freeze-thawing, cell culture l ysates were c larified by centrifugation at 500 g for 10 minutes. The supernatants were collected and ultra-centrifuged a t 1 80,000 g for 2 hours over a I -ml cushion of 45% w/v sucrose. The pel lets were resuspended in 100 f.ll of distilled H20 (dH20) overnight at 4 QC and used for negative staining. Formvar-coated, 400 mesh copper grids were placed coating side down onto 50 f.ll drops of 1 % bovine selUm albumin (BSA) ( 10 seconds), sample (40 seconds) , dH20 ( 10 seconds) and 2% phosphotungstic acid pH 7.0 (PTA) (40 seconds) i n the described order. Between each transfer, excess fluid was blotted away and the grid was thoroughly dried before p lacing i n a grid holder. The stained grids were examined using a Philips 20 le electron microscope. Histologic staining of fixed cells Cells for histologic staining were grown in eight-wel l chamber s li des (Tissue Tek, Nunc). When cell s reached approximately 80% confluency, they were inoculated with 75 f.ll of cell lysate from the third passage. After 4 to 5 days incubation at 37 QC in 5% CO2 atmosphere, the cell s were fixed overnight in Bouin's fixat ive, washed i n 70% ethanol, stained with haematoxylin and eosin CH & E), and examined under a l ight microscope. 3.2.5 Polymerase chain reaction Samples for peR EHV-2 and EHV-5 peR: With some exceptions, PCR reactions using primers specific for EHV -2 and EHV -5 were routinely performed on all the samples that showed herpesviral CPE in cell culture. On most occasions, where a CPE was observed on - - - -- - �-------� -- � - - - - EHV- 114 5,'erology 73 RK- 1 3 cel l s i t was also present in the EFK cells . In such cases, occasionally, i f PCR specific for either EHV -2 or EHV -5 performed on lysate from one cell culture was positive, the corresponding cell culture was not checked for the presence of the same virus. Al l the cell cultures that were positive for EHV -5 were also checked for the presence of EHV -2 DNA. Also, on a few occasions, PCR was performed on cell lysates from cultures that did not show CPE. This happened mostly, but not exclusively, when CPE was present in a different cell l ine inocul ated with the same sample. EHV-J and EHV-4 peR: The following samples were checked for the presence of EHV- 1 and EHV-4 DNA: all PBL cultures on EFK cells , PBL cultures on RK- 1 3 cell s collected from routinely sampled foals a t the t ime when they showed serological evidence of recent EHV - 1 /4 infection, and all cell cultures positive for EHV -5. Additionall y, PCR with primers specific for EHV - 1 and EHV -4 were performed directly on all nasal swab fil trates. peR reactions The PCR primers and programs used i n this study are l isted in Table 3 . 1 . The reaction mix consisted of 0.2 mM of each dNTP, 1 IlM of each primer, and 1 .5 units of Taq DNA polymerase in I x Taq PCR reaction buffer ( 1 0 mM Tris-HCl, 1 .5 mM MgCb, 50 mM KCI , pH 8 .3 ) in a 25 IJ.l total volume. All PCR reagents were purchased from Roche, and primers were custom synthesised by Gibco, BRL. Reactions were performed i n thin-walled PCR tubes (Biological™ Continental Laboratory Products Inc . ) in a Perkin Elmer PCR 9600 thermocycler. For multiple reactions, a PCR master mix was prepared. PCR reagents were stored and used in a designated clean room, using dedicated pipettes, pipette tips and reagents. Preparation and addition of samples was performed in an area separated from the clean room and from the room where gel analysis of PCR products was performed. Chapter 3 74 Table 3. 1 : peR primers and programs used in the study PCR Primers Program' Target Product Re! gene size Forward: 5' -AGAAAATGGCACAGAGCCAG-3' -95 °C-5 min EHV-2 - 35 cycles: gB 257 bp Reverse: 5'-TGGCAATAAAATGGAGACTGC-3' 95 °C - 60 s Forward : 60 °C - 90 s 5' -GAGACCACGTTGTCCCCG-3' 72 °C - 6 0 s EHV-5 gB 25 1 bp Reverse: _4 °C 5' -GCTTCAAGTCCCTCA TG AGC-3' Forward: 5' -GCGAGA TGTGGTTGCCT AA TCTCG-3' -94 °C-5 min EHV- I E H V - 1 14 rever e2: gC 649 bp - 30 cycles: 5'-GAGACGGTAACGCTGGTACTGTTAA-3' 94 °C - 75 s EHV4 forward: 60 °C - 90 5' -ACGCACGAACAACTCAACCGATGT -3 ' 72 °C - 90 EHV-4 EHV - 1 14 rever e2 : gC 507 bp _4 °C 5 ' -GAGACGGT AACGCTGGTACTGTT AA-3' I B lack dots indicate steps in a PCR program 2 EHV- I and EHV-4 peci fic PCR reactions were performed as mul tiplex PCR reaction with al l three pri mers used in one tube 3 Reubel et al. ( 1 995 ) 4 Lawrence et al. ( 1 994) Preparation of samples for peR 3 4 DNA was not extracted for routine PCR screening. After freeze-thawing, aliquot of infected cell culture ly ate ( 1 00 �) were tran ferred to Eppendorf tube and incubated at 95 QC for 20 minutes. Four � of this preparation was used in a 25-� PCR reaction. On a few occasions, DNA was extracted from samples that were negative on PCR despite producing CPE in cell culture. In these cases a DNA extraction kit (QIAamp blood kit, QIAGEN) was used according to the manufacturer' instructions . Positive and negative controls were included with every run of the test. Positive controls consi sted of cell cultures infected with respective viruse and prepared in the same way as survey samples. Negative control included equine DNA (non-infected EFK treated in an identical manner to the urvey samples) and cel ls infected with EHV-2 (for EHV-5 PCR), EHV-5 ( for EHV-2 PCR), EHV- I ( for EHV-4 PCR) and EHV-4 ( for EHV- l - -- - -- -- - - �------------- EHV- 114 Serology 75 PCR) . Thus, EHV- l and EHV-4 infected cultures served as both negative and positive controls in EHV - 1 /4 multiplex PCR. Analysis of amplified DNA Aliquots ( 10 !J,l) of PCR products were subjected to electrophoresis through a 1 .5% ethidium bromide (EtBr) stained agarose gel (Gibco, BRL) in TBE buffer at 100V for 40 to 60 minutes, visual ised under UV l ight and photographed using polaroid black and white photographic film (Polaroid 667) . A molecular size marker (fX 1 74 RF DNNHae III fragments, Gibco BRL) was included for reference on every gel . The results were confirmed by dot blot and Southern hybridisation with digoxigenin (DIG) labelled probes specific for individual herpesvi ruses. A sample was considered posit ive i f a product of the correct size was visible on a gel and its specificity was confirmed by Southern hybridisation. Samples that were positive on a dot blot, but negative on a gel were also considered positive, as the detection limit of Southern hybridisation wi th DIG-labelled probes i s higher than the detection l imit of EtBr staining (Holtke et al. 1 995) . The PCR test was considered val id if the positive and negative controls showed expected results. Preparation of dot blots After gel analysis, PCR reaction tubes were placed again in a thermal cycler, heated to 95 QC for 1 0 minutes, and quickly chilled on ice. One � of each PCR product was spotted on a pre-marked membrane (Hybond N+, Amersham) , and the membrane was allowed to dry. The DNA was fixed to the membrane by UV cross li nking, by placing the membrane wrapped in a Saran Wrap, DNA side down, on an UV transi l luminator for 4 minutes. The blots were stored dry at 4 QC until use. Preparation of probes Virus speci fic probes were prepared by random primed labell ing of the ampli fication products from PCR reactions performed as described above with the reference v iruses used as target DNA. The fol lowing reference viruses were u sed: EHV -2 86, EHV -5 2- 1 4 1 , EHV - 1 Durham, and EHV -4 Homer. The identities of amplified products were confirmed by sequencing before they were labelled and used as probes. For label l ing, PCR products were gel purified after electrophoresis through a 1 .5% EtBr stained low melting point agarose gel (Seaplaque) in T AE buffer at 1 00V for 60 minutes. Gel s lices Chapter 3 76 containing PCR products were melted at 70 °C in the presence of 4 � of GELase buffer per 200 mg of a gel s lice (GELase™, Epicentre Technologies) . After equilibration, 1 unit of GELase enzyme per 200 mg of gel was added and the mixture incubated at 45 °c for 45 minutes. Finally, the DNA was ethanol precipitated with 5 M ammonium acetate, the pel let washed with 70% ethanol, dried under vaccum for 5 minutes (Savant Speedvac SC I OO) and resuspended in 10 � dH20. A small aliquot of this preparation was run on a 1 .5% EtBt stained agarose gel in TBE buffer, and the amount of recovered DNA estimated by comparison of the intensity of the band with the DNA mass ladder included on the gel (Gibco, BRL). Approximately 1 80-360 ng of DNA was used in the subsequent l abell ing reaction. Labell ing with DIG was performed using DIG-High Prime (DIG labell ing and detection kit, Roche) according to the manufacturer' s instructions. Briefly, DNA templates di luted to 1 6 � with dH20 were heat denatured by boil ing for 1 0 minutes and quickly chi ll ing on ice, before 4 � of DIG-High Prime was added to every reaction, and the tubes incubated at 37 °C overnight. Reactions were stopped by adding 2 � of 200 mM EDTA, pH 8.0. The yield of labelled DNA was estimated by spotting appropriate dilutions of the labelled products on DIG quantification teststrips and comparing the intensity of dots obtained after immunological detection wi th the i ntensity of corresponding dots on the control strips according to the manufacturer' s instructions (Roche) . For hybridisation, probes were used at a concentration of 2.5 ng/m! (EHV -5) or 5 ng/ml (EHV-2, EHV- l , and EHV-4) . Sequencing Sequencing of the PCR products was performed using AmpJiCycle ™ Sequencing Kit (Perkin Elmer) according to the manufacturer' s instructions. Four sequencing reactions, one with each of the four termination mixes, were performed for every template. The termination mixes consisted of 22.5 jlM 7-deaza-dGTP, 10 j.1M solution of each dATP, dCTP, dTTP, and 600 j.1M of one of the four ddNTPs (ddATP, ddTTP, ddCTP, ddGTP). The sequencing reactions consisted of 0.5 jlM of primer, 2 jlCi [a-33P] -dCTP, 50 mM Tris-HCl, pH 8.9, 10 mM KCI , 2.5 mM MgCh, 0.025 % v/v Tween® 20, 1 unit of AmpliTaq® DNA polymerase (cycle sequencing), 40 ng of template DNA, 0.5 j.1M of additional dA TP/dTTP and 2 � of one of the four termination mixes. The cycl ing - - - - -- - -- - - - �- - - �------------- - - - - - EHV- J/4 Serology 77 temperatures were as follows: denaturation at 95 °C (60 seconds) fol lowed by 25 cycles of denaturation at 95 °C (30 seconds), annealing at 60 °C (30 seconds) and elongation at 72 °C (60 seconds) . At the end of the nm, the samples were cooled to 4 °C, removed from the thermal cycler and 4 � of stop solution (95% Formamide, 0.05% Bromophenol Blue, 0.02% Xylene Cyanole FF, 20 mM EDT A) was added to every tube. The reactions were either analysed immediately or stored at -20 °C for up to one week. A 6% polyacrylamide/urea sequencing gel was prepared according to the standard protocol (Sambrook et al. 1 989). The amplification products were denatured by boil ing for 5 minutes and quickly chill ing on ice. Then, 3 � of the denatured DNA from each of the four termination reactions ( in the order: G, A, C, T) of one template was loaded into the wells of the gel . The well s were thoroughly rinsed immediately prior to loading and the samples subjected to electrophoresis at 70 W for 285 minutes. After the first 240 minutes, the electrophoresis was stopped and 3 �l of each of the termination reaction mixes loaded again into the additional wells, before sequencing products were subjected to electrophoresis for an additional 95 minutes. Thus, long and short runs were performed for every sample. Fol lowing electrophoresis, the gel was d isassembled from the gel sandwich, fixed with gel fixing solution (5% methanol, 5% glac ial acetic acid in dH20) for 15 minutes, transferred to 3MM Whatman fil ter paper and dried for 40 minutes in a gel dryer (BioRad, model 583) . The dried gel was exposed to an X-ray film for 1 2-24 hours at RT, developed, and the sequence read manually from the autoradiogram. Hybridisation with specific probes The following hybridisation temperatures were used: EHV-2 EHV-5 EHV- l EHV-4 45 °C The membrane was pre-hybridised in 20 ml of hybridisation buffer (DIG Easy Hyb, Roche) per 1 00 cm' of the membrane, at the appropriate hybridisation temperature, for at least 1 hour. Directly before use, the relevant probe was heat denatured by boil ing for 1 0 minutes in a water bath and quickly chilling on ice. The denatured probe was then diluted in DIG Easy Hyb to the desired concentration (see "probe preparation") and Chapter 3 78 added to the hybridisation bag, from which the pre-hybrid isation solut ion had been removed. The hybrid isation was performed overnight. After hybridisation, the membrane was washed twice in 2 x wash solution (2 x SSC, containing 0. 1 % SDS) for 5 minutes at RT, and then twice i n 0.5 x wash solution (0.5 x SSC, containing 0. 1 % SDS) for 1 5 minutes at 68 QC in order to remove unbound probe. The hybridisation solution containing the probe was saved for re-use. Re-used probes were stored at -20 QC and denatured at 68 QC for t o minutes prior to use in any subsequent hybridisations. Detection of DIG labelled probes DIG-labelled probes were detected colorimetrical ly using a DIG labell ing and detection kit according to the manufacturer 's instruction (Roche) . The reagents llsed in the detection procedure are l isted in Appendix E. All incubation steps were performed at RT with agi tation. Amounts of solutions given are for detection of a 1 00-cm3 blot. After hybridisation and hybridisation washes, the membrane was equilibrated in washing buffer for 1 minute. The membrane was then incubated in a series of solutions as follows: • 1 00 ml of blocking solution for 30-60 minutes • 30 ml of alkaline phosphatase conjugated anti-DIG antibody, di luted in blocking solution to a concentration of 1 50 mU/ml for 30 minutes • 1 00 ml washing buffer for 1 5 minutes, twice • 20 ml detection buffer for 2-5 minutes After equ i libration in the detection buffer, the membrane was sealed in a plastic bag wi th 1 0 ml color substrate solution, and incubated in the dark, without agitation, until the desired intensity of spots was reached (usual ly 3 to 6 hours) . The reaction was stopped by washing the membrane in dH20, and results documented by photocopying the wet fil ter. Usually, two to three membranes were developed at the same time. Re-probing Color precipitate was removed by incubating the membrane at 55-60 QC in dimethylformamide (Sigma) for several hours . After the blue colour was no longer visible, the membrane was washed in dH20, and incubated twice, 1 0 minutes per i ncubation, in alkaline-probe stripping solution (0.2 M NaOH, 0. 1 % SDS) at 37 QC. The EHV-l/4 Serology 79 membrane was then washed in 2 x s s e , and pre-hybridised before hybridising with the next probe. 3.3 RESULTS The only viruses identified were the herpesviruses EHV-2, EHV-5 and, on one occasion, EHV -4. The peR reactions used for identification of individual herpesviruses worked well. The positive control reactions resulted in the ampl ification of the products of the expected sizes and negative controls were consistently negative (Figure 3 . 1 , Figure 3 .3 ) . The peR results were further confirmed by Southern hybrid isation with target specific probes. Sequencing results confirmed that the peR products used for preparation of the probes contained expected sequences. 3.3. 1 Foals The results of v irus isolation from foals fol lowed on a monthly basis are shown in Figure 3 . 2. Blood samples for v irus isolation were not collected from the foals from group A in November and December 1 995. At all other sampling occasions, EHV -2 was isolated from PBL of nearly 1 00% of foals sampled. Additional ly, at t imes, EHV -2 was i solated from the nasal swabs of some of the foals. Generally, EHV -2 was i solated from both nasal swab and PBL samples of some, or all , of the foals sampled over a period of 2 to 4 months from January to April (group A), March to April (group B), or May to July (group e ) . Then, EHV -2 could no longer be isolated from the nasal swab samples, while it continued to be present in the PBL. Equine herpesvirus-5 was i solated from 1 5 foals on 32 sampling occasions (Table 3 .2 , Figure 3 .2) . The v irus was isolated from both nasal swabs and PBL samples of two foals on five occasions, from the nasal swab only from one foal on one sampling occasion, and from PBL samples only of each of 1 5 foals on at least one sampling occas ion. W ith one exception, all the samples that were positive for EHV -5 were also positive for EHV-2. The only exception was the nasal swab sample collected from foal B 1 3 i n April . This sample was positive for EHV-5 only. However, EHV-2 was i solated from PBL of this foal at the same sampling time. Thus, although not from the same sample, both EHV -2 and EHV -5 were recovered from this foal on the same sampling occasion. Equine herpesvirus-5 was often i solated from the same foals over a period of t ime. For Chapter 3 80 example, EHV-S was isolated from foal AS over a period of 1 1 months, from FeblUary to December (Table 3 .2 ) . Table 3.2: EHV -5 isolation from foals. Foal Feb Mar Apr May Jul Aug Oct Nov Dec A I • • • • • A3 • AS • • • • • • • • • • • A6 • A7 • B l • B4 • • • B6 • B7 • B8 • • • B9 • B l 2 • B I 3 • • • B I 6 • • C3 • • EHV -5 isolation from nasal swabs ( . ) and from PB L ( . ) M 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 2 3 4 EHV-l probe 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 � 2 3 : 6 J EHV-4 probe 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 • • • � . - Figure 3.1 : Results of EHV-1I4 PCR visualised on a 1 .5% EtBr stained gel and a corresponding dot blot probed with either EHV- l or EHV-4 probe as indicated. Lane M: molecular size marker fX174 RF DNAIHae III fragments; lanes 1 - 12: cell Iysates from survey samples; line 12: Vero cells inoculated with PBL sample from foal BT7; lane 13: EHV- l control; lane 14: EHV-4 control; lane 15: EFK negative control; lane 16: EHV- l + EHV-4 control. Expected products: EHV- l 649 bp, EHV -4 507 bp. EHV- /14 Serology A '" 6 iii .E 5 '0 4 .8 3 E ::I 2 c: 0 B 1 9 1 8 1 7 1 6 15 '" 14 iii 1 3 .E 1 2 - 1 1 � 10 .8 9 E 8 ::I 7 c: 6 5 4 3 2 1 0 C '" � 4 '0 3 ... .. � 2 � 1 0 E HV-2 '" 6 iii .E 5 '0 4 I I I .8 3 E ::I 2 c: • • 0 7 7 6 2 2 Nov Dec Jan Feb Mar Apr May Jul Aug Oct Nov Dec months 1 9 18 1 7 16 15 '" 14 iii 1 3 .E 1 2 - 1 1 � 10 1 1 1 1 I .8 9 E 8 ::I 7 c: 6 5 4 3 2 1 0 9 9 9 8 7 Mar Apr May Jul Aug Oct Nov Dec months '" 1 � 4 '0 3 .8 2 E � 1 0 5 4 4 Apr May Jun Jul Aug Oet Nov Dee months • isotation from PBL was done from 4 foals only Vi rus i solation from: _ nasal swab PBL 8 1 E HV-5 - - - 1 • • 1 • • • 7 6 3 2 2 2 Nov Dec Jan Feb Mar Apr May Jul Aug Oct Nov Dec months I - I . - - -1 9 1 1 10 9 9 9 8 7 Mar Apr May Jul Aug Oet Nov Dee months • • 5 5 5 5 5 5 4 Apr May Jun Jul Aug Oet Nov Dec months _ nasal swab + PBL Figure 3.2: Viruses isolated from foals followed on a monthly basis: group A (A), group B (B), and group C (C). Numbers above the X-axis indicate numbers of foals sampled. Chapter 3 82 Table 3.3: Viruses isolated from horses from 3.3.2 Outbreaks outbreaks of respiratory disease EHV-2 · EHV-5 . Horse TAl TA2 TA3 TA4 TA5 TA6 TA7 TA8 TA9 TA 10 H I * H2* H3* H4* H5* H6 H7 BT I BT2 BT3 BT4* BT5 BT6 BT7 BT8 BT9 BT I O BT l l BT L 2 BT I 3 BT l 4 BT I 5 S S I SS2 SS3 SS4 SS5 SS6 SS7 SS8 F I F2 F3 F4 F5 EHV-5 . + EHV-2 . EHV-2 .+ EHV-4. Virus isolated from: Swab PBL • • • • - / - - / - - / - - / - - / - • • . / . • • • • • • • • • 1 2 3 o • • • • • • • • • • • • • • • • nd • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 3 1 2 7 1 The results of virus isolation from horses from outbreak of respiratory disease are shown In Table 3 .3 . Equine herpesvirus-2 was isolated from PBL from 39 of 43 (90.7%) horses sampled, including seven samples from which EHV -2 was isolated concurrently with EHV -5, and one sample from which both EHV -2 and EHV -4 were isolated (foal BT7) . Equine herpe virus-2 was also the most commonly isolated virus from the nasal swab samples. Equine herpesvirus-5 wa isolated from 1 3 samples col lected from 1 2 hor es. Equine herpesvirus-5 was isolated from PBL of eight hor es, from nasal swabs of three horses and from both nasal swab and PBL samples of one horse. Only twice was EHV -5 i olated without concurrent isolation of EHV-2 from either nasal swab or PBL sample collected from the same horse on the ame sampling occasion (horses T AS and H6). Although EHV -5 alone wa isolated from the nasal swabs of three further horse , EHV -2 was isolated from PBL samples of these horses on the same sampl ing occasion. * A nasal or nasopharyngeal (outbreak H ) wab was collected on two different occasions; nd - not done; '-' denotes lack of isolation of any v irus; gray fields - healthy horses; yel low fields - horses showing c l in ical signs of respiratory di ea e . EHV- J/4 Serology 83 Table 3.4: Viruses isolated from yearlings from the sales Yearling Virus isolation from: Swab PBL SA l. • SA2 • SA3* - I . • SA4 • • SA5 * ? I - • SA6 • • SA7 • • SA8 • • SA9* - I ? • SA I D • SA i l • • S A I 2 * - I - • • SA I 3 • • • SA I 4* - I - • SA I 5 • T I • T2 • T3 • T4 • T5 • T6 • T7 • T8 • W I • W2 • W3 • W4 • W5 • W6 • W7* - I ? • W8 • W9* - I - • W I D ? • W l 1 • W I 2 W I 3 • • W I 4 • • • • • EHV-2 . 2 30 EHV-5 . 0 0 • • 2 6 ? 4 0 3.3.3 Yearlings The results of virus isolation from samples coJlected from the yearl ings from the yearl ing sales are presented in Table 3.4. Equine herpesvirus-2, ei ther alone or together with EHV- 5, was isolated from 36 of 37 (97 .3%) PBL am pies col lected. Six PBL samples that were positive for EHV-5 were also posit ive for EHV-2. Herpesviruses were i olated from 8 of 43 nasal swab samples col lected. Two of these isolates were EHV-2 only, two samples were positive for both EHV-2 and EHV-5, and four isolates were negative with all the primers used, despite producing herpesviral CPE in cell culture. 3.3.4 Other viruses None of the infected cell culture Iysates tested showed any haemagglutination activity with the RBC used, indicating that neither reoviruses nor PJ-3 virus were growing in the cultures. 3.3.5 Isolates negative by PCR Thirteen isolates that produced herpe viral CPE did not react with any of the herpe virus type speci fic primers used (Table 3 .5 ) . These isolates were either not investigated further, or EM and histologic staining were u ed to confirm that the isolate was a herpesvirus. The true numbers of PCR negative, CPE positive reactions may have been higher than this, since PCR reaction were not performed on every sample showing a herpesviraJ CPE (3 .2 .5) . * A nasal swab was collected on two different occasions; ' - ' denotes lack of isolation of any virus; ' ? ' herpesviral CPE seen but no reaction in PCR; gray fields - heal thy hor e ; yel low fields - horses with c l in ical signs of respiratory d isease; white field - no c l in ical data avai lable ( see table 2 . 2 for detai ls) . Chapter 3 Table 3.5: Isolates negative by peR Horse Date of Isolate: sameJing Source, cell line A5 Jan PBL, EFK A5 Jan PBL, RK- 1 3 A5 Feb Swab, RK- 1 3 [/J .....l B l4 Mar PBL, RK- 1 3 .I... B9 Apr PBL, EFK B l2 Apr PBL, EFK C3 Oct PBL, RK- 1 3 SA3 Jan PBL RK- 1 3 [/J SA3 Mar Swab, RK- 1 3 0 z ...... SA5 Jan Swab. EFK .....l cG SA9 Mar Swab, RK- 1 3 J..l W7 Mar Swab, EFK >-- W 1 0 Jan Swab, RK- 1 3 CPE herpes herpes herpes herpes herpes herpes herpes herpes herpes herpes herpes herpes herpes H&E . . I stallllng + + + + + + nd + + + + nd nd 84 EM Other virusei herpes EHV-2, swab herpes EHV-2, swab nd' EHV -2, swab (EFK) EHV -2/5, PBL nd EHV-2, PBL (EFK) nd EHV-2, PBL (RK- 1 3) EHV-2, swab nd EHV-2, PBL (RK- 1 3) nd nd nd nd EHV -2, swab (EFK) nd EHV-2, PBL nd herpes nd EHV-2, PBL I Other v iruses identified from the same sample i noculated on a different cel l l i ne or from a d ifferent sample col lected from the same horse on the same samp l i ng occasion. 2 Presence of intranuclear i nc lusion bodies. 3 Not done. 3.3.6 Primary isolation EHV-2: Equine herpesvirus-2 was isolated from nasal swab samples on 57 occasions, and from PBL samples on 229 occasions. Most of the time, EHV-2 grew on both RK- 1 3 and EFK cells. The i solation of EHV -2 on both cell l ines was confirmed by peR on 1 75 occasions. The remaining 1 1 1 samples included samples that produced ePE on only one of the two cel l l ines, but also samples that produced ePE on both RK- 1 3 and EFK cells , where the isolate from only one cell l ine was typed . EHV-5: Equine herpesvirus-5 was isolated from nasal swab samples on 1 2 occasions, and from PBL samples on 46 occasions. The virus was most often isolated on RK- 1 3 cel ls (45 isolations), fol lowed by EFK cells (7 i solations) and both RK- 1 3 and EFK cells (6 isolations) . With two exceptions, EHV -5 isolation was always accompanied by the isolation of EHV -2 from the same horse a t the same sampling occasion, although not necessarily from the same sample. The two exceptions were EHV -5 isolates from PBL samples of horses T AS and H6 from outbreaks of respiratory d isease (Table 3 .3 ) . EHV-114 Serology 85 EHV-4: Equine herpesvirus-4 was i solated on only one occasion, on Vero cel ls . Both RK- 1 3 and EFK cells i nocu lated with the same sample were negative for the presence of EHV -4 DNA. All peR reactions performed directly on nasal swab filtrates were negati ve for the presence of EHV - I and EHV -4 DNA. J -4 � 6 7 9 11 12 U 14 IS t6 I ' • • 4' () ' 8' 9' 1 0' 1 1 ' et et • • • • • l Y 1 .t' 1 :;' 1 (;' • 2 -' .I 5 I) 8 9 • • I I 1 2 13 14 tj 1 6 I ' 2 ' .t' 5' 6' 7' 8' 9' 10' 1 1 ' 13' 1 4' 15' 1 6' Figure 3.3: An example of amplification products from a peR with EHV -2 (upper part of a gel ­ samples l ' to 16') and EHV-5 (lower part of a gel - samples 1 to 16) primers, visualised on 1 .5% ErBr stained gel (a) and a corresponding dot blot probed with either EHV -2 probe (b) o r EHV-5 probe (c). Lanes M : molecular size marker fX174 RF DNAlHae III fragments; lanes 1 - 13 and l ' - 13' : samples from horses 1 - 13 (these numbers do not correspond to any numbers used to identify horses in the text and serve only to explain the interpretation of this figure); lanes 14, 14 ' : EHV-2 control; lanes 15, 15 ' : EHV-5 control; lanes 16, 16 ' : EFK negative control. Expected products: EHV-2 275 bp, EHV-5 251 bp. Horses 1 , 2, and 4 are positive for both EHV-2 and EHV -5; horses 10, 12 and 13 are negative for both viruses; horses 3, 5, 6, 7, 8, 9, and 1 1 are positive for EHV·2 only (note that bands 9' and 1 1 ' are very weak, but they are clearly positive on the dot blot). 3.3.7 Comparison of cell culture and peR results In general , the peR results correlated wel l with the cell culture results . Most of the v iral i solates that showed herpesviral ePE in cell culture were also posit ive for one or two equine herpes viruses by peR. Also, most of the peR reactions performed with lysates of cell s that were negative for ePE did not result in any specific ampli fication. However, there were 1 2 isolates that produced typical herpesviral ePE in cell culture, and did not react with any of the herpesvirus specific primers used (Table 3 .5 ) . Also, 1 2 Chapter 3 86 of 92 l ysates from cultures without visible CPE gave positive results when checked by PCR: seven were positive for EHV-2, two for EHV-5, and three for both EHV-2 and EHV-5. 3.3.8 Association with clinical signs The rates of i solation of EHV-2 and EHV-5 from horses from the outbreaks of respiratory disease and from yearlings from the yearling sales are shown in Table 3 .6 . EHV -2/5 detection in PBL The i solation of EHV -2 or EHV -5 from PBL samples could not be correlated with the presence or absence of cl inical s igns in the animals sampled (Table 3 .6) . Close to 1 00% of PBL samples collected were posit ive for EHV -2. Of 3 1 EHV -5 i solations made from foals sampled on a monthly basis, 23 (74%) were made from healthy foals, and 8 (25.8%) were made either from foals that showed respiratory signs (group A) or at the time when most foals on a given farm experienced respiratory problems, although i ndividual cl inical data were not available (group B ) ( section 2 .2 . 1 , Figure 3 .2 ) . Of horses from outbreaks of respiratory disease and yearl ings from the yearling sales, EHV-S was i solated from PBL of 4 of 2 1 ( 1 9%) healthy animals , 9 of S2 ( 1 7% ) sick animals, and 2 of 8 (2S%) animals for which i ndividual c l inical data were not available (Table 3 .3 , Table 3 .4) . Thus, EHV-S isolation from PBL was not significantly higher in horses showing signs of respiratory disease in comparison with healthy horses (OR 0.9, p = O.S) . Table 3.6: Herpesvirus (EH V -2, EHV -5, or both) isolation from horses from outbreaks of respiratory disease and yearlings from the yearling sales. Unidentified herpesviral isolates were counted as EHV -2 (see section 3.4.2), Horses Isolations f!'om nasal swabs lsolations jjom PBL orfoafs Outbreaks Yearlinss T0s.ether Outbreaks Yearlings Together Healthy 33% (2/6) 0% (O/ \ S ) 9 .5% (2/2 1 ) 830/, (S/6) 1 00% ( 1 S/ l S) 95% (2012 1 ) Sick 340/, ( 1 31 1 8) 36<,1'1' (S/ 1 4) 35% ( 1 8/52) 970/, (37/38) 93% ( 1 3/ 1 4) 96% (50/52) Data not 37.5% (3/8) 37 .S% (3/8) available 1 00(1<:) ( 8/8) 1 00% (8/8) Together 34% ( 1 5/44) 22% (8/37) 28% (23/8 \ ) 95% (42/44) 97% (36/37) 96% (78/8 \ ) EHV-114 Serology 87 EHV -2/5 detection in nasal swabs From foals , EHV -2, EHV -5 or both viruses were i solated from nasal swab samples during the time when most foals from groups B and C showed some respiratory signs. However, the i solation of EHV-2 and EHV-5 from nasal swabs of foals from group A was usually not accompanied by any c linical signs of respiratory disease (section 2 .2 . 1 , Figure 3 .2) . The isolation of EHV -2/5 from nasal swabs from yearlings from the sales was statistically associated with respiratory disease (corrected OR 34.6, P = 0.02) , as all i solations were made either from horses showing respiratory signs or from horses for which clinical data were not available (Table 3 .6) , and not from healthy horses. From outbreaks, herpesviruses were isolated from nasal swabs of 4 of 8 (50%) sick horses from outbreak TA, from 8 of 1 2 (67%) sick foals from outbreak BT, one of 5 (20%) horses from outbreak F and none of the horses from outbreak SS . 3.4 DISCUSSION 3.4.1 Lack of isolation of viruses other than herpesviruses Herpesviruses were the only viruses isolated during the survey, despite serological evidence that equine adenovirus and equine rhinoviruses were also active among the horses sampled (see Chapters 4 to 7) . There may be several explanations for this . Firstly, nasal swabs rather than nasopharyngeal swabs were collected. This may have intluenced the results. However, the processing of the samples and cell culture techniques used supported the isolation of EHV -2 and EHV -5. Therefore, it is unlikely that l ack of i solation of other viruses was due to inappropriate collection or processing of the samples. This is a particularly valid comment for the isolation of EHV - 1 and EHV -4, but may be less valid for rhinoviruses (see later this section) . Secondly, herpesviruses present in the samples may have interfered w ith growth of other v iruses. Although several horses showed serological evidence of recent EHV - 1 /4 infection, EHV - 1 was not isolated from any samples, and EHV -4 was i solated only once, from a PBL sample. Interestingly, this isolation was made in Vero cells , and not in EFK cells in which EHV-4 is usuall y grown (Crabb & Suddert 1 996). Equine herpesvirus-2 was isolated from the same sample on RK- 1 3 and EFK cell s , but neither of these cell cultures were positive for EHV -4 DNA. Chapter 3 88 These results may i ndicate the existence of some interference between EHV - 1/4 and EHV -2/5 in vitro. In agreement with this view, Dutta et al. ( 1 986) reported the inhibitory effect of EHV -2 infection on the propagation of EHV - 1 in equine dermis and lymphocyte cell cultures. In another study, EHV- J and EHV-4 were detected by PCR in t issue samples collected from one horse that had been experimental ly infected with EHV - 1 . However, when the same tissue samples were u sed for virus i solation in cell culture, only EHV-2, and neither EHV- l nor EHV-4, was isolated (Welch et al. 1 992) . On the other hand, both EHV -2 and EHV - 1 were isolated from several t issues collected from other EHV- l infected horses i ncluded in the same study. Additionally, EHV-2 was reported to be able to trans-acti vate immediate early genes of EHV - 1 in vitro (Purewal et al. 1 992), suggesting that it may activate rather than i nhibit EHV - 1 replication . Thus, the exact i nteractions between EHV -2 and EHV - 1 /4, both in vitro and in vivo, are not ful ly understood and need further investigation. These i nteractions, however, could have been the reason for the lack of i solation of EHV - 1 and EHV -4 from all but one of the samples processed. It has been suggested that EAdV infection may interfere with EHV -2 replication in vivo (Gleeson et aI. , 1 977) . These authors i solated EHV -2 from a foal experimentall y infected with EHV -2 in utero, from the nasal swab samples collected between birth and when the foal was 65 days of age. At that time the foal was experimental ly infected with EAdV. Equine herpesvirus-2 was no longer recovered from any of the nasal swabs collected from this foal after experimental i nfection with EAdV. The final outcome of i nteractions between two viruses in vitro may depend on several factors including the dose of i nfecting virus, competition in using host cell components, or cell culture conditions. If the results of that experiment were interpreted simply as an indication of the existence of some interference between EAdV and EHV-2, the lack of i solation of EAdV in our study might have been due to such interactions. Thirdly, it is also possible that in dual infection of EHV-2/5 and any other v irus, both viruses were replicating, but herpes viral CPE overshadowed the ePE produced by the other virus. In this case, the sample would have been classified as positi ve for EHV -2 or 5 only, whereas in fact, i t might have been positive for both EHV -2/5 and the co­ infecting virus. EHV-JI4 Serology 89 Also, the cell culture conditions may have not been optimal for growth of some viruses. For example, the important difference, in terms of in vitro cultivation, between rhi noviruses and other equine respiratory viru ses concerned in this study is the aci d sensitivity of the former. For virus i solation, cells were cultured in a C02 atmosphere i n medium of approximately pH 7.0. However, after a few days in culture, the pH of the medium often became lower due to the products of cell metabolism (Freshney 1 994) . Considering that rhinoviruses are extremely sensitive to low pH and become unstable at pH lower than 6 .5 (Studdert 1 996b), it is possible that rhinoviruses were not i solated during this study because the conditions used were not optimised for i solation of these viruses. Alternatively, rhinovi ruses may have been i solated, but did not produce any visible CPE. In this case the growth of rhinoviru ses would have not been recognised by the methods used. I n one study, ERh V - 1 was detected by immunofluorescence i n cell s i noculated with nasopharyngeal swab samples and the presence of ERh V - 1 i n the swabs was further confirmed by PCR. In none of the cell cultures, however, was any CPE observed (Li et al. 1 997) . Despite the serological evidence of their presence (see Chapter 5) , equine rhinovirus isolation has never been reported in New Zealand, further indicating that isolation of these viruses may be difficult . On the contrary, 28 and 1 9 ERh V -2 isolates obtained during studies conducted i n Switzerland and America, respectively, were isolated using standard cell culture techniques (Carman et al. 1 997; Steck et al. 1 978) . Finally, i t i s possible that viruses other than EHV-2 and EHV-5 were not present in the samples collected. The success of virus i solation depends to a great extent on the t iming of sampli ng, and many i nfections are diagnosed only retrospectively based on serology results. The fact that none of the nasal swab samples checked directly for the presence of EHV- l /4 DNA yielded positive results supports the view that neither EHV- J nor EHV -4 were present in the samples collected. Alternatively, only low levels of the virus were present, below the detection l imi t of the PCR assay used. 3.4.2 Unidentified viral isolates During the present study, 1 3 viral i solates were not identified (Table 3 .5 ) . Al l of these isolates were shown to be herpes viruses based on CPE produced in tissue culture and, for some, histologic staining and EM results. However, they did not react with any of the type specific primers u sed. It is most probable that these i solates were EHV -2. A Chapter 3 90 large degree of genomic heterogeneity has been reported for different EHV -2 i solates (Browning & Studdert 1 987b) . Thus, it is possible that not all EHV -2 isolates reacted with the primers used, as the design of primers was based on the publ ished sequence of the reference strain of EHV -2 (Reubel et af. 1 995) . Moreover, the majority of unidentified i solates came from animals from which EHV-2 was i solated from a different sample or from the same sample, but on a different cell l ine. Multiple infections of one horse with several different genotypes of EHV -2 have been reported by others (Browning & Studdert 1 987 a) . 3.4.3 Clinical significance of EHV -2 and EHV -5 infections The significance of EHV -2 and EHV -5 i nfection has not been ful ly evaluated. Due to the widespread d istribution of EHV -2 i nfection among horses, and i solation of this vims from both healthy and cl inically sick animals, it was not considered to be important in equine respiratory disease. In the present study, there was no association between EHV-2 and EHV-5 i solation from PBL samples and the presence of clinical signs (Table 3 .6) . However, EHV -2 or EHV-5 isolation from nasal swabs of yearl ings from the sales and horses from outbreaks of respiratory disease was statistically associated with development of cl inical signs of respiratory d isease. Also, both viruses were i solated from nasal swabs of foals followed on a monthly basis at the time when some of the foals experienced respiratory problems (Figure 3 .2) . These results might suggest some association between active EHV-2 and EHV-5 i nfections and the presence of cl inical signs. However, EHV-2 and EHV -5 were also i solated from nasal swab samples of healthy foals from group A, and two of the i solations from nasal swabs of horses from outbreaks came from healthy foals (BT lO , BT l l ) . Thus, if EHV-2 or EHV-5 play any role in equine respiratory disease, the causation is not straightforward since i nfection is not always associated with cl inical signs. Both EHV -2 and EHV -5 have been recently re-classified as y-herpesvimses (Telford et af. 1 993) . Thus, they belong to the same family as the human pathogen EBV. Interestingly, the epidemiology of EHV -2 seems to be similar to that of EBV (Agius & S tuddert 1 994) . In humans, most EBV infections occur within the first three years of l i fe and are usually asymptomatic. However, primary infection in adolescence often EHV-J/4 Ser% gJ' 9 1 leads to i nfectious mononucleosis (IM) . The disease i s characterised by fever, headache, pharyngitis, lymphadenopathy, and general malaise, which can last for several weeks or even months. It has been postulated that IM symptoms are caused by immune responses to EBV infection, rather than viral replication per se (Rickinson & Kieff 1 996) . If so, the severity of disease could be related to differences in genetic predisposition and also to the magnitude of the immune response in relation to the dose of infecting v irus. S imilar to the situation observed for EBV, most horses become infected with EHV-2 early in l ife . Also, l ike EBV, the i nfection does not always lead to development of cl inical s igns, but it has been associated with upper respiratory disease, chronic pharyngitis, keratoconjunctivitis, lower respiratory d isease, general malaise and poor performance (Murray et al. 1 996; Nordengrahn et al. 1 996; Fu et al. 1 986; Bel- - 0 .... Q) .0 E :l c: recent ERhV-2 + EHV- 1 /4 infections recent ERhV-2 in fection recent EHV- 1 /4 infection no indication of recent viral i nfection 1 6 1 4 1 2 1 0 8 6 4 2 0 healthy showing respi ratory signs yearl ings data missing* Figure 8.2: Association between the presence of clinical sings and recent viral infections in yearlings from the yearling sales. EHV -2/5 isolation from PBL is not incorporated into this graph, because rates of isolation of both EHV -2 and EHV-5 from PBL of healthy and diseased foals or horses were similar (Chapter 3). * indicates horses from which either individual clinical data were not available or from which the second blood sample for serology was not collected. 8.2.2 Yearlings from the yearling sales The sampling of yearl ings from the yearl ing sales was implemented in order to overcome the difficulties in being able to col lect the samples soon after development of cl inical signs. Recent viral infection was only sl ightly associated with development of respiratory signs when all viruses were considered (adjusted OR 1 .3 ), although this result was not statistical ly significant (p = 0.5) . However, EHV -2/5 infection (defined by virus isolation from nasal swabs) was strongly associated with the development of Chapter 8 1 60 respiratory signs (OR 34.6, P = 0.02) . The results obtained for the remain ing vimses were not statistical ly significant. Nonetheless, recent ERh V -2 infection, e ither alone or in combination with other infections, was positively associ ated w ith c linical signs of respiratory disease (OR 2.2 , P = 0.4), while EHV - 1 14 infection, either alone or i n combination with other infections, was most often detected among healthy horses (OR 0.3, P = 0.2) (Figure 8 . 1 , Figure 8 .2) . These results may indicate that EHV -2/5 and ERh V -2 infection are more l ikel y to be associated with c l inical signs than EHV - 1 /4 infections. 8.2.3 Foals followed on a monthly basis A number of viral infections were diagnosed among foals fol lowed on a monthly basis . (Figure 8 .3 ) . The majority of foals from group A remained healthy, while most of the foals from groups B and C showed respiratory signs at some stage between April and June. Considering that similar v iral i nfections were observed in foals from groups A and B in March - May, additional factors must have i nfluenced the presence or absence of cl inical signs in these two groups of foals. Foals from both groups were weaned at the end of March. For foals from group A, this involved grouping the foals with one mare as a leader, i n a paddock, away from the foals ' dams. By the t ime they were weaned, the foals from this group were already used to being frequently handled and were not afraid of people. The foals from group B were not handled before weaning. Weaning of this group i nvolved keeping four to five foals in a box for about a week, before they were released into the paddock. During this t ime the foals were halter-broken and branded. Thus, foals from group B were probably more stressed by the experience, in comparison with foals from group A. Additionally, the boxes in which they were confined were possibly dustier and more contaminated than pasture. Foals from group C showed evidence of infection with the same viruses as foals from groups A and B, but about 2 months later, in May and July. Most of these foals showed respiratory signs sometime during May and June, and thus, some of the c l inical s igns observed may have been attributed to viral infections . However, foals from this group were weaned between March and April , so approximately 2 months before first viral infections were detected. Thus, stress of weaning was unlikely to play a predisposing The Survey - General Discussion -------- - - -- ----- - - - 1 6 1 role for viral infections i n this group of foals . One explanation for this may be that these foals were not confined in boxes for weaning. Also, they were handled before weaning, and graduall y prepared for the separation from their dams. Several authors indicated the importance of environmental conditions in equme respiratory disease (Clarke 1 987) . Burrell et af. ( 1 996) reported that the duration of respiratory d isease in horses housed on straw in loose boxes was significantly longer in comparison with horses kept on shredded paper. Poorly ventil ated boxes have been shown to have much more respirable dust, which is heavi ly contaminated with fungi , when compared to a 'clean ' , well-ventil ated environment (Clarke et al. 1 988; C larke et al. 1 987). A poorly venti lated environment was associated with an i ncreased amount of mucopus observed in tracheas of horses over that seen in a group stabled in well venti lated boxes (Clarke et al. 1 987) . During an outbreak of EHV - 114 infection, horses from both groups showed increased amounts of mucopus in their tracheas, but this increase was greater for horses in poorly ventilated boxes than for the other group (Clarke et a/. 1 988) . Another possibility is that the respiratory signs observed in some of the foals were due to bacterial infections rather than viral ones. The data of Hoffman et al. ( 1 993) implicated the predominant role of primary bacterial infections in respiratory d isease i n foals . These authors d id not i solate any viruses from 10 1 cases of respiratory d isease in foals. Also, only twice was seroconvertion to EHV- 1I4 detected among 47 randomly selected, paired serum samples. These results are in contrast to the results of the present study. Although the occurrence of bacterial infections in foals was not monitored, there was a clear indication that several respiratory viru ses were active among foals included in the study. However, it was not poss ible to correlate the presence or absence of c l in ical signs with infection with any specific viruses. 8.3 TIME OF VIRAL INFECTIONS I N FOALS Foals from group A were the only foals that were sampled from their first month of l ife . The mean titres to EHV- 1 I4, EAdV- l , ERhV- I and ERhV-2 i n this group were high at the first sampling time, and decl ined gradually until March-April , presumably reflecting declining levels of maternally derived antibodies (Figure 8 .3 ) . In April , the mean Chapter 8 1 62 antibody t itres to EHV - 1 , EAdV - 1 and ERh V -2 increased i ndicating that some of the foals became infected with these viruses around that time. Foals from group B were not sampled from the first month of age. Therefore, it could not be concluded whether titres observed during the first sampling i n March represented passive or act ive immunity. The March sampling of foals from group B took place at the end of March-beginning of April . S ince the passive antibody titres for foals from group A were very low or not detectable at the beginning of March, it seems l ikely that at least some of the t itres present in group B foals at the end of March represented act ive immunity. In any case, the mean titres to EHV - 114 and EAdV - 1 i ncreased i n April and May, indicating that some of the foals from this group showed serological evidence of infection with these viruses at the same time as foals from group A (Figure 8 .3 ) . Equine herpesviruses 1 14, EAdV - 1 and ERh V -2 were also found to circulate among foals from group C (Figure 8 .3) . However, these foals seemed to become infected later than foals from groups A and B . The reasons for this remain unclear. Foals from group C were not protected by longer lasting or h igher-levels of maternal antibodies, as they were negative for antibodies to all the viruses tested, except for ERh V -2, during the first sampling in April . The foals remained seronegative for EHV - 1 /4 and EAdV - 1 during the fol lowing 2 to 3 months . The fact that they did not show evidence of any viral infection, except for EHV -2 i nfection, in April and May supports the view that other environmental or husbandry factors may be important in the spread of respiratory viruses among foals. In all groups of foals , the isolation of BHV -2 and EHV -5 from nasal swabs preceded the serological evidence of i nfection with other respiratory viruses. This may support the predisposing role of EHV -2/5 infection to secondary infections (Chapter 3 ) . Alternatively, i t could be due to the fact that EHV -2/5 infections were detected by virus isolation, whereas infections with other respiratory viruses were detected only serologicall y. Serological diagnosis, especial ly in young animals, is less rel i able than virus i solation, because residual levels of maternal antibodies can i nterfere with mounting an active humoral response. Therefore, it is possible that infections with other viruses occurred earlier, but they were either not detected serologically or detected at a l ater stage. - - - - - - - - �--�---------------------- -------- The Survey - General Discussion 1 63 8.4 SUMMARY Several respiratory viruses were found to be active among New Zealand horses including EHV- 1I4, EHV-2, EHV-5, ERhV- l , ERhV-2, and EAdV- l . Foals were shown to possess detectable levels of maternally derived antibodies up to 5-6 months of age. Most foals became infected with one or more of the viruses investigated within their first year of l ife. Although the activity of different v iruses was recorded around the t ime that some of the foals experienced respiratory signs, infection was not always accompanied by the development of disease. Probably other factors, such as environmental conditions, husbandry practices, exposure to stress, individual genetic predisposition, or secondary bacterial infections, are l ikely to influence the outcome of infection. Equine herpesvirus-2, EHV-5 and ERhV-2 infections appeared to be associated with development of c l in ical signs in yearli ngs from the yearling sales, although these results were s igni ficant only for EHV-2/5 , and not ERhV-2. However, s ince none of the foals or horses sampled was examined endoscopical ly, it is possible that a number of lower airway i nfections were not recognised. The most common infection among horses with respiratory signs from outbreaks, for which paired serum samples were available, was EHV -2/5 infection (30.4%), fol lowed by ERh V -2 ( 1 3 .0%), ERhV- l (4.3%), and EHV- 1I4 (4.3%) infections. A large number of horses from outbreaks for which no recent viral infection was detected could reflect primary bacterial i nfection. Alternatively, it could support the role of v iruses as predisposing agents, rather than strong primary pathogens. It i s l ikely that equine respiratory disease is a multifactorial disease, with different pathogenic organisms and the environment influencing the final outcome of infection. The Survey - General Discussion A " � 2 z If) i 1 i i lOO 70 t!- I o .5 months Qct Nov Jan Fab Mat Apt Qct Nov Oec i i i months .. ·B 2 z If) o � , iv v (; 4 Qcl Nov Jan Feb Mar Apt May Jun Jul Aug � Qcl Nav Dec months Qct Nov Jan Fab Mar AiJr ::;:;:: I�:�: �,�� months _ EHV·2 I = EHV.2 and � - - - - - - - [] Qct Nov Jan Feb Mar Apt May � Jut Aug � Qcl Nov Dec months B � >;; i i .. 'S :;: 3 I ELlSA I • SN 2 � • • • Ma. Ap' May i i i " � z If) 0 � I -=- � -=- Ma. Ap' May V � 4 1 I (; B 3 E � c: Ma. Ap' May • • June Ju� Aug months � . - - 1 65 c lOO I ELlSA I • SN lOO � I months i i l . i � J. J. I 2 :;: 0 l ' .I. _ � @.i] Oct Nov Doe Ap, May Ju, Jul Aug Ocl Nov Doe months i i i .. � 2 i i i z If) 0 ,;; I SI J. J. � � - - � • • Ocl Nov Doe Ap' May Ju, Jul Aug � Oc, Nov Doe months V _EHV·2 _EHV·2 =EHV·5 !!1 4 I =EHV·5 .2 (; 3 B E I � c: Oc, Nov Doe ApI May Oc, Nov Doe Figure 8.3: Foals group A (A), 8 (8) and C (C) : mean titres to EHV- 1 I4 (i) , EAdV- 1 ( i i ) , ERhV-2 (iii), ERhV-1 (iv), and virus isolation from nasal swabs (v). Error bars show standard deviations. Shaded boxes indicate months when foals were not sampled. Virus isolation from P8L is not shown, as close to 100% of foals were positive at any sampling time. Part 2 : Some aspects of the b io logy of EHV-2 and EHV-5 CHAPTER 9: GENOMIC COMPARISON OF EHV-S ISOLATES 9.1 INTRODUCTION Equine herpe virus-5 has only recently been characterized as a species distinct from EHV-2 (Browning & Studdert 1 989; Browning & Studdert 1 987b). Equine herpesviru - 2 comprises a group of very heterogeneous viruses, although they show a high degree of homology in Southern hybridi ation studies (Browning & Studdert 1 987b). In contrast, the four EHV -5 isolates examined by Browning & Studdert ( 1 989; 1 987b) showed little cro s-hybridisation with the reference EHV -2 strain and appeared to be homogeneous. Until recently, the only identified EHV -5 isolates were the four isolates originally described by Browning & S tuddert ( 1 987b). Although several papers describing genetic and antigenic characteristics of the reference strain of EHV -5 have been published (section 1 .3 . 1 ) , there are no epidemiological data available with regard to EHV-5 infection in horses. Also, the data regarding the apparent homogeneity of EHV-5 isolates was based on comparison of only four isolates avai lable at the t ime. As a result of the respiratory virus survey, several EHV -5 isolates were obtained from a number of horses (Chapter 3 ) . S ince these represented the first EHV-5 isolates identified after the original discovery of the virus in Austral ia, I decided to examine these isolates in more detai l . The aim was to compare the New Zealand EHV -5 isolates with the prototype Australian strain, as well as with each other, in order to gain more knowledge about the biology and epidemiology of this recently recognized virus. Glycoprotein B (gB) gene was chosen to use in this study, as it codes for the most highly conserved glycoprotein among herpesviruses. Glycoprotein B plays a role in virus entry and spread between cel ls , and has been shown to be a major target for the immune response (Neubauer et al. 1 997; Pereira 1 994). Restrict ion enzyme digest of gB has shown the existence of polymorphism in several herpesviruses. Franti et al. ( 1 998) Chapter 9 1 70 described three different restriction fragment length polymorphism (RFLP) profiles for human herpesvirus-7 gB from 1 08 samples and four distinct profiles of human cytomegalovirus gB have been described (Chou & Dennison 1 99 1 ). 9.2 MATERIALS AND METHODS 9.2.1 Viruses Seventeen EHV-5 isolates were chosen for comparison with the reference Australi an strain EHV-S.2- 1 4 1 (Table 9. 1 ) . Table 9.1 : Details of the source of the EHV -5 isolates used in the study. Isolate Horse Age Clinical signs Site of Cell line isolation (E.assage #) 5 months healthy nasal swab RK 13 (2) 2 9 months nasal discharge PBL RK 1 3 (2) 3 1 1 months A5 healthy PBL EFK (3) 4 1 2 months healthy PBL RK 1 3 (3) 5 1 3 months healthy nasal swab EFK (3) 6 1 3 months healthy PBL RK 1 3 (2) 7 nasal swab RK 13 (2) A I 5 months healthy 8 PBL RK 13 (2) 9 1 2 months healthy PBL RK 1 3 (3) C3 1 0 1 3 months healthy PBL RK 13 (3) 1 1 TA5 3 years history of nasal d ischarge PBL EFK (3) 1 2 EFK (3) TA8 yearling enlarged lymph nodes nasal swab 1 3 RK 1 3 (3) 1 4 TA9 5 years sudden onset of i nappetence, watery nasal swab RK 1 3 (3) nasal discharge 1 5 SA4 yearl ing healthy PBL RK 1 3 (3) 1 6-3 RK 1 3 (3) SA7 yearling sl ight nasal d i scharge PBL 1 6-6 RK 1 3 (6) 1 7 SA I 3 yearling data not available nasal swab RK 1 3 (2) EHV-5 2- 1 4 1 1 EFK (24) I EHV -5 : 2- 1 4 1 was kindly supplied by Prof. M. J. Studdert. Details of this isolate can be found i n Turner & Studdert ( 1 970). The selected EHV-5 isolates represented i solations from groups of horses of different ages, with varied clinical manifestations of infection and varied geographical locations. Genomic Comparison of EHV-5 Isolates 1 7 1 Isolates from individual horses, obtained either from different cell l ines, or at different sampling times, were also included. 9.2.2 DNA extraction DNA was extracted (blood DNA isolation kit, QIAGEN) from 200 Ml of freeze-thawed EHV -5 positive cell culture lysates. The EHV -5 DNA was e luted in 1 05 III of Buffer EB ( 1 0 mM Tris-HCl , pH 8.5) and the concentration of the extracted DNA was checked spectrophotometrical ly (GenQuant DNA/RNA calculator, Pharmacia) . 9.2.3 Polymerase chain reaction Based on EHV-2.86/67 and EHV-5 .2- 1 4 1 gB sequences, EHV-5 speci fic primers were designed to amplify the entire EHV-5 gB. The primers used were: forward primer (fp) : 5' - AAAGGAGTGGGGGATCGCT - 3 ' reverse primer (rp): 5 ' - TTGTGTGAGTCATGAAGAAACCAG 3 ' The predicted size of the ampli fied product was 2738 bp. PCR reactions were performed using Perkin Elmer 9600 thermocycler in a 50-Ill reaction containing 1 III target DNA ( 1 2-240 ng), 3.5 units of Expand High Fidelity enzyme mix (Roche) and final concentration of 350 IlM dNTP and 0.6 IlM of each primer in 1 x Expand HF buffer with 1 .5 mM MgCb. The reactions were overlaid with mineral oil (Sigma), denatured for two minutes at 94 QC fol lowed by 1 0 cycles of 1 5 seconds denaturation at 94 QC, 30 seconds annealing at 60 QC and two minutes elongation at 68 QC l inked to 20 cycles with the same denaturation and anneali ng conditions, but with 20 seconds added to the elongation step in each successive cycle. Seven minutes of prolonged elongation time at 72 QC was added at the end of the run and samples were kept at 4 QC until analyzed on ethidium bromide stained 1 % agarose gel . 9.2.4 Cloning peR products EHV-5 gB PCR products were purified from agarose gel s using Qiagen Gel Extraction Kit (Qiagen) according to the manufacturer' s instruction. Briefly, the DNA fragment was excised from a gel and the gel s l ice i ncubated for 1 0 minutes at 50 QC in the presence of three volumes ( l OO mg corresponded to 1 00 Ill ) of Buffer QG. After solubil ization of agarose, the mixture was transferred to the QIAquick column and centrifuged at 1 0 000 g for 1 minute. The DNA adsorbed to the membrane was washed Chapter 9 1 7 2 once with 500 III of Buffer QG and then with 750 III of buffer PE. After the last wash, the QIAquick column was centrifuged for an addit ional 1 minute in order to remove the residual ethanol. DNA was eluted from the column in 1 00 III of Buffer EB into a fresh Eppendorf tube by 1 minute centrifugation at 10 000 g. For cloning, DNA was ethanol precipitated in the presence of 1 / 1 0 volume of 3 M sodium acetate, pH 5 .2, pelleted by 20 minutes centrifugation at 15 000 g, washed in 70% ethanol, pelleted again, and re­ suspended i n 10 III Tris buffer, pH 8 .5 . Purified PCR products were blunt-ended by treatment with Klenow fragment (Promega), phosphorylated by T4 polynucleotide kinase (PNK) (Gibco BRL), cloned into EcoRV site of pBluescript KS( +) cloning vector (Stratagene) using Rapid DNA Ligation kit (Roe he ) and transformed into competent E. coli cell s . For Klenow reaction, 3 III of a Klenow reaction master mix was added to 1 0 III of each purified PCR product. The amounts of reagent used to prepare 9 III of master mix were as follows: 0. 1 38 III H20, 3 .9 III 1 0 x Klenow buffer, 0.3 1 2 III 5 mM dNTP, 3 .9 III BSA ( l mg/Ill), and 0.75 III Klenow enzyme (5 unitS/il l ) . The reaction mixes were incubated at RT for 1 5 minutes and the reactions stopped by heating the mixtures to 75 QC for 1 0 minutes . Then, 1 III of PNK reaction master mix was added to every tube and reaction mixes incubated at 37 QC for 1 hour. The fol lowing components were used to prepare 3 III PNK master mix: 1 .23 III H20, 0.6 III 5 x Forward Reaction Buffer, 0.42 III ATP ( l OO mM) and 0.75 III T4 PNK ( 1 0 unitS/ill) . The PNK reactions were stopped by heating to 65 QC for 20 minutes, recovered on ice and either used directly for l igations or stored at 4 QC for no longer than one week. The l igation reactions were performed according to the manufacturer' s instructions (Rapid DNA Ligation Kit, Boehringer Mannheim), with some modification of the amounts of reagents used. Each l igation reaction consisted of 4 III insert DNA, 1 III vector DNA, 1 III 5 x DNA dilution buffer, 5 III T4 DNA l igation buffer, and 0.5 III T4 DNA l igase. The l igation reactions were allowed to proceed for at least 2 hours, after which t ime the mixtures were used for transformation into competent E.coli cel l s without any further purification. Genomic Comparison ()lEHV-5 Isolates 173 For transformation, 2 .S I-l I of each l igation reaction was added to an Eppendorf tube and put on ice. Then, SO I-lI of competent XLI -Blue MRF' cells was added to each tube and the mixtures incubated on ice for 30 minutes. The cell s were heat-shocked by incubation at 42 QC for 1 minute, recovered on ice for 2 minutes, d i luted with 4S0 I-lI of SOC broth, and incubated at 37 QC with shaking at approximately 1 80 rpm for an hour. Following incubation, the entire volume was spread onto LB agar plate containing 1 00 mg/ml ampicil l in, and incubated inverted overnight at 37 QC. 9.2.5 Colony screening Single colonies were picked from a plate into Eppendorf tubes containing 20 I-lI of LB broth each and allowed to grow at 37 QC for 1 -4 hours. Colonies contain ing a gB i nsert in the reverse orientation were identified by PCR using the vector T7 primer and EHV-S rp. DNA ampli fication was performed in a 2S-l-ll reaction containing 1 III of colony broth, 0.6S units of Expand High Fidelity enzyme mix and final concentrations of 200 f!M dNTP and 0.4 mM of each primer in I x Expand HF buffer with 1.5 mM MgC}z using the same cycling conditions as for the EHV -5 gB PCR. 9.2.6 RFLP of glycoprotein B gene From each original EHV -5 isolate, five positive colonies were chosen for further examination. Amplification products (9.2 .5) were purified directly from PCR reaction mixtures using QIAquick spin columns (QIAGEN) according to the manufacturer' s instructions. This involved several steps during which DNA was bound to a s i l ica membrane, washed, and eluted into a fresh Eppendorf tube in 34 I-lI of Buffer EB ( 1 0 mM Tris-HCI, pH 8 .S) . An aliquot of 8 I-lI of each purified PCR product was digested with 1 I-lI Bfa 1 (New England B iolabs) for 4 hours at 37 QC i n a supplied buffer i n a total volume of 10 I-ll . The digested PCR products were subjected to electrophoresis in 1 0% polyacrylamide gels ( lOOV, 1 00 min), stained with gel star nucleic acid stain (FMC Bioproducts) and photographed using a gel star photographic filter (FMC Bioproducts) . The obtained patterns were compared to the pattern produced by EHV- 5.2- 1 4 1 prepared in the same way. Chapter 9 9.2.7 Sequencing and sequence comparison 174 Eight c lones were sequenced from T7 primer on an ABI Prism 377 DNA sequencer using the dye terminator cycle sequencing ready reaction kit (Perkin Elmer) . Five hundred base pairs from the 5' end of each insert were compared to each other and to corresponding published sequences of EHV-5 .2- 1 4 1 (GenBank accession number AF05067 1 ) and EHV-2.86/67 (GenBank accession number U20824). The obtained nucleotide sequences were translated (Anon.a) and the predicted amino acid sequences, corresponding to the N terminus of gB, compared with each other. DNA and protein sequence comparison was performed using the OClustal wO computer program. 9.3 RESULTS The PCR with the EHV -5 gB pnmers resulted i n amplification of products of a predicted size (Figure 9. 1 ) . From cloned products, five colonies containing a gB insert i n the reverse orientation were bp 3 054 1 636 M 1 $B, \� \� lEt(� 2 3 4 5 � "- - .. - - . o 6 randomly chosen from the colonies positive for PCR with EHV -5 rp and vector T7 primer. An example of a gel with amplification products Figure 9.1 : Amplification products of PCR with primers (rp + fp) specific for EHV -5 gB. Lane M: 1 kb molecular ladder (Gibco BRL), lanes 1 - 6: amplification products of isolates 10, 9, 4, 14, and EHV -5 : 2-141 (lanes 1 , 2, 3, 4, and 6, respectively). Lane 5: isolate not included in the study. from colony screening PCR i s shown in Figure 9 .2 . The predicted RFLP profi le of the amplification product generated by PCR with EHV -5 rp and vector T7 primer bp M 1 2 3 4 5 6 7 8 9 1 0 with plasmid containing the 3 054 2 036 Figure 9.2: Amplification products of PCR colony screening with EHV -5 rp and T7 primer: lane M : 1 kb molecular ladder (Gibco BRL); lanes 1-10: clones 1-10 from isolate 9. reference EHV-5 :2- 1 4 1 gB used as target DNA is shown in Figure 9 .3 . I t contains 1 2 Bfa 1 cutting s ites resulting in generation of 1 3 bands ranging in size from 6 to 553 bp. Bands 6 bp and 52 bp are derived from the vector. By comparison, the similar product containing the relevant part of the reference Genomic Comparison of EHV-5 Isolates 1 75 EHV -2 genome would contain only four Bfa 1 restriction sites (Figure 9 .3) . The RFLP pattern of gB of the reference EHV-5 : 2- 1 4 1 showed the predicted pattern. All other clones examined showed vector derived bands as well as bands 1 34 bp, 20 1 bp, 472 bp and 544 bp. The number and size of the remaining bands varied between clones from different EHV -S isolates (Figure 9.4, Table 9.2) . EHV-5 5' I � -l I> S ) I n I I I � 3' I 52 I 6 I 134 544 I 201 I 96 I 180 1 59 I 66 I 90 I 275 I 553 I 472 T7 EHV-5 fp EHV-5 rp II--vector-II-----==----------------------------------------------insert--------------------------.-------------------------------------1 1 EHV-2 5' I 52 I 6 I 607 3' 650 1486 Figure 9.3: The position of predicted Bfa 1 sites in a cloned EHV-S rp-T7 primer amplification product (yellow) and in a corresponding fragment of EHV -2 sequence (white). Twelve Bfa 1 cutting sites are numbered 1 to 1 2. Not to scale. 9.3. 1 EHV-5 isolated from different horses had different RFLP profiles All EHV -S isolates cultured from different horses had different RFLP profi les, with the exception of isolates obtained from horses TA8, TA9 and SA4 (Figure 9.4) . Clones ' B­ A' I and ' 1 4 ' deri ved from EHV -S isolated from horses T A8 and T A9, respecti vel y, showed identical RFLP profi les. Also, clones ' 1 2 ' and ' I S ' derived from EHV-S isolated from horses T A8 and SA4 had the same Bfa 1 cleavage patterns. Additional ly, the gB cleavage pattern of clone ' I T, derived from a nasal swab isolate of yearling SA l 3 , was nearly identical to the pattern of the prototype strain E H V-5: 2- 1 4 1 . The only difference observed between the two profi les was that the 66-bp fragment of EHV­ S : 2- 1 4 1 was sl ightly bigger in isolate 1 7 (Figure 9.4). 9.3.2 RFLP profiles of EHV -5 from the same horses were identical Apart from EHV -5 isolates 1 2 and 1 3 , cultured from the nasal swab of the horse TA8, al l isolates from the same horse had identical gB cleavage patterns . This was J Throughout this chapter ' x ' i nd icates al l c lones with the same RFLP pattern, derived from isolate x. Chapter 9 1 76 particul arly evident for foal A5. Al l EHV -5 isolates cultured from foal A5 over a period of nine months from April to December, from both nasal swabs and PBL, in either cell l ine, had identical RFLP profiles. S imilarly, isolates 7 and 8 from foal A I , and isolates 9 and 1 0 from foal C3, had identical RFLP profiles (Figure 9.4, Table 9.2) . � "f � N lci � ill C"l <0 > � � N ;:! ;:! "f '!2 cb cb r-- I � � � � UJ Figure 9.4: Bfa 1 digest of EHV -5 rp-T7 primer amplification products. Numbers on the top represent clones derived from isolates listed in Table 9.1 . Lane M: 50 bp molecular ladder (the lowest visible band corresponds to 50 bp). Table 9.2: Comparison of RFLP patterns obtained for different EHV -5 isolates. Isolates Horse Bfi l ' . . ) a sIte mlssmg Additional Bla 1 .life' 2- 1 4 1 , 1 7 Prototype strain , S A 1 3 I I TA5 6, 8 7 . 8 A l 6, 1 1 I to 6 A5 6, 7, 8 Between 6 and 8 1 6-3, 1 6-6 SA7 6, 7 , 8 , IO 9, I O K3 6, 7 , 8 , 1 1 1 4, I 3-A TA9, TA8 6. 7 , 8, I O, 1 1 I 3-B TA8 6, 7 , 8 , 1 0. I 1 Between 9 and 1 2 1 2, 1 5 TA8, SA4 6, 7 , 8 , I O, I I Between 6 and 8 I EHV-5 Bfa I sites Figure 9.3 numbered 1 to 1 2 With one exception, all five c lones from any EHV -5 isolate showed identical restriction patterns. The single exception was for isolates obtained from horse T A8. Isolates 1 2 and 1 3 were obtained from the same nasal swab of horse T A8, but grown on different cell l ines (Table 9. 1 ) . For isolate 1 2 all clones showed an identical restriction pattern. ----- --- --- -- - - Genomic Comparison qf EHV-5 Isolates 1 77 However, for i solate 1 3 four of five c lones showed an i dentical restriction pattern ( 1 3- A) that varied from that obtained for i solate 1 2 . Furthermore, clone 1 3-B, had an additional restriction site, resulting in the presence of two shorter bands of approximately 270 bp and 650 bp in size, i nstead of one approximately 900 bp band seen in the other four clones derived from the same isolate (clone ' 1 3-B' in Figure 9.4) . As such, the isolates from horse T A8 appeared to be a mixture of three different genotypes. By comparison, c lones ' 1 6-3 ' and ' 1 6-6' represent different cell passages of the same i solate. They had identical cleavage patterns. 9.3.3 Sequence comparison Comparison of the 500-bp sequence from the 5' end of the EHV -5 insert of eight of the clones showed a high degree of homogeneity in this region (Figure 9 .5) . The percent identity scores ranged from 97 to 99% between different EHV-5 clones and the publ ished EHV -5 sequence (Appendix C) . The scores between any of the c lones sequenced and the publ ished sequence of the reference EHV -2 strain were lower than 67-68%. Most of the time the observed nucleotide variation did not result in any changes to the predicted amino acid sequence. However, nucleotide changes that led to amino acid changes were also present (Figure 9 .6) . All clones sequenced from EHV-5 i solated from foals ( isolates 1 , 2 , 6 and 9) had an addit ional serine residue at codon 33 . For isolates 9, 1 2, 1 5 , 1 7, a C-to-G substitution at codon 27 induced an aspartic acid to a glutamic acid change, i solate 9 had an additional proline to serine substitution at codon 34, and isolate 6 had a glutamic acid to glycine change at codon 74. 9.4 DISCUSSION Equine herpesvirus-5 i s an equine y-herpesvirus c losely related to EHV-2. I n contrast to the heterogeneity reported for different EHV -2 isolates, the small number of EHV-5 isolates examined previously appeared to be genomically homogeneous (Browning & Studdert 1 989; Browning & Studdert 1 987b). In this study, RFLP comparison of the gB gene of EHV-5 showed marked fragment pattern heterogeneity. Ten c leavage profiles were identified among 1 7 i solates from nine horses and only on two occasions did i solates obtained from different horses have the same RFLP profiles. This heterogeneity is considerably greater than that observed in most other herpesviruses (Franti et al. 1 998; Chou & Dennison 199 1 ), but similar to the genomic variab il ity described for EHV-2 (Browning & Studdert 1 989; Browning & Studdert 1 987b) . Also, other workers Chapter 9 1 78 reported a simi larly high degree of nucleotide sequence variation between different isolates of other y-herpesviruses including EBV (Triantos et al. 1 998), HVS (Desrosiers -IV 4 AAA GA _ JG�A EHV- S : 2- 14 1 1 . . . . . . . . . . . . . . Clone 1 1 Clone 2 1 . Clone 6 1 . Clone 9 1 . . Clone 12 1 Clone IS Clone 17 • AAAA 'AA:;A er MM EHV- 5 : 2 - 1 4 1 61 Clone 1 61 • • Clone 2 61 - - - - . . Clone 6 61 - - - - , . _ . Clone 9 61 - - - . • • . Clone 12 61 - - - • . • • . Clone 15 61 - - - - . • . Clone 17 61 - - - • . • . . f;'HV 1 I ;T j r'T H , EHV- S : 2- 14 1 1 1 2 Clone 1 112 'AA . . , G TCC. A A1"I' C . . . . Clone 2 112 . . . G . . Tee . . c Clone 6 112 . . . G. . Tee . . C Clone • 112 . . . G . TCC'T.C . Clone 12 112 . . . . - - . A . G . . Clone 15 112 . . . . -- .1\ . G Clone 17 112 . . . , - - .A . G . \' 4 � 'AAJ. A '.AA EHV- 5 : 2- L4 1 167 Clone 1 170 Clone 2 170 Clone 6 170 Clone • 170 Clone 12 167 Clone 15 167 Clone 17 167 . . . . . . . . . . . . . FHV H, r,HV , , A', 'A' ,. A,A • AAJV> EHV- 5: 2 14 1 22' A . Clone 1 227 A Clone 2 227 A Clone 6 227 A .G Clone • 227 A. Clone 12 22. Clone 15 22. A. Clone 17 22. . . A A . . G . . . . G . . G • G . • . • • • G . . . . . G, . . . . • G • o • • G . . .G . . . . G . . A A '1\ AAA 'AT A ;.A A , . . . . . A A AA A A . C . . . G . . . . C . . ' . G . . . C . . . . G . A G . G . G G , A. ;.G}. .... 7l' A A IV' '" 'AA' ATA EHv- S : 2- 1 4 1 284 Clone 1 2.7 Clone 2 2.7 Clone 6 2.7 Clone 9 2.7 Clone 12 2 •• Clone 15 2 •• Clone 17 2 •• fll A, T !IV " 4 /l. '1\;AAA ::'TGG 'A '1\ 'A A A AA EHV- S : 2- 14 1 344 Clone 1 347 Clone 2 )47 Clone 6 347 Clone • )47 Clone 12 )44 Clone 15 ) 4 4 Clone 17 )44 E . . . . . . . . . . . A CA -l A 'A A '1\ '1\ � ATG EHV- S : 2 - 1 4 1 404 Clone 1 407 . Clone 2 Clone 6 407 . _ • . . _ . . . 407 _ . . Clone 9 407 . Clone 12 404 Clone 15 404 Clone 17 4 0 4 -" . . . ;A 1::1·" ; 1 4 A ;A ::-TA 'AA :A A '1\ ,A '1\ ;AAAA EHV- S : 2 14 1 464 Clone 1 '67 Clone 2 .67 Clone 6 '67 Clone 9 .67 Clone 12 '6' Clone IS '6' Clone 17 '6' A A A A .T . .T . . T A • Figure 9.5: Comparison of the nucleotide sequences of the EHV -5 inserts from the clones listed on the left. Published sequences of EHV -5 and EHV -2 are shown in red. Blue arrows points at the start of the amino acid alignment (Figure 9.6). Only the differences are shown. & Falk 1 982), and bovine herpesvirus-4 (Bublot et al. 1 99 1 ) . Amplification of tissue culture isolates, rather than viruses directly from clinical sample , might have influenced the results. Passaging in cel l culture has been shown to select for specific genotypes, different from the original virus ( Bonass et al. 1 994; Studdert et al. 1 986; AlIen et al. 1 983) . However, most of the EHV -5 isolates were not passaged in cell culture for more than one or two times before they were used for ampl ification of gB. Also, there was no difference in the RFLP patterns of clones ' 1 6- 3 ' and ' 1 6-6' . These clones represented different cell passages of the isolate obtained from the same cl inical sample. Additional ly, different isolates from foal A6 cultured on either RK- 1 3 or EFK cel ls had the same RFLP profiles. Together, this suggests that the RFLP patterns obtained reflected the characteristic of the infecting viru rather than genotypes selected for by cell culture. Genomic Comparison of EHV-5 Isolates 1 79 The use of Expand™ high fidelity polymerase reduced the possibi l i ty of errors being introduced during PCR. However, assuming the error rate of Expand TM polymerase as 8.5 x 1 0 -6 per nucleotide, the expected rate of errors in 30 PCR cycles would be 2.55 x 1 0 -4 per nucleotide, which corresponds to approximately 0.56 artifacts per every cloned PCR product. Cloning and sequencing also provides an opportunity for introduction of artefactual diversity among homogeneous sequences (Smith et al. 1 997). This should be kept in mind while interpreting the results. The single clone ( 1 3-B) with a different RFLP pattern in comparison with the remaining four may represent such PCR or cloning artifact. Similarly, the glutamic acid to glycine substitution observed at codon 74 for clone 6, whkh resulted from only one nucleotide change, may represent an introduced error. Nonetheless, the presence of an additional serine in clone I , 2, 6 and 9 and the substitution of aspartic acid by glutamic acid in c lones 9, 1 2 , 1 5 and 1 7 are more l ikely to be true findings. EHV 5 2 141 1 MVAWFGLWGFARLMATLALLCGRVALDESSAT PS IPPTHKPAVHHEDNTTNPFLLFRVCGASPTG EIFRFPLEENCPNTEDKEHVEGI EHV-S , 2 · 1 4 1 . . . . . . . . S . . . . . . . S . . . Clone 1 Clone 2 Clone 6 Clone 9 Clone 12 Clone 1 5 Clone 17 EHV 2 86 . . . . . . . . . S . . . . . • • . . . • . . . . . . . . G . . . . E . . . . . SS . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . E . . . 1 • • . . • • . . • • . • • . . . • . . . • . . . . . E . . 1 • • . . • • . . • • . • • • • • • • . • • • . . . . E . . . . • - • • • • • . . • . . . . . • • • • . • • • . • . • . • • • . . • • - . . • 1 GVGG PRVVLC WCVA QC QEVVAE TTPFA R E VA E PA P G EHV S " 14 1 89 LLIYKTNIVPYIFNVRKYRKLVTSTTI YKGWSQDAITNQYTSSFAMPLWEARLVOYNYECYNGIQVTEN EHV-S , 2 - l4l 89 • . . . • . . . . . . Clone 1 90 . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clone 2 90 90 . . . . . . . • . . . . • . . . 90 . . . • • . . • • . . . • . . . Clone 6 Clone 9 Clone 12 Clone 1 5 Clone 17 EHV 2 86 89 . . . . . . . . . . . . . . . . . . . • • . . • . . . . . . 89 . . . • . . . • • . . . • . . . . . 89 . . . . • . . • • . . • • . 90 A V . . IM E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H R Y V Y VQMM . HY Q FSAV N G . . . S . . . . . D I Figure 9.6: Comparison of the predicted amino acid sequences from the N terminus of gB. Published sequences of EHV -5 and EHV -2 are shown in red. The predicted sequences of clones listed on the left are shown in black. Only the differences are shown. Only one of five clones derived from a given PCR product was sequenced. Therefore, for the reasons mentioned above, no conclusions could be made regarding EHV-5 diversity based on the single nucleotide substitutions observed (Figure 9 .5) . The sequencing was performed as a part of this study to confirm the identity of the isolates as EHV -5 rather than to examine diversity between different EHV -5 isolates. The latter aim was addressed by RFLP analysis of c loned gB products. The Bfa I restriction enzyme was chosen to use in this study because it cuts at sites that are not conserved between different herpesviruses and that differ in publ ished sequences of EHV-2 and EHV-5 gB (Figure 9 .3) . Chapter 9 1 80 With the exception of clone ' 1 3-A' and 1 3-B, all five clones obtained from any EHV-S isolate had identical RFLP patterns indicating that, with the exception of the horse TA8, all horses were each infected with a single genotype of EHV -S. As only five clones from any EHV -S isolate were examined, the possibil ity of smaller numbers of other genotypes being present in a sample cannot be excluded. Horse TA8 was infected with at least two different genotypes of EHV-S (clone ' 1 2 ' and ' 1 3-A' ), also present in horses SA4 and TA9 (clones ' I S ' and ' 1 4' ), respectively. As horses T A8 and T A9 were from the same outbreak of respiratory disea e, these results suggest that lateral spread of EHV-S between these two horse may have occurred. Also, the identification of at least two different genotypes from the na al swab of the horse T A8 suggests that multiple infections with di fferent genotypes can occur. Browning & Studdert ( 1 987a) reported multiple infections of one horse with several different genotype of EHV -2. Triantos et aL. ( 1 998) found a high degree of EBV intra-host diversity in specimens col lected from pat ients infected with human immunodeficiency virus (HIV) . Others reported a higher incidence of infections with more than one strain of EBV in patients presenting with infectious mononucleosis-like ymptoms and in HIV positive individuals than in other patients (Rickinson & Kieff 1 996). By extrapolation, multiple genotypes of EHV -S may be more commonly detected in horses presenting with cl inical signs or in animals whose immune system was compromised by stress or infection with other pathogens than in healthy horses. The finding of two, or possibly three EHV-S genotypes in horse T A8 supports this hypothesis. This horse presented with enlarged lymph nodes, was concurrently infected with EHV -2, and had recently been taken to the yearl ing sales, a situation commonly regarded as stressful to horses. With the exception of horse T A8, discussed above, all EHV -S isolated from any one horse at different t imes, from both PBL and nasal swab , in either cel l l ine had identical RFLP profiles. The fact that there was no change in the cleavage pattern over t ime in foals followed on a monthly ba is suggests that the repeated isolation of EHV -S from these foals reflected the establi shment of latent or persistent infections rather than repeated infection with different viruses. ---- - -- ---- - ----- Genomic Comparison of EHV-5 Isolates 1 8 1 The heterogeneity observed between EHV -5 isolates from different horses and the apparent homogeneity of EHV -5 isolates obtained from individual horses is interesting. The conservation of patterns obtained from EHV -5 cultured from individual horses suggests that the differences observed between different horses are not s imply the result of spontaneous point mutations, but may have been driven by some unidentified selection pressure. S imil ar results were obtained during a study of the diversity of naturally occurring EBV isolates, in which no two patients examined had identical EBV banding patterns. However, the pattern of the isolates obtained from one patient at different sampling times did not change over a period of one-year (Triantos et al. 1 998). Attempts to correlate EHV -2 genomic diversity with plaque size, anatomical site of isolation or epidemiological associations have been unsuccessful (Browning & Studdert 1 987b). There is some correlation between genomic variation and cross-neutralisation data, which suggests that immune selection may be the major force behind EHV-2 heterogeneity (Browning & Studdert 1 989; Plummer et al. 1 973) . As gB was shown to be a major target for the immune response to both EHV -2 and EHV -5 i nfections (Hollow ay et al. 1 998; Agius et al. 1 994) , the observed heterogeneity of RFLP patterns of EHV -5 from different horses could reflect the effects of the diversity of the immune response between individuals. The role of immune selection on the evolution of vIruses within a host i s well documented with respect to RNA viruses, including lentiviruses and i nfluenza viruses (Burns & Desrosiers 1 994; Webster et al. 1 982) . Whether the same can occur with more genetical ly stable DNA viruses is not known. In general, genetic adaptation to the hosts' immune system resulting in the evolution of many quasispecies during infection with HIV and other RNA v iruses does not occur with DNA viruses, which are believed to have more stable genomes due to the proof reading properties of DNA polymerases. However, there are many difficulties involved in measuring virus mutation rates and the estimated frequencies of mutation for RN A viruses differ up to 100 fold depending on the methodology used (Smith et al. 1 997; Smith & Inglis 1 987) . On the other hand, the observed frequencies of neutralization-resistant variants or variants bearing a defined nucleotide substitution in some DNA viruses i ncluding herpes simplex virus, canine parvovirus or SV40 viruses, lay well within the range reported for different RNA viruses (Smith & Ingl i s 1 987) . These high frequencies of mutation for SV 40 and parvovirus are observed despite the fact that these viruses use host repl icating enzymes Chapter 9 1 82 with the highest known proof reading properties (Smith & Inglis 1987). Similarly, while human polyomaviruse are genetical ly stable in vivo, they show a very high mutation rate when cultured in vitro in human cells (Shadan & Villarreal 1 996). Taken together, the stabi l ity of DNA viruses is more l ikely to reflect the greater complexity of their interaction with the host rather than to be simply the consequence of using proof reading replication enzyme (Smith et al. 1 997). Viral antigens are recognized by the host immune system in conjunction with MHC molecules. Indirect evidence for the possibil ity that herpes viruses are able to adapt to their host over t ime was provided by an epidemiological study of EBV infection in different populations (de Campos-Lima et al. 1994; de Campos-Lima et aL. 1993) . Epstein-Barr virus isolate from Caucasian and African donors, where the MHC- l 1 allele is present, but reasonably rare, had a conserved nuclear antigen 4 epitope that was bound by MHC- I 1 and recogni ed by MHC- l l restricted CTLs. However, EBV isolates from populations in New Guinea and China, where A l l allele is unusual ly common, had a mutation in the relevant part of the genome that prevented the same antigen from being bound by A- I l glycoprotein. The structure and function of the MHC of the horse imi lar to those in other specie (Antczak 1992). Therefore, the diversity of MHC genes and resulting host- pecific CTL responses are one likely explanation for the observed heterogeneity between EHV -5 isolates from different horses in contrast to the high degree of homogeneity between isolates from the same horse. Another possibi l ity i that the existence of many different EHV -5 genotype reflects the existence of many EHV -5 viruses with sl ightly different biological properties. Based on the lack of correlation between results obtained using different tests to diagnose EBV infect ion, Johannessen et a L . ( 1 998) suggested the existence of 'a previously unrecognised degree of heterogeneity in the B cel l population in which EBV resides'. I t has also been suggested that gB sequence variation of human cytomegalovirus could correlate with changes in tis ue tropism (Chou & Dennison 199 1 ) or pathogenicity (Shepp e t aL. 1 996; Fries et aL. 1 994). Several l ines of evidence suggest that EHV -2 and EHV -5 may cause immunosuppression predisposing to secondary viral or bacterial infections (Chapter 1 , Chapter 10). It i s also possible that EHV -5 acts as a co-factor i n mixed infections Genomic Comparison ofEHV-5 Isolates 1 83 exacerbating d isease signs. Further i nvestigation i s needed i n order to assess the impact of EHV -S i nfection on the health and performance of horses. This study demonstrates that EHV-S isolates are more heterogeneous than previously thought. Thus, RFLP of the gB gene may provide a useful epidemiological approach to gain more knowledge about the biology of EHV -S. 9.5 SUMMARY Equine herpesvirus-S is a recently recognized y-herpesvirus. It is closely related to EHV -2, which constitutes a group of very heterogeneous v iruses. In contrast, the only four EHV -S i solates described to date appeared to be genetical ly homogeneous. The aim of the present study was to compare 1 7 New Zealand EHV -S isolates to each other and to the Australi an prototype strain. PCR primers were designed to ampli fy EHV -S gB gene and RFLP was used to detect d ifferences between cloned PCR products. Equine herpesvirus-S isolated from different horses showed a high degree of heterogeneity. However, EHV -S isolated from individual horses remained homogeneous when examined over a period of time or i solated from different sites. A single EHV -S gB RFLP was detected in each individual horse but one. Two, or possibly three, different genotypes of EHV -S were detected from cultures inoculated with a nasal swab of this horse. An EHV -S isolate with a gB restriction pattern identical to one of the three genotypes was isolated from another horse during an outbreak of respiratory disease, suggesting that the lateral spread of the virus may have occurred between these horses. These results correct an earlier view, based on the examination of only four i solates, that EHV -S comprises a homogeneous population of viruses. The heterogeneity observed between EHV -S isolates from different horses suggests that the use of RFLP may provide a useful epidemiological approach to gain more knowledge about the biology of EHV-S. CHAPTER 10: REPRESENTATIONAL DIFFERENCE ANALYSIS OF EHV-2 INFECTED EQUINE LEUCOCYTES 1 0. 1 INTRODUCTION Herpesviruses are well adapted to their hosts. They rarely cause serious diseases In adult, immunocompetent individuals, and can establish latent or persistent infection that may last for a l ifetime. The importance of herpes viral infection may be difficult to assess based on observation of disease signs in experimental ly infected animals, as in a field s ituation, disease may be caused by increased susceptibility to secondary infections, rather than be a direct con equence of primary herpesviral infection. In contrast, interactions that occur on a molecular level between a virus and the infected cell may provide more rel iable information about the relationships between a microorganism and its host. Several epidemi ological studies have indicated that EHV -2 may be involved in equine re piratory di ease and poor performance (Borcher et al. 1 997 a; Murray et al. 1 996; Schlocker et al. 1 995 ; Fu et al. 1 986). In this chapter, an attempt to elucidate the influence of EHV -2 infection on the immune status of infected horses is presented. Equine her pesvirus-2 has been shown to be latent in B Iymphocytes (Drummer et al. 1 996) . The ability to establish infection in cells central ly involved in the immune response clearly has potential to modulate these responses. Addit ionally, the examination of the genome of EHV -2 revealed that it codes for several proteins that have potential to influence host immune responses. These proteins include an interleukin- I O like protein and two, or possibly three, proteins that have a structure characteristic of GPCR (Chapter I ) (Telford et al. 1 995) . In another study, EHV-2 vaccination prevented the occurence of Rhodococcus equi pneumonia in foals, which further indicated that EHV -2 might interact with the host immune defences and influence susceptibility of infected foals to secondary infections. Chapter 10 1 86 The research of this thesis concentrated on interactions between EHV -2 and infected equine PBL. The approach taken was to look for differences, at a molecular level , between equine PBL infected with EHV -2 and those adsorbed with inactivated EHV -2. The novel technology, representational difference analysis (RDA) (Shi e t al. 1 997) was used to detect genes that were differentiall y expressed i n these two populations of cells . 10. 1 .1 Approaches to investigate differentially expressed genes In the past, identification of differentially expressed genes rel ied mostly on subtractive hybridisation . However, this technique was unreliab le, difficult to perform, and uti l ised large amounts of RNA (Mil ler et al. 1 999; Wieland et al. 1 990) . Subsequently, other methods have been developed, some of which have been recently reviewed by Carul l i e t a l . ( 1 998). The main concepts behind the two most commonly used techniques, differential display and representational difference analysis, as well as the newest and most advanced DNA chip technology, are outlined below. Differential display (DD) The principles behind the differential display technique have been described (Liang e t al. 1 993 ; Liang & Pardee 1 992) . Briefly, short fragments of the mRNA from two samples of i nterest are ampl ified by RT -PCR. Several pairs of primers are used. In every pair, one primer is an anchored oligo-dT primer consisting of 1 1 or 1 2 Ts with two additional 3' bases that provide speci ficity, and the second one is an arbitrary 1 0- mer that randomly anneals to the RNA template. The PCR reactions are performed in the presence of radiolabeled dA TPs. As a result, a number of fragments varying in size are generated. These fragments are separated on a sequencing gel and the patterns produced by every primer pair compared. The bands that differ between the two samples of i nterest are recovered from the sequencing gel , re-ampl ified with the same primer pair that was originally used for generation of the fragments, cloned and analysed (Liang e t al. 1 993 ; Liang & Pardee 1 992) . The advantage of the DD system i s its abi l i ty to detect both up- and down-regulated sequences i n a s ingle experiment . The main d isadvantage is a large number of false positives, sometimes in the range of 85 - 90% (Miller et al. 1 999; Shi et al. 1 997) . -----�----------- -- - ---- Representational Difference Allalysis ()f EHV-2 Illfected Equille Leucocytes 1 87 Representational difference analysis Representational difference analysis was original ly developed as a tool for comparison of complex DNA genomes (Lis i tsyn 1 995 ; Lisitsyn et al. 1 993) . The technique i nvolves generation of representat ive samples of the two genomes of interest by digestion with a restriction enzyme, l igation to nucleotide adaptors, and PCR ampli fication using the same adaptor as a primer. This results i n generation of short PCR fragments that are representative of the whole genome, but have reduced sequence complexity in comparison with genomic DNA. Enrichment for sequences specific for the sample in question (tester) is achieved by several rounds of sub tractive hybridisation and selective enrichment for specific sequences by PCR ampl ification. Before each round of hybridisation, all tester fragments are digested with the appropriate restriction enzyme and annealed to a new oligonucleotide adapter. Hybrid isation is performed with a large excess of driver DNA, where driver comprises a sample that is identical to the tester, except for the differences imposed by the factor that is investigated. Most of the tester sequences that are common to both populations would anneal to complementary driver sequences. However, the sequences present only in the tester population would self­ anneal, resulting in double stranded DNA fragments with the oligonucleotide adapter on both ends. Only these fragments will be exponential ly amplified in PCR with the same oligonucleotide used as a primer, leading to selective enrichment for specific sequences. After three rounds of subtraction and PCR ampl ification, the tester popul ation i s enriched more than 1 07 -fold for speci fic sequences not present in the driver (Lis itsyn et al. 1 995) . The technique has been successful ly used to generate probes for detection of DNA alterations in cancer cell s (Lisitsyn 1 995) , to d iscover new pathogens, such as Kaposi ' s sarcoma associated herpesvirus (Chang et al. 1 994) , or to detect genetic polymorphism between closely related organisms (Calia et al. 1 998). Subsequently, the system was adapted for use with cDNA for identifying differences in gene expression between two populations of cell s (Hubank & Schatz 1 994) . The main difference between cDNA RDA and DD is the fact that, while DD amplifies randomly selected fragments from the entire mRNA population, cDNA RDA amplifies only the sequences that are different between the two populations. The cDNA RDA technique has been successful ly applied by several research groups to identify both known and novel genes that are transcriptionally regulated by a variety of conditions (Familari & Giraud 1 998; Lucas et al. 1 998; Kibel et a1. 1 998; Chu & Paul 1 998; Feldman et a1. 1 998; Morris et al. 1 998; Chu & Paul 1 997; Dron & Manuelidis 1 996). The technique, its advantages Chapter 10 1 88 and l imitations were reviewed by Frazer et al. ( 1 997). Recently, other conceptual ly similar techniques were described (Diatchenko et al. 1 999; Diatchenko et al. 1 999; Yoshida et al. 1 999), and a commerci al system uti l is ing some aspects of cDNA RDA became available (PCR-select cDNA subtraction kit, Clontech). DNA chip technology DNA chip technology is the newest and most promlSlng technique that combines traditional molecul ar b iology with physics and computing. The result is a novel technology offering speed, sensitivity and extremely high throughput of analysed sequences. The expression of many genes can be monitored in parallel by hybridisation of the entire population of fluorescently labelled mRNA or cDNA to high-density arrays of oligonucleotide probes (de Saizieu et al. 1 998; Lockhart et al. 1 996). The oligonucleotide probes are designed based on the DNA sequence of the organism in question. They are mounted on a s i licon surface i n a precise location using l ight directed combinatorial chemistry (Varga et al. 1 997). The process involves attaching synthetic l inkers modified with photochemical ly removable protecting groups to the glass surface. The oligonucleotide probes are then built on the l inkers. This involves incubation of the chip with one of the A, C, T, or G deoxynucleosides coupled to the l ight-sensitive protective groups. A given base is added to the growing probe one at a t ime, and only at specific sites. These sites are selected with high precis ion by producing localised photodeprotection by directing l ight to speci fic areas using photolithographic masks, in the same way as they are used i n semiconductor factories. The process i s repeated with a series of masks until the chip is covered with oligonucleotide arrays, each usuall y 20 bases in l ength. At present, a 1 .6-cm3 chip can contain approximately 400,000 probes (Varga et al. 1 997). When a chip is flooded with labelled mRNA from cel l s of interest, the mRNA hybridises to complementary sequences on the chip. The position and intensity of fluorescence can then be measured using laser con focal fluorescence scanning. Because the preci se position of each probe on the chip is known, the i dentity of differentially expressed genes and their rel ative abundance can be deduced directly from the fluorescence patterns and i ntensities, w ithout the need for additional cloning and sequencing (de Saizieu et al. 1 998; Lockhart et al. 1 996). The disadvantage of the chip technology is the requirement to know the DNA sequence of the organism of i nterest in order to design suitable oligonucleotide probes. This does not seem to be a major hurdle, as the sequences of many organi sms are now avail able. -� --- ------ ----- � --- Representational D!fference Analysis of EHV-2 Infected Equine Leucocytes 1 0.2 MATERIALS AND METHODS 10.2.1 Equine herpesvirus-2 1 89 The EHV -2 used was isolated from the PBL sample of a foal showing respiratory disease (foal F4 in table 2 . 1 ). The v irus was i solated on EFK cells . Passage seven i n EFK cells was used in the present experiment. The v irus was grown i n one 595-cm3 flask of EFK cell s unti l all cell s showed CPE. The supernatant was c larified by centrifugation at 700 g for 1 0 minutes, collected, and the virus was pelleted by u ltracentrifugation at 1 00,000 g for 90 minutes through a 25% sucrose cushion. Pel lets were resuspended in 500 f! l of PBS overnight at 4 QC, pooled, aliquoted and frozen at - 70 QC. The titre of this preparation was determined by titration in a 96-well microtitre plate on EFK cells . Virus inactivation Half of the virus preparation was inac tivated by i ncubat ing at 56 QC for 30 minutes, fol lowed by UV treatment for 1 5 minutes. Two I -week passages i n EFK cel l s were performed to confirm that inactivation was successful . Leucocyte cultures Twelve tubes of blood on heparin were collected from a healthy, adul t Thoroughbred horse "Travolta". The horse was negative for EHV-2 on several occasions prior to the experiment, as checked both by PCR and co-cultivation of PBL with EFK cells . Additional l y, the SN t itre of the serum sample collected at the time of blood collection for use in RDA, against the EHV -2 isolate used in the study, was less than 4. Leucocytes were separated as described in Chapter 2, adsorbed with either 1 0s TCIDso of EHV-2 (+ sample) or the equivalent volume of inactivated EHV-2 (- sample) for 90 minutes at 37 QC, washed once i n PBS , and seeded i nto two small flasks at a concentration of 1 .3 x 1 07 cell s/ml in 10 ml of RPMI (Gibco) medium supplemented with 10% autologous serum, 1 % PSK, 1% glutamax (Gibco) and 1 5 mM HEPES buffer, pH 7 .0. The cell s were i ncubated for 4 days in humidified, 5% CO2 atmosphere at 37 QC. The media were half-changed on the third day of i ncubation. Chapter 10 1 90 Mononuclear cells (MC) from 4-day-old cultures were separated on lymphoprep (Pharmacia) according to the manufacturer's instructions. Cell suspensions from each flask were topped up with RPM I medium to a final volume of 1 2 ml and layered over lymphoprep (3 ml of lymphoprep + 6 ml of cell suspension in a 1 5 ml centrifuge tube) . The tubes were centrifuged for 30 minutes at 2000 g. Lymphocytes were collected from the interface, washed once i n PBS and resuspended i n I ml of PBS. An aliquot of 2 10 III of this preparation was saved for inoculation of EFK cells, and RNA was extracted from the remaining volume. EFK inoculation Equine foetal kidney cells , grown in a 24-well plate, were infected with 1 00 III (approximately 2 x 1 06 cell s ) of either fresh or freeze-thawed purified MC from either the (+) or (-) sample. Two I -week passages were performed, and cell s were observed dail y for the presence of herpes viral CPE. 10.2.2 RNA extraction RNA was extracted using Trizol LS reagent (Gibco) according to the manufacturer' s instructions. The RNA pel let was dissolved in 1 05 III o f DEPC-treated H20. An aliquot (5 J.lI) was used to spectrophotometrical ly estimate the amount of extracted RNA. The remaining 1 00 J.lI was used for mRNA i solation using oligotex technology (mRNA i solation kit, QIAGEN). B riefly, RNA dissolved in 1 00 J.1l DEPC-treated water was mixed with 1 00 J.ll of 2x binding buffer and 6 III oligotex suspension. The mixture was transferred to a spin column, washed with 400 III of wash buffer, and finally RNA was eluted with 1 2 J.lI elution buffer ( 10 mM Tris-HCl, pH 8 .5) . 10.2.3 cDNA synthesis First and second strand cDNA was synthesised on the RNA template according to the manufacturer' s i nstructions (SuperScript Choice system for cDNA synthesis, G ibco BRL). The reactions were cleaned up using a PCR purification kit (QIAGEN) and cDNA was eluted i n 90 J.1l of 1 0 mM Tris-HCI, pH 8 .5 . -------------------- - - -- Representational Difference Analysis of EHV-2 Infected Equine Leucocytes ",RNA - -- tester tester-tester hybrid tester-driver heterohybrid ss-tester exponential amplification linear amplification Dirrerence I'roduct (DI') ds cDNA --- --- driver digest with Dpn 1 1 ligate RI2124 adapters PCR with R24 primer to generate amplicons driver-driver hybrid ss-driver no amplification depleted digest with Dpn 1 1 ligate J 12124 adapters to tester only PCR with J24 primer digest with Mung Bean Nudease PCR with J24 primer 1 9 1 Figure 10. 1 : Schematic diagram of cDNA RDA (adapted from Frazer et al. 1 997). Preparation of only one sample is depicted. Usually RDA is performed in both directions, so a mRNA sample is used both as a tester and a driver. For simplicity, only one direction is shown in this diagram. Tester sequences are depicted as green lines, driver sequences as blue lines, R 12/24 adapters as red lines, J 1 2/24 adapters as grey lines. Fill-in ends create the primer binding sites, so that the DNA fragments can be amplified with the appropriate primer. 10.2.4 Preparation of driver and tester Representational difference analysis was performed as described (O'Neill & Sinclair 1 997; Frazer et al. 1 997; Hubank & Schatz 1 994) _ The detailed protocols were obtained from these authors_ The RDA principle is depicted in Figure 1 0_ 1 . Chapter 10 1 92 The fol lowing 1 2-mer and 24-mer oligonucleotides were obtained (desalted) from a commercial source (Gibco BRL) : R12 5 '-GATCTGCGGTGA-3 , JI2 5'-GATCTGTTCATG-3' NI2 5'-GATCTTCCCTCG-3' R24 5' -AGCACTCTCCAGCCTCTCACCGCA-3' J24 5'-ACCGACGTCGACTATCCATGAACA-3' N24 5' -AGGCAACTGTGCTA TCCGAGGGAA-3' According to RDA terminology, a sample containing unique sequences that are the focus of interest is referred to as ' tester' , whereas 'driver' constitutes a sample containing sequences common to both tester and driver. During the RDA procedure, driver is used to subtract common sequences from the tester on one hand, and sequences unique to the tester are progressively ampl ified on the other hand). Thus, after three rounds of subtraction and amplification the tester sample should be greatly enriched for unique sequences. The representative populations of tester and driver are called 'amplicons ' . In the present study, RDA was performed in both directions, so that each sample was used both as a driver and as a tester. Peripheral blood leucocyte samples that had been adsorbed with EHV -2 were regarded as (+) samples, whereas PBL that had been adsorbed with i nactivated EHV -2 were regarded as (-) samples. S imil arly, amplicons generated by subtraction of (+) sample were referred to as (+) amplicons, whereas amplicons generated from subtraction of (-) sample were referred to as (-) amplicons. Dpn 11 digest of cDNA: cDNA was digested with 2111 Dpn 11 ( 10 u/Ill ) (New England B iolabs) in a supplied buffer in a total volume of 20 III at 37 QC for 2 hours. The digest was washed three times with 500 �l dH20 on a microcon 30 (Amicon) . The final spin concentrated the cDNA to a 1 6 �l volume for l igation. Ligation to R adaptors R24 ( 1 mg/ml) and R 1 2 ( 1 mg/ml) oligos were annealed to each other in a total volume of 12 �l in a PCR machine. The mixture was heated to 70 QC for 2 minutes, and then cooled to 10 QC at 1 QC/minute. The annealed oligos (2 Ill) were added to a l igation mixture consisting of 1 6 !-Ll of Dpn 11 digested cDNA, 2 III of 1 0 x l igation buffer and 1 �j of T4 DNA l igase (5 u/Ill ) (New England B iolabs), and i ncubated at 1 6 QC overnight. Representational Difference Analysis ofEHV-2 b(fected Equine Leucocytes 1 93 The ligation reactions were washed three times with 500 III of H20 on a microcon 30 and brought up to a final volume of 50 Ill . Primary and secondary peR to generate tester and driver representations (R amplicons) Four primary PCR reactions were performed for each sample. Each reaction consisted of 3 III of R l igated cDNA, 10 Jll l Ox Expand buffer with 1 5 mM Mg2+ (Roche), 2 Jll of 1 0 mM dNTP mixture (Roche) , 0.8 Jll of R24 primer ( 1 mg/ml ) , and 0.7 Jll of the Expand polymerase mix (3 .5 u/Jll) (Roche) in a final volume of 1 00 Jll. Following initial incubation at 72 QC for 5 minutes (to melt away 1 2mer and fill in ends), 20 cycles of denaturation at 94 QC for 1 minute and annealing/extension at 72 QC for 3 minutes, with the final extension at 72 QC for 1 0 minutes, were performed. Secondary PCR was performed as for primary PCR, but 5 Jll of primary PCR product was used as target DNA. Forty-eight secondary PCR reactions were set up for each sample. The PCR products were combined (eight per one Eppendorf tube) , extracted once with phenol/chloroform, once with chloroforrnlisoamyl alcohol (IAA), and isopropanol precipitated with 1 / 1 0 volume of 3M sodium acetate, pH 5 .2 . The DNA pellet was washed once with 70% ethanol, dried, and resuspended in 50 � of TE buffer pH 8.5 . Amplicons from the same RNA sample were pooled and DNA concentration determined spectrophotometrically . Removal of R adaptors from amplicons Approximately 1 10 Ilg of each amplicon was digested with 25 Jll Dpn II in a supplied buffer in a total volume of 800 III at 37 QC for 2 hours. The digests were extracted once with phenol/chloroform, once with chloroforrnlIAA and isopropanol precipitated with sodium acetate, pH 5 .2 . Pel lets were washed once with 70% ethanol , dried and resuspended in 102 III TE buffer pH 8.5 . An aliquot (2 Jll) was used to check the concentrations spectrophotometrically. The remaining 1 00 Jll constituted the driver. Concentrations of both (+) and (-) amplicons were adjusted to 6 10 Ilg/ml . Each tester sample was prepared from an aliquot (5 Jll ) of each driver. The tester samples were purified away from the cut adaptors by three washes with 500 Jll dH20 on Chapter 10 1 94 a microcon 30, and resuspended in 50 � dH20. The concentration of each tester was determined spectrophotometric all y. 10.2.5 First round of amplification - generation of the first difference product (DPl) Ligation of testers to J adaptors Oligos J 1 2 and J24 were annealed to each other as described for R oligos. An aliquot ( 3 Ill) was used for l igation to 500 ng tester cDNA using 1 � T4 DNA l igase in a 20 � final volume in a supplied buffer. The l igations were performed overnight at 1 4 QC. Subtractive hybridisation Tester cDNA (250 ng) was combined with driver cDNA (25 Ilg) , purified by phenol/chloroform and chloroformlIAA extractions, and ethanol precipitated with ammonium acetate at -80 QC for 10 minutes, fol lowed by 2 minutes at 37 QC to minimise salt precipitation. The pellet was washed twice with 70% ethanol , dried, and resuspended in 4 III 3 x EE buffer (Appendix E) by pipetting up and down for three minutes, i ncubating at 37 Q C for 5 minutes and vortexing, after which the DNA was centrifuged briefly and transferred to a thin-walled PCR tube. The mixture was overlaid with 35 III paraffin oil, heated to 98 QC for 5 minutes, cooled to 67 QC and immediately 1 III 5 M NaCl was added. The hybridisation was allowed to proceed at 67 QC for 20 hours. Primary peR After hybridisation, oil was removed from the tube, 8 !-LI of TE with 5 mg/ml yeast RNA was added, the contents mixed, and additional 67 III of TE was added. Finally, 320 III of dH20 was added for a final volume of 400 !-L l . Eight primary PCR reactions were performed. Each reaction consisted of 20 !-L I DNA from hybridisation reaction, 2 !-L l dNTP ( 10 mM), 10 !-L l Expand l Ox buffer, and 0 .7 !-L l Expand enzyme mix in a total volume of 99 .2 !-L l . The reactions were heated to 72 0 C for five minutes to fil l i n ends. Then 0.8 !-L l of J 24mer ( l mg/ml) was added and 1 0 cycles of 94 QC ( l minute) and 70 QC (3 minutes) were performed, followed by final extension -- -- -------- Representational Difference Analysis of EHV-2 Infected Equine Leucocytes 1 95 at 72 QC for 10 minutes. The reactions were combined, phenol/chloroform extracted, fol lowed by chloroform/IAA extraction and ethanol precipitation with sodium acetate (pH 5 .2) and 2 III glycogen carrier (20mg/ml) . The pellet was washed once with 70% ethanol , dried and resuspended in 40 �l H20. Mung bean nuclease digest An aliquot (20 � l ) of primary PCR product was digested with I � l ( 1 0 u/�l ) of mung bean nuclease (New England Biolabs) in a supplied buffer in a total volume of 40 � l , at 37 QC for 30 minutes. The nuclease was inactivated by boil ing for 5 minutes after addition of 1 60 Jll 50 mM Tris (pH 8.9) to the reaction mixture. Secondary PCR Eight secondary PCR reactions were performed. Each reaction consisted of 20 � l of DNA from the Mung Bean Nuclease reaction, 2 �l dNTP ( lOmM) , 10 � l lOx Expand buffer, 0.8 �l J24 ( 1 mg/ml) in a 95-�1 final volume. The reactions were heated to 94 0 C for 1 minute, cooled to 80 0 C, and 2 .5 units of Expand enzyme mix was added i n a 5 -�1 volume to each tube. Eighteen cycles of denaturation at 94 QC ( 1 minute) and annealing/extension at 70 QC (3 minutes), fol lowed by 10 minutes of final extension at 72 QC, were performed. The reactions were combined, phenol/chloroform and chloroform/IAA extracted, fol lowed by i sopropanol precipitation with sodium acetate (pH 5.2) . The pellet was washed once with 70% ethanol, dried and resuspended i n 50 Jll TE buffer (pH 8 .5) . The concentration of the product was determined spectrophotometrically. This preparation represented Difference Product One (DP 1 ). 10.2.6 Second round of amplification - generation of the second difference product (DP2) Changing adaptors Ten �g of DP 1 was digested with 2 .5 � l Dpn 11 in a supplied buffer in a total volume of 1 00 �J . The digest was washed on a microcon 30 three times in 500 � l dH20. After the final spin, digested DNA was brought to a final volume of 50 � l with dH20. The concentration of this preparation was determined spectrophotometrically . Chapter 10 1 96 Ligations to N adaptors were performed at 1 6 °C overnight. Al l other manipulations were performed as described for 1 adaptors. Subtractive hybridisation The l igation reaction was di luted to 1 .25 nghd and 3 1 .25 ng of N-ligated tester was mixed with 25 Ilg of driver (hybridisation ratio 1 : 800). All fol lowing hybridisation and subtraction steps were performed as described in section 1 0.2 .5 except that the annealing/extension step during primary and secondary PCR reactions were performed at 72 °C instead of 70 qc. 10.2.7 Third round of amplification - generation of the third difference product (DP3) The third round of subtraction/amplification was performed as described for the second round with the fol lowing changes: 1 . For l igations, 1 oligos were used and l igations were performed at 1 4 °C. 2. l- ligated DP2 was di luted to 10 pg/J.il and 62.5 pg l-ligated DP2 was mixed with 25 Ilg driver ( 1 :400,000 hybridization ratio) . 3 . 70 ° C anneal ing temperature was used for primary and secondary PCR with 124 as the primer. Final amplification was performed for 22 cycles. 10.2.8 Cloning of the DP3 amplicons Third difference products, DP3( +) and DP3( -) , were cloned into pBluescript KS+ plasmid vector. The insert DNA was blunt-ended by treatment with Klenow enzyme and T4 PNK. For the Klenow reaction, 1 III of either DP3 (+) or (-) DNA, 6 J.i l of dH20 and 3 II I of Klenow master mix (3 II I l Ox buffer, 0.3 J.il 10 mM dNTP, 3 J.il 1 mg/ml BSA, 0.75 II I Klenow fragment, 1 .95 J.i l H20), were mixed and i ncubated at RT for 1 5 minutes. The Klenow enzyme was inactivated by incubation at 75 °C for 1 0 minutes. Then, 1 III of PNK master mix (0.3 II I l Ox PNK buffer, 0.3 �t1 1 00 mM A TP, 0.8 II I T4 PNK, 1 .6 II I H20) was added to each tube, and PNK reactions were allowed to proceed at 37 °C for 60 minutes, followed by enzyme inactivation at 65 °C for 20 minutes. For l igation, 1 � of this preparation was mixed with 0.4 � of vector DNA ( 1 50 ng/�) . Ligation reactions were performed for 30 minutes at RT, using a rapid DNA l igation kit --------------�- - Representational Difference Analysis of EHV-2 Infected Equine Leucocytes 1 97 (Roche) according to the manufacturer' s i nstructions, i n a total reaction volume of 1 0 Il l . An aliquot (2.5 � ) of each l igation reaction was used for transformation of competent E. coli cells according to standard l aboratory procedures (Sambrook et al. 1 989). Transformed colonies were grown on LB/ampicil l in plates at 37 QC overnight. 10.2.9 Dot blots For colony dot blots, 96 white colonies from either DP3( +) or DP3( -) plates were p icked and grown for 4 hours at 37 QC i n a 96-well microtitre plate in 1 00 III TB with 1 00 Ill/ml ampici l l in . An aliquot (2 Il l ) from each well was transferred onto each of four nylon membranes. Each membrane was then placed, DNA side up, onto each of the three stacks of filter paper (in the described order) saturated with: 1 . denaturation solution (O.5M NaOH, 1 .5M NaCI, 0. 1 % SDS) for 5 minutes, 2. neutrali sation solution (0.5M Tris , 1 .5M NaCl, pH 7 .5 ) for 5 minutes 3 . 2 x SSC for 1 0 min Dried membranes were placed on a UV transi lluminator for 4 minutes in order to fIx DNA. Finall y, each membrane was incubated at 37 QC for 1 hour with 1 ml (2 mg/ml) protei nase K solution (Roche) distributed evenly onto the surface, incubated, blotted between two sheets of filter paper saturated with distil led water, dried and stored at 4 QC until use. For dot blots of difference products, approximately 500 ng of either (+) or (-) amplicon (R representations, DP I , DP2, and DP3) were spotted on four identical membranes. 10.2.10 Preparation of probes Probes were prepared using a DIG PCR probe synthesis kit (Roche) using a 1 :4 ratio of labelled to unlabel led dUTP. PCR reactions to generate DP3( +) and DP3( -) probes were performed identically to the reactions used to generate R representations ( 1 0.2 .4), with the difference that 124 primers were used with an annealing temperature of 70 QC, and 30 cycles PCR were performed. One III of 1 : 200 dilution of either DP3( +) or DP3( -) DNA was used as target DNA in a 1 00-� reaction. For preparation of R amplicon probes, 5 � of primary PCR product was used in a I OO-� reaction, and 30 PCR cycles were performed. Chapter 10 1 98 Individual probes were prepared from colonies DP3(+)A 1 2, DP3(+)C9, DP3(-)E 1 2 and DP3( -)F7 . Following in i ti al denaturation at 95 °C for 2 minutes, 10 cycles of denaturation at 95 °C ( 10 seconds) and annealing/elongation at 70 °C (2 .5 minutes) were performed, followed by 20 cycles with an additional 20 seconds of elongation added to each successive cycle, and a final elongation step at 72 °C (7 minutes) . Approximately I ng of template plasmid was used as target DNA. After label l ing, PCR probes were digested with 4 � of Dpn II in a 200-� final reaction volume at 37 °C for 3 hours, washed three times on a mirocon WO, and resuspended i n 60 f..Ll o f dH20. For hybridisations, 6 � per 1 m l o f hybridisation solution was used. 10.2.1 1 Hybridisation Colony hybridisation with DP3( +), DP3 (-) , R( +) and R( -) probes were carried out overnight at 42 °C. Five ml of hybridisation solution was used per bag. Two membranes, DP3( +) and DP3( -) were hybridised together in each bag. Dot blots of ampl icons were hybridised with the individual probes prepared from colonies DP3(+)A 1 2, DP3(+)C9, DP3(-)E 1 2 and DP3(-)F7. Hybridisation was performed overnight at 42 °C for both DP3( -) probes, or at 50 °C for both DP3( +) probes. 10.2. 1 2 Selection of clones for further analysis Dot blots of c loned DP3 products were probed against homologous and heterologous probes prepared from R and DP3 amplicons. The clones were chosen for further analysis if they reacted with the homologous, but not with the heterologous, DP3 probe. The blots probed against R amplicon probes provided an addi tional ' selection ' l eve1. Clones reacting with both (+) and (-) R amplicon probes were excluded from further analysis . However, clones that reacted with neither of the R amplicon probes were not excluded from further analysis, if they showed the expected binding pattern against DP3 ampl icon probes. -------- ----- --- - Representational Difference Analysis of EHV-2 Infected Equine Leucocytes 10.2.13 Sequencing and molecular analysis 1 99 Selected colonies were grown overnight i n 4 ml of TB medium supplemented with 1 00 Ilg/ml ampici l l in . Plasmid isolation was performed using a plasmid i solation kit (Tri­ pure plasmid i solation kit, Roche) accordi ng to the manufacturer' s instructions. Sequencing reactions were performed according to the manufacturer' s i nstructions (Dye termination kit, Perkin Elmer) using approximately 500 ng of plasmid DNA as a template, and the products were separated on an automated ABI prism sequencer (Perkin Elmer). The computer search for sequence homology i n GenBank was done using the BLAST program. 1 0.3 RESULTS The titre of the EHV -2 preparation was calculated to be 1 055 TCID50/ml. Inactivation of EHV -2 was successfu l , as no CPE was observed in EFK cells adsorbed with inactivated EHV -2 after two I -week passages. Leucocyte cultures were successful ly infected with EHV -2, as determined by re­ isolation of the virus by co-cultivation with EFK cells . Cytopathic effect was observed on the fourth day after i noculation of the EFK cells, only i n well s inoculated with fresh MC from (+) samples. The CPE was extensive, with most cell s showing a round, refractile appearance by the seventh day of i ncubation. However, on the second passage, several plaques were also present in wells inoculated with freeze-thawed MC from (+) samples. No CPE was observed in cell s i noculated with either fresh, or freeze-thawed MC from (-) samples, further confirming the success of EHV -2 inactivation. Approximately 2 .25 x 1 07 and 2.5 x 107 MC cell s were obtained after separation on lymphoprep from the (+) and (-) samples, respectively. Thus, approximately 2 x 107 cel l s were used for RNA isolation. Approximately 5 ng of total RNA was i solated from each of the purified MC samples. S ince these amounts were not enough to perform RDA as original ly described by Hubank & Schatz ( 1994), the protocol was adapted as described by Frazer et al. ( 1 997). Instead of setting up many PCR reactions to generate R amplicons, only four primary reactions were set up. The amplification products of these primary reactions were re- Chapter 10 200 amplified using the same cycling conditions, as for primary PCR ( 1 0.2 .4) . This allowed generation of approximately 220 �g of each R representation, which was sufficient for RDA to continue. Subsequent hybridisation reactions were performed with 25 �g of driver and an appropriate amount of tester DNA, depending on the desired driver:tester ratio. Approximately 0.5- 1 �g of each amplicon was run on a gel (Figure 1 0.2) . A B C D Figure 10.2: Approximately 0.5 - 1 Ilg of R representations (A), DP1 (B), DP2 (C), and DP3 (D) run on 1 % agarose gels and stained with gel star nucleic acid stain (FM C). Lanes: M - 50 bp molecular ladder (Gibco), 1 - (-) amplicons, 2 - (+) amplicons. All ONA preparations throughout the experiment were of high quality, with the A26()i A280 absorbance ratios greater than 1 .S . The fol lowing amounts of OP amplicons were generated: 3 1.4 �g DP l (+), 33 .2 �g DP l (-) , 43.4 �g OP2(+), 40.6 �g DP2(-), 1 4.5 �g DP3(+), and 1 2.7 �g DP3(-) . Cloning of the entire DP3 DNA resulted in generation of approximately 1 20 colonies on each plate (either DP3(+) colonies or DP3(-) colonies) . There were no blue colonies observed. Patterns obtained after hybridisation with either homologous or heterologous DP3 probe are shown in Figure 1 0.3 . Clones DP3( -) : A2, B3 , B4, B5 , C4, C7, E5, E 1 2, F7, F l l , F 1 2, G l O and OP3(+) : A 1 2, B6 , CS, C9, OS, 09, E9, H I , H3, H5, HS were selected for further analysis, as they reacted with homologous DP3 probes, but not with - .. _.--------- Representational Difference Analysis (if EHV-2 Infected Equine Leucocytes 20 1 the heterologous DP3 probes (Figure 10 .3) , None of the selected clones reacted with either homologous or heterologous R amplicon probes . Cl) .c 2 c.. ... (;) Il. C A B C 0 E F G H A B .8 c o � 0 ..-. -±.. E C') Il. C F G H DP3(-) colony blot 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 A B Cl) C .c 0 ... 0 c.. ,...... * E c.. 0 F G H A B Cl) C .c 2 0 c.. +' c;; E c.. 0 F G H DP3(+) colony blot 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 • Figure 10.3: Colony dot blots of DP3(-) or DP3(+) products, probed with either DP3(+) or DP3(-) probes, as indicated. Colonies that reacted with homologous probes, but not with heterologous probes were regarded as 'interesting' and re-amplified for further analysis. All 23 clones were re-amplified using J24 primers (Figure 1 004). Of these, c lones DP3(-) : B3, C4, C7, E5, E 1 2, F7, F 1 2, G l O and DP3(+) : A 1 2, B6, C5, C9, D8, D9, E9, H3, H5, H8 were sequenced. The GenBank sequences with significant homology to the sequenced clones are l isted in Table 10. 1 (see also Appendix D). As expected, clones from DP3(+) sample contained EHV-2 sequences. Seven of eight DP3(-) clones sequenced contained a sequence homologous to equine monocyte chemoattractant protein- l (MCP- I ), and for one clone no homologous sequences were identified. Thus, these results suggested that EHV-2 infection possibly inhibits mRNA expression of MCP- t . Chapter 10 DP3- bp 800- 350- 50- 800- 350- DP3+ 202 C\J M ,- CD lO Ol CO Ol Ol ,- C') lO CO « Q) () () O O W I I I I Figure 10.4: Re-amplification products of PCR using selected DP3(- ) amplicons (top) and DP3(+) amplicons (bottom) as target DNA. PCR reactions were performed as described in section 1 0.2.5. Table 10. 1 : GenBank sequences with significant homology to the listed clones. Clone GenBank sequence with significant Blast score homology (E value) Equus cabal/us mRNA for monocyte 704 DP3(- ) : B3, C4, C7, ES, E 1 2, F 1 2, G 1 0 chemoattractant protei n- I ( MCP- I ) (0.0) DP3(-)F7 No blast re u l t I EHV-2 749 DP3(+) : C9, H3 ( 1 34322 bp - 1 34703 bp) (0.0) EHV -2 ORF36, 850 DP3( +) : A 1 2, B6, CS, 08, D9, E9, HS, H8 (virion protei n k i na e) (0.0) I No equences produced al igment with a score higher than 1 00 Representat ive clones (DP3(+)A 1 2, DP3 (+)C9, DP3(-)E 1 2, DP(-)F7) were PCR label led and used for probing against dot blots of DNA from R, DP I , DP2, and DP3 amplicons (Figure 1 0.5 ) . As expected, DP3( +) probe reacted more strongly with (+) amplicons, wherea DP( - ) probe reacted preferentially with (-) amplicons. -- - - -�- �-- � �� ---�-- ----- -- Representational Difference Analysis of EHV-2 Infected Equine Leucocytes ( + ) s am pl e s ( -) samples (+) samples ( -) samples D P3(+)A1 2 probe OP R 1 2 3 D P3(+)C9 probe OP R 1 2 3 D P3(-) E 1 2 p robe OP R 1 2 3 D P3(-) F7 probe OP R 1 2 3 _ . l" igure 10.5: Approximately 500 ng of R representations, DP1, DP2, and DP3 products, obtained after subtraction of either (+) or (-) samples, probed with DIG-labelled probes, as indicated. 1 0.4 DISCUSSION 203 Herpesviruses are among the many pathogens that have evolved variable and sophisticated ways of interacting with the immune response of their hosts (Brodsky 1 999; Banks & Rouse 1 992). Equine herpesvirus-2 is probably not an exception (section 1 .3 . 1 ) . Several strategies used by herpesviruses to escape host immune defences include latency, infection of privileged sites, epitope mutation, inhibition of antigen processing and presentation by class I and class II MHC molecules, inhibition of natural killer cell mediated kill ing, inhibition of apoptosis, molecular mimicry of chemokines and their receptors, inhibition of cytokine receptor signaling pathways, infection of lymphoid cells , and immunosuppression (Farrel l et al. 1 999; Brodsky 1 999; Hil l & Masucci 1 998; Davis-Poynter & Farrel l 1 998; Kieff & Shenk 1 998; Krajcsi & Wo1d 1 998; Davis­ Poynter & Fat'rel l 1 998; Banks & Rouse 1 992). V iral-induced suppresion of the host immune responses results in a better chance of surv ival of infected cells . At the same time, however, infected hosts may become more susceptible to other infectious agents that are present in their environment. In such individuals , the observed clinical signs may be due to infection with other pathogens, Chapter 10 204 yet herpesv irus-induced immunosuppression i s the primary, underlying cause of the di sease. The aim of the present study was to determine differences in gene expression between two populations of equine PBL: infected with EHV -2 and adsorbed with inactivated EHV -2. Cells adsorbed with the inactivated virus, rather than uninfected cells , were used in order to el iminate differences between infected and uninfected cell s that may have resulted from physiological reaction to stimulation of immune cells with EHV-2 antigen. The results of this thesis suggest that EHV -2 i nfection of equine PBL may down­ regulate the transcription of a gene coding for MCP- I . Monocyte chemoattractant protein- I is a p-chemokine that is a strong chemotactic factor for monocytes and natural k i l ler (NK) cel ls , both in vitro and in vivo. It also regulates the expression of cell surface antigens and cytokines IL- l and IL-6 (Van Coil l ie et al. 1 999). Additional ly, MCP- l was also shown to be a strong mediator of inflamatory responses in cells not belonging to the immune system. In one study, the inflamatory response of endothelial cell s to bacterial i nfection was abrogated by anti-MCP- l antibody, indicating that most of the chemotactic activity of these cells was due to MCP- l expression (Van Der Voorn et al. 1 999). Monocyte chemoattractant protein- l not only attracts monocytes, but also mediates differentiation of monocytes into macrophages (Fantuzzi et al. 1 999). S ince macrophages play a key role in inflamation, antigen presentation and development of cell mediated immune responses, i nhibition of MCP- l expression and the resulting failure to activate monocytes has a potential to affect host defence mechanisms. Clones specific for DP3( +) ampl icon contained EHV -2 sequences derived from a gene coding for virion protein kinase and a part of the EHV -2 genome between ORF 56 and 57. The significance of identification of these particular sequences is yet to be determined. Identification of only four genes that may be differential ly expressed in cell s infected with EHV -2 or adsorbed with inactivated EHV -2 may reflect the fact that the samples were collected at only one time-point, 4 days after infection. The few differences found between the two populations may reflect the true s ituation at that particular time point. Representational D(fIerence Analysis of EHV-2 Infected Equine Leucocytes 205 It i s possible that collection of samples at other time points would result i n identification of other genes . Alternatively, only a fraction of differentially expressed genes may have been identified. Failure to isolate a fragment from a specific gene does not prove that this gene was not differential ly expressed. Only genes that possess at least two Dpn II restriction sites could be i dentified. It has also been suggested (Hubank & Schatz 1 994) that the ampli fication of a few predominant products may hinder amplification of less abundant sequences. The latter problem can be overcome by ' spiking' the driver sample with the sequences known to be differential ly expressed in the tester, so that other, not yet identified, sequences could be amplified. Identification of only four sequences in the present study could also indicate that the RDA system was not well optimised. Optimisation of RDA condi tions for use with specific source material can be performed by supplementing the tester population with different amounts of known DNA or RNA. Isolation of appropriate sequences not only verifies that the system performed well , but also estab l ishes detection l imits. Such preliminary experiments were not performed due to financial constraints. Nonetheless, no identical sequences were identified as differential l y expressed in both (+) and (-) samples. Also, EHV -2 derived sequences were, as expected, ampl ified from the (+) sample, and the results from subtraction of the (-) sample correlated well w ith those obtained by others in studies on HCMV infection (Hirsch & Shenk 1 999) (see below) . Representative c lones were used a s probes against the amplicon blots (Figure 10.5) . The probes prepared from sequences identified after subtraction of the (+) sample reacted strongly with the (+) amplicons only, while sequences identified after subtraction of the (-) sample reacted strongly with (-) amplicons only. Low levels of signal observed after hybridisation with heterologous ampJicons were probably due to non-specific background binding. To some extent, these findings provided validation of the RDA performance. None of the 23 clones selected for further analysis reacted with either homologous or heterologous R amplicon probes. This probably indicated low sensitivity of R amplicon probes, so that only reasonably abundant sequences may have been detected. Also, the selected clones may have represented relatively rare transcripts in the original RNA samples . Chapter IO 206 Although RDA provides a good screening method, in order to show conclusively that the identified sequences are d ifferential ly expressed in the two cell popul ations a further step is required. Definitive conclusions would need more strict proof, such as Northern blot or RNase protection assays (Shi et al. 1 997). These were not performed due to insufficient amounts of starting material . Thus, the conclusions drawn from this experiment need to remain speculative. Despite this l imitation, the results obtained provided some validation of the performance of the system (see above) and suggested that EHV -2 i nfection may inhibit transcription of MCP- l mRNA. Herpesviruses, and also members of other v irus famil ies, have been shown to have the abi lity to i nterfere with cytokine signal ling pathways (Ploegh 1 998; Krajcsi & Wold 1 998) . Interactions between chemokines and their receptors are thought to be a major mechanism of controlled recruitment of specific cells required for both inflammation and host defence mechanisms (Price et al. 1 999; Ramshaw et al. 1 997; Splitter 1 997). Down-regulation of MCP- l expression was reported i n HCMV (Hirsch & Shenk 1 999; Bodaghi e t al. 1 998) and HIV (Genin et al. 1 999) infected cells . B locking of MCP- l gene expression in HCMV infected cell s occurred at a transcriptional level, and could not be reversed by induction with TNF-ex or IL- I p , known inducers of MCP- l . The block of MCP- I expression was not observed unti l 8 hours post infection and required v iral gene expression, s ince the inhibitory effect was not observed in control cell s adsorbed with inactivated HCMV. The authors speculated that the inhibition of MCP- l transcription was induced either by one or more HCMV gene products produced soon after infection or, alternatively, by some inhibitory factor(s) present within the viral tegument (Hirsch & Shenk 1 999). It has been postulated that this i nhibitory effect was dependent on expression of HCMV gene US28 (Bodaghi et al. 1 998). This gene codes for a functional p-chemokine receptor, which has been shown to bind MCP- l , RANTES, macrophage inflammatory protein (MIP)- 1 et, and MIP- l p (Gao & Murphy P .M. 1 994). However, the association between expression of US28 and down-regulation of MCP- I expression in HCMV infected cell s could not be demonstrated by others (Hirsch & Shenk 1 999). Conflicting results were also reported regarding the effect of HCMV infection on the expression of another p-chemokine - RANTES. It was reported to be down-regulated (Bi l lstrom et al. 1 999; Bodaghi et al. 1 998) or up-regulated (Michelson et al. 1 997) in HCMV -infected cells . Nonetheless, both Bi l lstrom et al. ----- �----- - - - -- Representational Difference Analysis of EHV-2 Infected Equine Leucocytes 207 ( 1 999) and Bodaghi et a!. ( 1 998) agreed that depletion of extracellular RANTES was attributable to intracellular accumulation through binding to viraUy encoded US28. Herpesviruses have also been shown to be able to modulate the expression of other chemokines in infected cell s and analysis of herpes viral genomes showed that these viruses code for their own versions of different chemokines and chemokine-receptors (Pelchen-Matthews et al. 1 999; Diaraghi et al. 1 998; Gosselin e t al. 1 992) . Equine herpesvirus-2 codes for a gene homologous to the HCMV US28 gene, and one, or two, other GPCR. This, together with the findings from the present study, i ndicate that modulation of the chemokine environment in infected cell s is l ikely to be one of the strategies used by EHV -2 to escape recognition by the immune system. The results of in vitro studies do not necessarily reflect the in vivo situation. For example, despite observed in vitro down-regulation of MCP- l expression in HCMV infected cells (Hirsch & Shenk 1 999; Bodaghi et (If. 1 998), elevated MCP- l levels have been reported in the cerebrospinal fluid of AIDS patients with HCMV encephalitis (Bernasconi et al. 1 996) . One explanation for these discrepancies is that the in vitro environment can never ful ly mimic the in vivo situation. For example, the effects of EHV -2 infection on gene expression in equine leucocytes may depend on whether this i nfection is l atent or active. In this study, the recovery of EHV -2 from fresh MC, but with no CPE observed during the first passage in cells inoculated with freeze-thawed MC from (+) samples, i ndicated that EHV -2 probably established latent infection in MC. However, the presence of several plaques i n wel ls inoculated with freeze-thawed (+) samples on the second passage i ndicated that some MC were actively infected w ith EHV -2 and were the source of infectious virus in the freeze-thawed preparation. Thus, the (+) sample probably represented a mixture of latently and actively infected cells, which may be different in natural infection. Another explanation for discrepancies between in vivo and in vitro effects of HCMV infection on expression of MCP- l is that HCMV infection may have different effects at different sites of infection. Chemokine receptors have a common structure of GPCR. Initiation of intracel lular signall ing occurs v ia association with heterotrimeric G proteins in response to binding of a chemoattractant, such as MCP- l . The same chemokine can exert different effects in a variety of cel l s . This i s due to the fact that G­ proteins are expressed in a t issue-specific manner, and thus coupling of the G-protein to Chapter 10 208 the GPCR may exert different biological effects in different cell types (Billstrom e t al. 1 998). Also, a chemokine can only activate cell s that express a receptor able to bind this chemokine. The number and kind of receptors expressed on the surface of leucocytes is not constant, but change according to the activation or differentiation state of cells , and hence, the susceptibility of cell s to the action of a given chemokine may vary considerably. For these reasons, in vivo effects of modulation of cytokine production in virally infected cel ls may be different to those observed in vitro. Additionally, the expression of US28 mRNA has been reported in cell s that do not support HCMV replication (Zipeto et al. 1 999). This raised the possibility that HCMV infection may influence the chemokine expression and transcriptional activity not only in actively infected cells , but also in non-permissive neighbouring cells . Equine herpesviru s-2 can infect a variety of cel l s (Browning & Agius 1 996). Thus, by extrapolation from HCMV studies, the biological effects of EHV -2 infection i n vivo may be different at different sites of infection. Also, the effects may depend on pre­ infection immune status, age of the animal or the presence of co-infecting pathogens, as these factors may influence the expression of chemokine receptors on the surface of leucocytes. Equine herpesvirus-2 has been implicated in the pathogenesis of COPD, as EHV-2 antigens were detected in pulmonary macrophages from horses with COPD, and not from healthy horses (Schlocker et al. 1 995) . Early stages of COPD are referred to as small airway disease (SAD). The characteristic feature of the syndrome is immune­ mediated inflammatory reaction characterised by neutrophil ic infiltration of small bronchioles, increased airway resistance due to airflow obstruction, bronchial mucus and hyper-responsivness to non-specific stimuli , such as histamine (Vie l 1 997). Franchini et al. ( 1 998) suggested that overstimulation of alveolar macrophages by dust particles leads to decreased phagocytic activity and increased production of IL-8 and MIP-2, potent chemoattractants for neutrophils, in susceptible horses. The levels of other chemokines were not measured in this study. Equine herpesvirus-2 has been shown to infect alveolar macrophages of horses (Schlocker et al. 1 995) . Thus, altered function of bronchoalveolar macrophages may play a key role in the development of COPD in horses. If this hypothesis were true, the possibility that EHV -2 can modulate the chemokine environment of infected cel ls may be important in predisposing to the development of disease. Representational Difference Analysis of EHV-2 Infected Equine Leucocytes 209 1 0.5 SUMMARY The results of the RDA study of equine PBL samples infected with EHV -2 and adsorbed with inactivated EHV-2 suggested that EHV-2 may downregulate MCP- l transcription i n infected cell s . These findings correlate well with, and support, similar findings described for HCMV and supports the view that EHV -2 may have the abi l i ty to modify the chemokine environment of infected cel ls . This may constitute an important feature of EHV -2 biology, because such an abi lity has a potential to compromise host defence mechanisms and predispose to infections with other pathogens. CHAPTER 11: CONCLUDING REMARKS One of the main research priorities identified by the equine industry is to minimise wastage among competing horses. Equine re piratory disea e has been regarded a an important cause of wastage (Chapter 1 ) . Despite considerable research effort put into identifying the main causes of equine respiratory disease, different researchers have reported different results, and often the conclusions were conflicting (Chapter 1 ) . The research of this the is concentrated on determining the viral causes of equine respiratory disease in New Zealand. New Zealand, as an island well separated from the rest of the world, is fortunate in the fact that several viruses, including equine influenza, are exotic to this country. This creates a unique environment in which some pathogens may prove to be more important than they are considered to be overseas. The purpose of the present study was to establ ish which viruses circulate among New Zealand horses and which are most commonly associated with development of re piratory signs. The results of the survey conducted indicated that most of the equine respiratory viru es reported to circulate among horses overseas are also present in New Zealand. The general discussion of these findings is presented in Chapter 8 . Thus, this chapter wil l focus mainly on a discussion of is ues regarding the establi hment of causative link between identified pathogens and disease, and also possible future directions for research into equine respiratory disease and its causes. Cel l culture and serology were used for identification of respiratory viruses in cl inical samples . There are several reasons why in vitro cultivation may fai l to identify organisms responsible for disease. First ly, not all microorganisms can be cultured in the laboratory. Several microbial agents are known to be able to induce re piratory igns in horses (Chapter 1 ) . However, the ident i fication of all of these agents has been based on the abi l ity to culture them in vitro. Simi larly, all the surveys reporting association between different viruses or bacteria and respiratory disease in horses rel ied on identification of culturable organisms. Recently, surveys of some aquatic and terrestrial ecosystems indicated that the diversity of microbial organism detectable at a molecular level was considerably greater than that assumed on the basis of cultivation tudie . It Chapter 1 1 2 1 2 has been estimated that up to 99% of bacteria from many terrestrial and aquatic ecosystems resist cultivation using traditional methods (Ward et al. 1 998; Relman, 1 999; Pace, 1 997; Hugenholtz et al. 1 998). By extrapolation from these studies, the ' in vitro culture' approach to identifying organisms causatively involved in equine respiratory disease i s l ikely to fai l to i dentify all organi sms present in a c l in ical sample. Although the majority of the studies c ited above investigated bacterial diversity, there i s no reason to assume that the situation is different with respect to v iruses. Recent discoveries of several new virus species (Relman 1 999; Fredericks & Relman 1 996) support this conclusion. Thus, i t is possible that some pathogens causatively i nvolved i n equine respiratory disease have not yet been discovered. Secondly, the orgamsms that are cultured from diseased horses, whether they are viruses, bacteria or other pathogens, may not play a causative role i n the development of disease. Yet, they may sti l l be present in the majority of diseased animals due to a favourable environment created for them by the causative agent. Thirdly, the time of sampling may influence the presence or absence of a pathogen. In some instances, the disease develops after the causative agent i s el iminated from the organism, as happens for example in d iseases caused by immunopathology. Another example of such s ituation may be secondary bacterial infections m immunocompromised hosts. Although the observed disease signs are due to bacterial infection, the real cause of disease is an agent that caused immunosuppression rather than the i solated bacteria. Serological diagnosi s is less dependent on correct timing than culture of the agent. However, demonstration of recent infection requires avai lab il ity of both acute and convalescent serum samples. Again - the immune response to the causative agent may have already developed after the acute serum sample is taken. Also, serological testing requires prior knowledge of what pathogens may be involved. Thus, while it provides a rel iable means of detection of a specific infection, i t does not have the abi lity to detect infection with new or unanticipated pathogens. In contrast, molecu lar methods that rely on identification of pathogen-specific nucleic acids directly in cl inical samples, rather than after in vitro cultivation, offer more Concluding Remarks 2 1 3 rel iabil ity and sensiti vity of diagnosis (Gao & Moore 1 996). With the invention of PCR, molecular techniques have been successful ly applied to discover new or uncultivable organi sms (Relman 1 999; Gao & Moore 1 996; Fredericks & Relman 1 996). Use of consensus sequence-based primers to identify viruses from a pruticular family, alongside further development and optimisation of species-speci fic PCR assays, may provide a more rel iable means of virus identification directly in cl inical specimens, without any b ias i ntroduced by cultivation. Additional advantages of PCR-based diagnosi s are speed and the abi l ity to detect viruses that are no longer viable due to improper transport conditions. Also, fragments of pathogen nucleic acid may sti l l be present in macrophages or lymphocytes of the host, when entire pathogens are no longer present. However, the same features that make molecular-based diagnosis so attractive (sensitivity, speci ficity, detection of non-viable pathogens), also provide additional difficulties in terms of establ ishing the disease causation (Fredericks & Relman 1 996) . Firstly, ampli fied DNA could originate from ' i rrelevant ' micoroorganisms e.g. those that were successful ly ki lled by the host immune system, 0ppOltunists not l inked to disease, or from laboratory contamination. Secondly, the v iable organism is not available for experimental i nfection, and thus, the third Koch' s postulate cannot be ful fi l led. According to Koch' s postulates for causation, a parasite is considered as the cause of disease if: • The parasite occurs In every case of the disease in question and under circumstances, which can account for the pathological changes and cl inical course of disease. • The parasite occurs in no other disease as a fortuitous and nonpathogenic parasite. • After being ful ly isolated from the body and repeatedly grown in pure culture, the parasite can induce the disease anew (Fredericks & Relman 1 996) . Clearly, none of the equine respiratory v iruses identified during the survey could ful fi l a l l three requirements, a s i nfection with none o f these viruses was identified exclusively in animals showing respiratory signs. However, Koch' s postulates were formulated in the nineteenth century. With the advances in today' s knowledge about the microbial world, the l imitations of Koch' s postulates become more and more apparent. In Chapter / / 2 14 particular, Koch ' s postul ates could not be applied to diseases caused by persistent infections, environmental factors, or immunopathology. They also did not take i nto consideration individual genetic predispositions. As Fredericks & Relman ( 1 996) noted: "Considerations of host, environment, microbial adaptation, and the complexities of host -parasite relationships suggest that we change our perspective on microbial causation . Defini tions should address the difference between 'necessary' and 'sufficient ' ; that is, the presence of a microbial pathogen or its products (at some point in time) may be necessary but not sufficient to produce disease in a given host". Subsequently, Koch' s postulates have been modified by several authors (Fredericks & Relman 1 996). The most apparent trends in the evolution of thought on disease causation was accepting the epidemiological associations and relative numbers of pathogens or pathogen-derived nucleic acids in diseased vems non-diseased hosts or tissues, rather than blindly adhering to their presence or absence alone. Equine herpesvims-2 (and possibly EHV -5) is one of the examples where disease causation has not been clearly estab l ished. Epidemiological studies have l i nked EHV-2 infection with several cl in ical signs including respiratory disease and poor performance (Chapter 1 ) , although i n only one study was EHV -2 detected exclusively i n diseased animals, and not in healthy ones (Schlocker et al. 1 995) . Additionally, several features of the EHV -2 genome suggest that this vims is l ikely to modulate the immune response of its hosts. Thus, most probably EHV -2 infection affects horses indirectly, v ia modulation of the their defence mechanisms rather than via direct pathology caused by viral repl ication. In Chapter 1 0, I showed that EHV -2 is l ikely to i nfluence the cytokine environment of infected cells. Other v imses (Krajsc i et aI. , 1 998), as well as bacteria (Hull inger et al. 1 998), also are able to modulate cytokine production in infected cells . Thus, i t may be that the association between EHV -2 i nfection and disease is even more complex, and the final outcome may depend not only on EHV -2 interactions with its host, but also on the presence or absence of other pathogens. S imi larly, the development of respiratory disease is probably not simply the result of i nfection with a particular pathogen or pathogens, but may depend on the complex i nterplay between these pathogens and the host ' s cells . If so, the i nvestigation of equine respiratory d isease should focus on a broader picture, rather than concentrate on the effects of infection with specific pathogens. Because of the complexities of these interactions, such a complex approach has not been feasible in the past. However, with the advancements in ----�-- - - Concluding Remarks 2 1 5 molecular biology techniques, such a task becomes more achievable. For example, Relman ( 1 999) proposed that it might be possible to d istinguish between different infectious agents and different pathogenic mechanisms by looking at the host gene expression patterns. In this approach, one would look for 'diagnostic signatures' that pathogens leave in their hosts rather than for pathogens themselves. Thi s may prove useful in identifying aetiologic agents in those cases where they cannot be found using traditional methods. The abi lity to screen thousands of genes simultaneously using DNA chip technology (section 10. 1 ) provides an exciting opportunity for examin ing many parameters at the same time. For example, a DNA chip containing representative sequences from the equine genome, as well as sequences derived from genes of selected respiratory pathogens, could be probed against mRNA obtained from either healthy horses or horses affected by respiratory disease. Comparison between the host gene expression, presence or absence of specific pathogen gene expression and the cl inical status of horses sampled could provide very comprehensive information not only on the association between infectious status and presence or absence of disease, but also on the influence of host responses or individual genetic predi spositions on the disease process. In conclusion, the research of this thesis supports the view that equine respiratory disease is multifactorial . It is probably complex interactions between different microorganisms, the environment and the host, that determines the presence or absence of disease. Therefore, future research should focus on elucidating such i nteractions rather than examining the influence of specific pathogens on the disease status outside the context of their natural existence. Recent developments i n molecular techniques open exciting possibilities for future research. Better understanding of complex dependencies between different respiratory pathogens and the horse would enable development of better means for control or prevention of disease, for example through uti l isation of cytokine therapy to neutrali se the detrimental effect of infectious agent on host defence mechanisms. Appendices Appendix A: Resu lts of the s u rvey Appendix B: Blocking E L l SA controls Appendix C : C l ustal W results Appendix D: BLAST results Appendix E: Buffers and solutions Appendices 2 1 9 APPENDIX A: RESULTS OF THE SURVEY Table A.I : Yearlings from the yearling sales Virus isolation) Serologl Horse Nasal PBL EHV- l/4 EhV- J EAdV- J ERhV.· ] ERhV-2 swabs SN EL/SA HI SN SN SAl EHV-2 <2 S 1.7 20 < 1 0 4 1 6 9 1. 1 < 10 < 1 0 1 6 SA2 EHV-2 4 68.9 < 1 0 < 1 0 8 8 72.4 < 1 0 < 1 0 64 SA3 EHV-2 EHV-2 <2 SO.9 < 1 0 < 1 0 1 6 <2 S6.9 < 10 < 10 32 SA4 EHV-2 <2 1 4. 1 < 1 0 < 1 0 4 EHV-S <2 4 1 .6 < 1 0 < 1 0 2 SAS ? EHV-2 <2 1 8. 2 < 1 0 < 1 0 16 <2 22.8 < 1 0 < 1 0 1 6 SA6 EHV-2 EHV-2 8 77.2 < 1 0 < 1 0 <2 8 76. 1 < 1 0 < 1 0 <2 SA7 EHV-2 2 32 . 1 < 1 0 < 10 <2 EHV-S 32 88.9 < 1 0 < 1 0 1 28 SA8 EHV-2 <2 S I .9 < 1 0 < 1 0 <2 EHV-S nd nd nd nd nd SA9 ? EHV-2 4 86.5 < 10 < 1 0 2 4 8S.7 < 1 0 < 1 0 1 6 SA I O EHV-2 4 26.0 nd nd nd nd nd nd nd SA I l EHV-2 1 6 69.3 < 1 0 < 10 1 6 EHV-S 8 63.7 < 1 0 < 10 1 6 SA I 2 EHV-2 2 38 .9 20 < 1 0 8 EHV-S 1 6 73 .5 1 0 < 1 0 8 SA I 3 EHV-2 EHV-2 <2 1 8.7 40 < 1 0 3 2 EHV-S <2 I S .4 20 < 10 32 SA I 4 EHV-2 8 83.5 1 0 < 1 0 32 1 6 89.6 < 1 0 < 1 0 1 28 SA1 S EHV-2 <2 S8 . 1 nd nd nd nd nd nd nd nd T l EHV-2 4 37.7 20 < 1 0 32 64 67.9 1 0 < 10 1 6 T2 EHV-2 8 95.3 < 10 < 10 1 6 64 94. 1 < 10 < 1 0 3 2 T3 EHV-2 4 7 1 .9 40 < 1 0 <2 8 83 .3 40 < 1 0 2 T4 EHV-2 2 33 .4 < 10 < 1 0 <2 4 65.4 < 10 < 1 0 8 T5 EHV-2 8 80. 1 20 < 1 0 2 1 6 78 .8 20 < 1 0 2 T6 EHV-2 8 68.8 < 1 0 < 1 0 8 32 96. 1 < 1 0 < 1 0 8 T7 EHV-2 8 26.5 40 < 1 0 2 4 24 .2 20 < 1 0 2 T8 EHV-2 2 8S.4 < 1 0 < 10 2 5 1 2 94.8 < 1 0 < 1 0 <2 W I EHV-2 8 66. 1 < 1 0 < 1 0 4 4 68.3 < 1 0 < 1 0 4 W2 EHV-2 <2 1 3.7 20 <10 <2 <2 27.8 1 0 < 1 0 64 Appendices 220 Virus isolation' Serolos./ Horse Nasal PBL EHV-JI4 EhV- J EAdV- J ERhV- J ERhV-2 swabs SN EL/SA HI SN SN W3 EHV-2 <2 53 .4 40 < 1 0 4 <2 43.0 20 < 1 0 8 W4 EHV-2 <2 49.9 10 <10 2 <2 4 1 . 1 < 1 0 <10 4 W5 EHV-2 8 70.7 < 1 0 < 1 0 8 8 73.7 < 1 0 < 1 0 1 6 W6 EHV-2 8 82 . 1 < 1 0 <10 <2 nd nd nd nd nd W7 ') EHV-2 <2 28 .8 < 1 0 < 1 0 <2 <2 3 1 .2 < 1 0 < 1 0 4 W8 EHV-2 2 45.0 < 10 <10 8 4 36.8 < 1 0 80 4 W9 EHV-2 1 6 85.6 80 < 1 0 <2 8 87. 1 40 < 1 0 <2 W I O ? EHV-2 8 48. 1 40 1 28 < 1 00 > 1 28 < 1 00 > 1 28 < lO O 1 28 Appendices 224 Virus isolation Serology � � Date Nasal EHV-l/4 EhV- J \:) PBL EAdV- J ERhV- J ERhV-2 ::t: swabs SN ELlSA 71 1 1 /95 nd 64 86.7 20 1 600 32 271 1 1 195 nd nd 88 .2 1 0 1 600 64 9/0 1 /96 EHV-2 EHV-2 1 6 6 1 .8 < 1 0 200 1 6 A6 9/02/96 EHV-2 EHV-2 4 37.9 < 1 0 100 2 6/03/96 EHV-2 EHV-2 4 2 1 .2 < \ 0 < \ 00 2 EHV-5 3/04/96 EHV-2 4 25.6 < 1 0 < 1 00 <2 7/ 1 1 /95 nd 256 95.6 40 < 1 00 64 2711 1 /95 nd 93.2 20 < 1 00 32 9/0 1 /96 EHV-2 64 80.9 1 0 < 1 00 2 9/02/96 EHV-2 EHV-2 65 .0 < 1 0 < l OO 1 6 A7 6/03/96 EHV-2 EHV-2 1 6 44. 1 < \ 0 < 1 00 nd 3/04/96 EHV-2 EHV-2 8 46.7 < 1 0 < l OO 8 7/05/96 EHV-2 4 52.7 < \0 < 1 00 64 1 5/8/96 EHV-2 64 7 1.4 < 10 < 1 00 64 EHV-5 1 1 / 1 0/96 EHV-2 8 33.7 < 1 0 < \ 00 1 6 29/03/96 EHV-2 4 46. 1 20 < 1 00 <2 29/04/96 EHV-2 8 72.0 < 1 0 < 1 00 2 25/05/96 EHV-2 4 55 .5 20 < 100 <2 1 0/07/96 EHV-5 2 54.5 < 1 0 < \ 00 1 6 EHV-2 B I 20/08/96 EHV-2 2 32 .3 < 1 0 < 1 00 8 1 4/ 1 0/96 EHV-2 2 49.2 < 1 0 < 1 00 8 1 8/ 1 1/96 EHV-2 4 42.9 320 < \0 0 8 1 71 1 2/96 EHV-2 4 44.4 320 < 1 00 8 EHV-5 B2 29/03/96 EHV-2 nd nd nd nd nd B3 29/03/96 EHV-2 nd nd nd nd nd 29/03/96 EHV-2 2 86.5 \ 0 < 1 00 <2 29/04/96 EHV-2 2 83.9 1 0 < \ 00 2 25/05/96 EHV-2 <2 73.4 20 < \ 00 <2 1 0/07/96 EHV-2 <2 60.6 40 < 1 00 2 EHV-5 B4 20/08/96 EHV-2 <2 47.4 40 < 10 0 4 1 4/ 1 0/96 EHV-2 <2 32.8 1 0 < 1 00 8 1 8/ 1 1 /96 EHV-2 <2 39.5 < 1 0 < 1 00 1 6 1 7/ 1 2/96 EHV-2 1 6 95 . 8 1 60 < 1 00 32 EHV-5 B5 3/04/96 EHV-2 EHV-2 nd nd nd nd nd 29/03/96 EHV-2 4 34. 1 < 1 0 < 1 00 <2 29/04/96 EHV-2 4 38 .2 < 1 0 < 1 00 2 25/05/96 EHV-2 <2 34.8 < 1 0 < 1 00 <2 1 0/07/96 EHV-2 <2 22.5 < 1 0 < 1 00 <2 B6 EHV-5 20/08/96 EHV-2 <2 1 8 .4 < 1 0 < 1 00 <2 1 4/ 1 0/96 EHV-2 32 6 1.4 < 1 0 < 10 0 1 6 1 8/ 1 1 /96 EHV-2 32 59.6 < 1 0 < 10 0 1 6 1 7/ 1 2/96 EHV-2 1 6 68.9 40 < 100 1 6 Appendices '" :::: Date C) ::J:: 29/03/96 29/04/96 25/05/96 B7 1 0/07/96 20/08/96 1 4/ 1 0/96 1 8/ 1 1 /96 1 71 1 2/96 29/03/96 29/04/96 25/05/96 B8 1 0/07/96 20/08/96 1 4/ 1 0/96 1 81 1 1 196 29/03/96 29/04/96 25/05/96 B9 20/08/96 1 4/ 1 0/96 1 8/ 1 1 /96 1 71 1 2/96 B 10 29/03/96 29/04/96 B l 1 3/04/96 29/03/96 B I 2 29/04/96 25/05/96 1 0/07/96 3/04/96 29/04/96 25/05/96 B I 3 1 0/07/96 20/08/96 1 4/ 1 0/96 1 81 1 1 196 171 1 2/96 B 1 4 3/04/96 B I 5 3/04/96 3/04/96 29/04/96 25/05/96 B I 6 1 0/07/96 20/08/96 1 4/ 1 0/96 1 81 1 1196 1 71 1 2/96 Virus isolation Nasal PBL swabs EHV-2 EHV-2 EHV-2 EHV-5 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-5 EHV-2 EHV-5 EHV-2 EHV-2 EHV-2 EHV-5 EHV-2 EHV-2 EHV-5 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-5 EHV-2 EHV-2 EHV-2 EHV-2 EHV-5 EHV-2 EHV-2 EHV-5 EHV-2 EHV-5 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-2 EHV-5 EHV-2 EHV-2 EHV-2 EHV-2 ------------------- -- EHV-JI4 SN 4 2 2 2 <2 32 8 4 8 64 64 4 4 2 32 4 4 4 2 64 4 2 4 1 6 nd 1 6 2 2 <2 4 2 8 4 2 1 6 2 2 nd nd 8 64 32 8 8 4 <2 4 225 Serology EhV- J EAdV- J ERhV- J ERhV-2 EL/SA 1 9 .2 < 1 0 < 1 00 <2 35.6 < 10 < 100 <2 22.7 < 1 0 < 1 00 <2 1 9.5 < 1 0 < 100 2 1 5 .8 1 0 < 1 00 <2 88.5 < 1 0 < 1 00 1 6 85 .8 < 1 0 < 1 00 1 6 85.6 20 < 100 32 73.7 20 < 1 00 <2 8 1 .5 1 0 < 100 <2 77.9 1 0 < l OO <2 73.0 < 1 0 < 100 1 6 69.4 < 1 0 < 1 00 8 63.5 20 < 1 00 8 92.2 1 0 < l OO 8 62.2 1 0 < 1 00 <2 59.8 20 < 1 00 <2 59.0 20 < 100 <2 67.0 1 0 < l OO 4 87.7 < 1 0 < 100 2 9 1 .5 < 10 < 1 00 2 89.7 < 1 0 < 1 00 4 26.3 < 1 0 < 1 00 <2 77.0 20 < 1 00 <2 nd nd nd nd 39.0 20 < 10 0 <2 45.3 1 0 < 1 00 <2 34.5 < 1 0 < 1 00 <2 29.7 20 < 1 00 <2 34.0 < 1 0 < l OO <2 35.3 < 1 0 < 10 0 <2 70.3 < 1 0 < l OO <2 69.3 < 1 0 < 100 8 5 1 .7 < t o < l OO 8 7 1.6 < 1 0 < lOO 64 57.6 20 < l OO 32 46.0 20 < lO O 1 28 nd nd nd nd nd nd nd nd 6 1 .0 < 1 0 < 1 00 2 86.4 < 1 0 < 1 00 <2 82.7 < 1 0 < 1 00 2 42. 1 < 1 0 < 1 00 1 6 48.2 < 10 < l OO 1 6 27.5 < 1 0 < 10 0 1 6 33.7 < 1 0 < l OO 1 6 27.4 < 1 0 < 10 0 32 Appendices 226 Virus isolation Serologv '" to Date Nasal EHV- l/4 EhV- J 0 PBL EAdV- J ERhV-J ERhV-2 ::t: swabs SN ELlSA B I 7 3/04/96 EHV-2 nd nd nd nd nd B 1 8 3/04/96 EHV-2 nd nd nd nd nd 3/04/96 EHV-2 EHV-2 4 1 6.4 < 1 0 < 10 0 <2 29/04/96 EHV-2 EHV-2 8 60,6 < 1 0 < 100 <2 B I 9 25/05/96 EHV-2 8 5 1. 7 40 < l OO <2 1 0/07/96 EHV-2 2 3 1.0 1 0 < 100 <2 20/08/96 EHV-2 2 27,9 < 1 0 < 1 00 2 1 4/ 1 0/96 EHV-2 8 79.2 < 10 < 1 00 8 3/04/96 EHV-2 <2 1 8,7 < 1 0 < 1 00 <2 1 6/05/96 EHV-2 EHV-2 <2 1 6.4 < 1 0 < 100 <2 1 0/06/96 EHV-2 EHV-2 <2 1 2 ,5 1 60 < 1 00 <2 C l 1 6/07/96 EHV-2 4 62, 1 80 < 1 00 <2 1 5/08/96 EHV-2 1 6 78 ,9 80 < 1 00 <2 1 4/ 1 0/96 EHV-2 8 95,6 20 < 1 00 <2 1 4/ 1 1 /96 EHV-2 8 9 1 .4 1 0 < 1 00 2 1 61 1 2/96 EHV-2 8 89,0 1 60 < 10 0 4 3/04/96 EHV-2 <2 1 7,7 < 1 0 < 1 00 <2 1 6/05/96 EHV-2 <2 1 0 .5 < 10 < 1 00 <2 C2 1 0/06/96 EHV-2 EHV-2 <2 1 0.9 < 1 0 < 1 00 <2 1 6/07/96 EHV-2 4 69,9 20 < 1 00 2 1 5/08/96 EHV-2 8 73 .0 < 1 0 < 1 00 2 1 4/ 1 0/96 EHV-2 1 6 86.3 < 10 < 1 00 <2 3/04/96 EHV-2 <2 1 2.0 < 1 0 < 1 00 <2 1 6/05/96 EHV-2 EHV-2 <2 4.5 < 1 0 < 1 00 <2 1 0/06/96 EHV-2 EHV-2 <2 1 9.4 < 1 0 < 1 00 4 1 6/07/96 EHV-2 256 8 1 .2 < 10 < 1 00 <2 C3 1 5/08/96 EHV-2 1 28 76,6 < 1 0 < 1 00 2 1 41 1 0/96 EHV-2 32 72.3 < 1 0 < 1 00 4 1 41 1 1 /96 EHV-2 32 69.9 < 1 0 < 1 00 4 EHV-5 1 6/ 1 2/96 EHV-2 1 6 74.3 80 < 100 2 EHV-5 3/04/96 EHV-2 <2 53 .2 < 1 0 < 1 00 2 1 6/05/96 EHV-2 <2 1 7 ,7 < 1 0 < 1 00 2 1 0/06/96 EHV-2 EHV-2 <2 1 0.5 < 1 0 < 1 00 <2 C4 1 6/07/96 EHV-2 <2 76.4 < 1 0 < 10 0 <2 1 5/08/96 nd <2 72 .8 < 1 0 < 1 00 <2 1 41 1 0/96 EHV-2 8 85.0 < 1 0 < 1 00 <2 1 41 1 1 /96 EHV-2 4 84.5 < 1 0 < l OO <2 1 6/ 1 2/96 EHV-2 2 86.0 80 < 100 <2 3/04/96 EHV-2 <2 1 4.5 < 1 0 < 1 00 1 6 1 6/05/96 EHV-2 <2 1 6.7 < 1 0 < 10 0 1 6 1 0/06/96 EHV-2 EHV-2 <2 1 1 .4 < 1 0 < 1 00 4 C5 1 6/07/96 EHV-2 EHV-2 8 69.6 < 1 0 < 100 <2 1 5/08/96 EHV-2 1 6 82.4 < 1 0 < 1 00 2 1 4/ 1 0/96 EHV-2 1 6 87.0 < 10 < 1 00 4 1 41 1 1 /96 EHV-2 32 90. 1 < 1 0 < 1 00 4 1 6/ 1 2/96 EHV-2 32 89.8 < 1 0 < 10 0 4 I Date of sampl ing nd not done Appendices 227 APPENDIX B : ELlSA CONTROLS Table B . l : Raw data Negative control Positive control Mean SD N CV Mean SD N CV O.D. O.D Plate I 1 .299 0.006 2 4.6% o.mS? 0.006 2 6.9% Plate 2 1 .228 0.023 2 1 .9% 0. 1 1 4 0.003 2 2 .6% Plate 3 1 .255 0.037 2 2.9% 0.09 1 0.007 2 7 .7% Plate 4 1 . 1 98 0.003 2 0.2% 0.096 0.002 2 2 . 1 % Plate 5 1 .267 0.036 2 2 .8% 0. 1 1 9 0.005 2 4.2% Plate 6 1. 1 88 0.0 1 9 2 1 .6% 0. 1 07 0.00 I 2 0.9o/c., Plate 7 1 . 1 42 0.000 2 09h 0.095 0.000 2 0% Plate 8 1 . 1 35 0.025 2 2.2% 0. 1 03 0.004 2 3 .9% Plate 9 1 . 1 60 0.059 2 5 . j f�) 0. 1 1 1 (W32 2 28 .9% Table B.2: Positive controls: transformed data O.D. readings % blockins. Mean % blocking SD CV 0.09 1 93 .0 Plate I 93.350 0.495 0.5% 0.082 93.7 0. 1 1 2 90.9 Plate 2 90.700 0.283 0.3l/c 0. 1 1 6 90.5 0.086 93 . 1 Plate 3 92.700 0.566 0.6% (W96 92.3 (W95 92. 1 Plate 4 92.350 0.345 0.4% 0.089 92.6 0. 1 22 90.4 Plate 5 90.650 0.354 0.4% 0. 1 1 5 90.9 0. 1 08 90.9 Plate 6 9 1 .000 0. 1 4 1 0. 1 % 0. 106 9L1 0.095 9 1 .7 Plate 7 9 1 .700 0.000 0% 0.095 9 1 .7 0. 1 06 90.7 Plate 8 90.950 0.354 0.4% 0. 1 00 9 1 .2 0. 1 33 88.5 Plate 9 90.450 2.758 3 .0% 0.088 92.4 N: number of replicates SD: standard deviation CV: coefficient of variation Between-plate variation: mean % blocking of positive controls = 9 1 .54%, SD = 1 .039% Appendices 228 APPENDIX C: CLUSTAL W RESULTS Table C.1 : Multiple sequence alignment of 500 bp from the 5' end of the gB gene from different EHV·5 isolates performed using Clustal W ( 1 .7) computer program. Clonel 1 2 6 9 1 2 I S 17 EHV-S EHV-S 2 1 002 6 99 99 9 99 99 98 1 2 98 98 98 98 I S 98 98 98 98 1 00 1 7 98 98 98 98 l OO 1 00 EHV-S 98 98 97 98 99 99 99 EHV-S3 97 97 97 98 98 98 98 99 EHV-24 67 67 67 67 68 67 67 68 68 I Details of the isolates from which clones were derived can be found in Table 9. 1 . 2 Numbers i ndicate 91.- identity scores. 3 EHV -S.2- 1 4 1 [GenBank accession number AFOS067 I J -I EHV-2.86/67 [GenBank accession number U20824 ] Table C.2: Multiple sequence alignment of the 157 amino acids from the N terminus of the gB gene from different EHV·5 isolates performed using Clustal W ( 1 .7) computer program. Clone I 2 6 9 1 2 I S 1 7 EHV-S EHV-S 2 1 002 6 99 99 9 98 98 98 1 2 98 98 97 98 I S 98 98 97 98 1 00 1 7 98 98 97 98 1 00 l OO EHV-S 98 98 98 97 99 99 99 EHV-S 98 98 98 97 99 99 99 1 00 EHV-2 S6 56 56 56 59 59 S9 58 58 I Details of the isolates from which clones were derived can be found i n Table 9 . 1 . 2 Numbers indicate % identity scores. 3 EHV -S.2- 1 4 1 [GenBank accession number AFOS067 I ) -I EHV-2.86/67 [GenBank accession number U208241 ---- - �-- -- -- - ---- Appendices 229 APPENDIX D: BLAST RESULTS >DP3 8 2 . GATCCTTGCA AGGACCCTCA ACACCATCCC AAGGGTAGAA CTGGGGTTCA CAGAGGAAAG CAGTTTGCTC AAGTCTCCAT ATCTCACAGA CACTAGTATT CGTGGAAGAT AAACTTTAAA CATCAATAAC TTAAATAAGA TTAACGCTAC TTAGGCATCA AGTTTCATGT CAATAAACAA AGTTCATACT CTTTGAAACT ATAATAAAAT AATATCAGGG GGCATTTAGG GAATGCTAGA AGACAATAAA TTAGCTTCAG ATTCTTGGCT TTTGGAGTAG GTGTTCAAGG CTTTGGAGTT TGGGCTTTCT TGTCCAGCTG CTTCACAGCA TCCTGGACCC ACTTCTTGCT CGGGGTCAGC ACAGATC Sequences produ c i ng s i gni f i cant a l ignment s : ( f i r s t f ive ) Score E ( b i t s ) Va lue gbnu : ECA2 5 1 I 8 9 Equus cabal I u s mRNA for monocyte chemoattractant . . . 7 0 4 0 . 0 gb : CFU2 9 6 5 3 Can i s f am i l ia r i s monocyte chemoatt ractant prot e i n - I . . . gb : BOVMOCHEM Bovine monocyte chemoat t ractant prote i n - l pre- curs . . . gb : BOVMCP I X Bovine monocyte chemoattractant prot e i n - l (MCP - I ) g . . . gb : S S PMCPl Sus sp . mRNA for monocyte chemo a t t ractant prot e in 1 . . . . >gbnu : ECA2 5 1 1 8 9 Equus caba l l u s mRNA for monocyte chemoa t t rac tant prote in - l ( mcp - l gene ) . Length = 7 6 7 Score 7 0 4 b i t s ( 3 5 5 ) , Expect = 0 . 0 I dent i t i e s = 3 6 5 / 3 6 7 ( 9 9 % ) , Gaps = 1 / 3 6 7 ( 0 % ) S t rand = P l u s / M inu s 2 0 0 1 5 1 1 5 1 1 0 3 Que ry : 1 ga t c c t t gcaaggacc c t caacaccatcccaagggtagaactgggg t t cacagaggaaag 6 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 e - 4 9 5 e 3 4 5 e 3 4 l e - 1 9 S b j ct : 6 3 8 ga t c c t tgcaaggaccc tcaacaccatcccaagggtagaactggggttcacagaggaaag 5 7 9 Query : 6 1 cagt t tgct caagt c t c catat c t cacagacactagtattcgtggaagataaac t t taaa 1 2 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj c t : 5 7 8 cagt t tgctcaagt c t c ca t a t c t cacagacactagtatt cgtggaagataaac t t t aaa 5 1 9 Query : 1 2 1 c a t caataact taaataagat taacgc tact taggcat caagt t t catgtcaataaacaa 1 8 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I Sbj c t : 5 1 8 catcaataact taaataaga t t aacgc t a c t t aagcatcaagtt tcatgt caataaacaa 4 5 9 Query : 1 8 1 agt t catac t c t t tgaaacta taataaaa taatatcagggggcat t tagggaatgc taga 2 4 0 I I I I I 1 1 1 I I I I 1 I I I I I I I I 1 1 I I I 1 I I I I 1 I I 1 I I I 1 I I I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S b j c t : 4 5 8 agt tcatac t c t t tgaaact ataataaaataatat cagggggcat t t agggaatgc taga 3 9 9 Query : 2 4 1 agacaataaa t t agct tcaga t t ct tggc t t tt ggagtaggtgt t caaggc t t t ggagtt 3 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 I 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S b j c t : 3 9 8 agacaat aaa t t agc t tcagat t c t tggc t t t tggagtaggtgttcaaggc t t tggagt t 3 3 9 Query : 3 0 1 tgggc t t t c t tgtc cagctgc t t cacagca t c c t ggacccac t t c ttgct cggggtcagc 3 6 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S b j c t : 3 3 8 tgggc t t t c t tgtccagctgc t t cacagcatcc tggacccac t t c - tgct cggggtcagc 2 8 0 Que ry : 3 6 1 acaga t c 3 6 7 1 1 1 1 1 1 1 S b j c t : 2 7 9 acagatc 2 7 3 Appendices >DP3 + 8 7 ( 3 8 2 l et t e r s ) GATCACAACA CCACCACCAC CACACACGCC CCCTGGAGGA AACAACCTAC GAGTTCTGGG CCGCCCGCCA GGCCCCCCAG GGCCAGACTA CCCCCGCGGG CCCTCCGTGT CCCACACGCC TCAACGGGGA CTACCCCCAG AGCCCGACCA GACGAGGAGC CCGTCCGAGA ACTCTACCTG GGGCAAGCAC GCCCTCCTAG AGCTCATCTC TGCAGACGAC GAGACTGATA CTGGACAAGA TCCCCCGCGG ACTCCGAGGA CCCGTCTTCC CATCGAGCTC CCGATGTGGA CACGAGGAAC CAACATACGC TC 230 GCAGAGAGCC GGACGAGTCC CCCCCGGCGC GAACACATCC GGAGGCCCTG CCGTCACCGA GCGCGAGTGG Score E S equences produc ing s i gni f i cant a l ignment s : ( bi t s ) Va l u e ( f i rs t f ive ) gb : EHVU2 0 8 2 4 Equ i ne he rpe svi ru s 2 , comp l e t e genome . >emb : EHU2 0 8 2 . . . gb : HS 9 5 C2 0 Homo sapiens DNA s e quence f rom PAC 9 5 C 2 0 on chromoso . . . gb : W2 9 3 1 3 mc 0 3 a 0 1 . r 1 Soares mou s e p3NMF 1 9 . 5 Mus musculus cDNA c . . . gb : AC0 1 1 5 3 0 Homo sap i ens chromosome 1 9 c l one LLNL - R_2 2 7G 9 , WORK . . . gb : ACO l 1 2 9 1 Homo sap iens c l one NH0 0 6 7G 0 7 , WORKING DRAFT S EQUENC . . . >gb : EHVU2 0 8 2 4 Equine herpesvirus 2 , comp l e t e genome . >emb : EHU2 0 8 2 4 he rpe sviru s 2 , comp l e t e genome . Length = 1 8 4 4 2 7 Score = 7 4 9 b i t s ( 3 7 8 ) , Exp e c t 0 . 0 I dent i t i e s 3 8 1 / 3 8 2 ( 9 9 % ) S t rand = Plus / P l u s Query : 1 gatcacaacaccaccaccaccacacacgcctcccccgcgggcagagagccccctggagga 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj ct : 1 3 4 3 2 2 gatcacaacaccaccaccaccacacacgcctcccccgcgggcagagagccccctggagga Query : 6 1 aacaacctacgagttctgggactccgaggaggacgagtccccgcccgccaggccccccag 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj ct : 1 3 4 3 8 2 aacaacctacgagttctgggactccgaggaggacgattccccgcccgccaggccccccag Query : 1 2 1 ggccagactacccgtcttcccccccggcgccccccgcgggccctccgtgtcccacacgcc 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj ct : 1 3 4 4 4 2 ggccagactacccgtcttcccccccggcgccccccgcgggccctccgtgtcccacacgcc Query : 1 8 1 catcgagctcgaacacatcctcaacggggactacccccagagcccgaccaccgatgtgga 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj ct : 1 3 4 5 0 2 catcgagctcgaacacatcctcaacggggactacccccagagcccgaccaccgatgtgga Query : 2 4 1 ggaggccctggacgaggagcccgtccgagaactctacctgcacgaggaacccgtcaccga 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj ct : 1 3 4 5 6 2 ggaggccctggacgaggagcccgtccgagaactctacctgcacgaggaacccgtcaccga Query : 3 0 1 gggcaagcacgccctcctagagctcatctccaacatacgcgcgcgagtggtgcagacgac 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj ct : 1 3 4 6 2 2 gggcaagcacgccctcctagagctcatctccaacatacgcgcgcgagtggtgcagacgac Query : 3 6 1 gagactgatactggacaagatc 3 8 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj ct : 1 3 4 6 8 2 gagactgatactggacaagatc 1 3 4 7 0 3 7 4 9 0 . 0 4 6 0 . 0 2 2 4 6 0 . 0 2 2 4 6 0 . 0 2 2 4 4 0 . 0 8 9 Equine 6 0 1 3 4 3 8 1 1 2 0 1 3 44 4 1 1 8 0 1 3 4 5 0 1 2 4 0 1 3 4 5 6 1 3 0 0 1 3 4 6 2 1 3 6 0 1 3 4 6 8 1 Appendices >DP3 + 2 8 ( 4 5 3 l e t t e r s ) GATCCGTTTT CACTTCCCCC CAGTTCAGCG CGGCCGCAAC TCTAAAGCAT CTCAACAGAA CGCACGCGGG CTTGTACGCG TCCTTGGACA CCATAAAGGG CCCCCGGTCC ACAAACATCT GATACAGCTG AGTTCCTTCG CTTGAAACAA AACTCACCCC GGTCTGCGGG TTCCCCGTGT GGGGAGTGGC AAGTCCCATG TCGGTCAGCA CCAACTTCCC CAAACCGGTT TCGGTCTGAG CCCTCTCCAC CAGAATGTTG CAGGGGCTGA TGTCAGAGTG AAACAGCCCG CACTCCTCGT TCAGAAAGAC CACCGCGTCC AGCAGGCTCT CGAAGCCCGC GACCAGGGGA GGGATGTTCT CGGGACCCCA GTGGGAAAAG TCATTCAGGG AACAGGAATA TCTGGGAAAG AACAAGCTGC TTGCAGGGCA TGCACGCGTC CACAAAAGAG ATC 23 1 Score E S equences produc i ng s ign i f i cant a l i gnment s : ( b i t s ) Va lue gb : EHVU2 0 8 2 4 Equ i ne herpe svirus 2 , comp l e t e genome . >emb : EHU2 0 8 2 . . . 8 5 0 0 . 0 gb : HS 4 5 5 J 7 Human DNA sequence f rom c l one 4 5 5 J 7 on chromosome I q . . . 4 2 0 . 4 2 gb : AQ0 4 1 2 6 2 C I T - HS P - 2 3 3 8L5 . TF C I T - HS P Homo sapiens genom i c c I on . . . 4 0 1 . 7 >gb : EHVU2 0 8 2 4 Equ i ne herpe svirus 2 , comp l e t e genome . >emb : EHU2 0 8 2 4 Equi ne herpesvi rus 2 , comp l e t e genome . Length 1 8 4 4 2 7 Score 8 5 0 b i t s ( 4 2 9 ) , Expect 0 . 0 I dent i t i e s = 4 4 8 / 4 5 3 ( 9 8 % ) , Gaps = 1 / 4 5 3 ( 0 % ) S t rand = P l u s / M inus Query : 1 gatccgt t t t c a c t t c c c c c cagt tcagcgcggccgcaac t c t aaagc a t c tcaacagaa 6 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj ct : 8 4 2 6 2 gatccgt t t tcacttccccccagt tcagcgcggccgcaactct aaagc a t c t c aacagaa 8 4 2 0 3 Query : 6 1 cgcacgcgggct tgtacgcgt cct tggacaccataaagggcccccggt c cacaaacatct 1 2 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S b j c t : 8 4 2 0 2 cgcacgcgggc tt gtacgcgtcct tggacaccataaaggggcc ccggtccacaaa c a t c t 8 4 1 4 3 Query : 1 2 1 gatacagc tgagt t c c t t cgct tgaaacaaaactcaccc cggt ctgcggg t t c c c cgtgt 1 8 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S b j c t : 8 4 1 4 2 gatacagc tgagt t c c t c cgct tgaaacaaaac t cacc c cggt c tgcgggt t ccccgtgt 8 4 0 8 3 Query : 1 8 1 ggggagtggcaagt cccatgt cggt cagcaccaac t t c c ccaaaccggt t t cggtc tgag 2 4 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj c t : 8 4 0 8 2 ggggagtggcaagt cccatgt cggt cagcaccaac t t c c ccaaaccggt t t cggtc tgag 8 4 0 2 3 Query : 2 4 1 c c c t c t c caccagaa tgttgcaggggctgatgt cagagtgaaacagcc cgcact c c t cgt 3 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S b j ct : 8 4 0 2 2 c c c t c t c caccagaatgttgcaggggctgatgt cagagtgaaacagcccgcactcc t cgt 8 3 9 6 3 Query : 3 0 1 t cagaaagaccaccgcgt c cagcaggc t c t c gaagcccgcgaccaggggagggatg t t c t 3 6 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 \ \ \ \ \ \ \ \ \ \ 1 \ \ I \ \ \ \ I \ I 1 1 \ I 1 1 \ 1 1 1 1 S b j c t : 8 3 9 6 2 t c agaaagaccaccgcgtccagcaggc t c t c gaagccagcgaccaggggagggatgt t c t 8 3 9 0 3 Que ry : 3 6 1 cgggaccccagtgggaaaagt c a t t cagggaacaggaatatctgggaaagaacaagctgc 4 2 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj c t : 8 3 9 0 2 cggggccccagtgggaaaagt catt cagggaacaggaatatc tgggaaagaac - agctgc 8 3 8 4 4 Que ry : 4 2 1 t tgcagggcatgcacgcgtccacaaaagagat c 4 5 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S bj c t : 8 3 8 4 3 t t gcagggcatgcacgcgtccacaaaagagat c 8 3 8 1 1 Appendices >DP3 - 6 7 ( 2 6 7 l e t t e r s ) GATCCTTTAT AGCACTTAGC ATGGTCCTGG TTATGTTGTA GATGAGAAAA CAACACTTGA TTTGCTTTTA TTTTTTTTAA TAGAGTCTAC CCATCACCAA CCAAATTAGA GGTGGTAGAG TTAGGCTCTG TTAAGATATA TTTTGCAGGA GCATCAGAGC TAGGAGTCAG AAGGATTGGA TTCTAGTTCT GTTTCCTTCA GTAATTAGCT ACATGACCCT AAACAAATGA CTTACATTCT TGTGTTTGTG TCATGATGTT TCAGATC S equences produc ing s i gni f i cant a l ignment s : ( f i rs t f ive ) gb : HSDJ2 6 1 K5 Human DNA s equence f rom c l one 2 6 1 K5 on chromosome . . . emb : HSDJ2 6 1 K5 Human DNA sequence * * * SEQUENCING I N PROGRESS * * * . . . gb : AI 0 6 4 4 9 5 GH0 4 9 4 1 . 5prime GH Drosoph i l a mel anoga s t e r head pOT2 . . . gb : AI 4 0 4 9 3 7 GH2 4 8 0 5 . 5pr ime GH Drosoph i l a mel anoga s t e r head pOT2 . . . gbhu : AC0 1 3 8 9 6 Drosoph i l a me l anoga s t e r , WORKING DRAFT SEQUENCE , Score ( b i t s ) 4 6 4 6 4 2 4 2 4 2 >gb : HSDJ2 6 1 K5 Human DNA s equence f rom c l one 2 6 1 K5 on chromosome 6 q2 1 - 2 2 . 1 . Cont a ins t he 3 ' part of the gene for a novel organ i c cat i on t ransporter ( BAC ORF RG3 3 1 P 0 3 ) , the DDO gene for D - a spartate oxidase ( EC 1 . 4 . 3 . 1 ) , ESTs , STS s , GSS s and two putat ive CpG i s l ands , comp l e t e sequence . >gbnu : HSDJ2 6 1K5 Human DNA sequence f rom c l one 2 6 1 K 5 on chromosome 6 q2 1 - 2 2 . 1 . Cont ains the 3 ' part of the gene f o r a nove l organic c a t i on t ransporter ( BAC ORF RG3 3 1 P 0 3 ) , the DDO gene for D - aspartate oxidase ( EC 1 . 4 . 3 . 1 ) , ESTs , STS s , GSS s and two putat ive CpG i s l ands , comp l e t e sequenc e . Length 1 3 1 9 7 4 Score = 4 6 . 1 b i t s ( 2 3 ) , Exp e c t I dent i t i e s = 2 6 / 2 7 ( 9 6 % ) S t rand = P l us / Plus 0 . 0 1 5 Query : 5 4 c a c t tgat t t gc t t t t a t t t t t t t taa 8 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj c t : 4 1 1 2 4 c a c t tgatttgc t t t t t t t t t t t t taa 4 1 1 5 0 > emb : HSDJ2 6 1 K5 Human DNA s equence * * * S EQUENCING I N PROGRES S * * * f rom c l one DJ2 6 1 K5 Length = 1 3 1 9 7 4 Score = 4 6 . 1 b i t s ( 2 3 ) , Exp e c t I dent i t i e s 2 6 / 2 7 ( 9 6 % ) S t rand = Pl us / P l u s 0 . 0 1 5 Query : 5 4 cact tgat t tgc t t t ta t t t t t t t taa 8 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sbj c t : 4 1 1 2 4 cact tga t t t gc t t t t t t t t t t t t taa 4 1 1 5 0 232 E Value 0 . 0 1 5 0 . 0 1 5 0 . 2 4 0 . 2 4 0 . 2 4 Appendices 233 APPENDIX E: BUFFERS AND SOLUTIONS ALSEVER'S SOLUTION Tri-sodium Citrate Citric acid NaCI Glucose 8.0 g 0.55 g 4.2 g 20.5 g Make up to l OOO ml with dH20, adjust to pH 6.0 and sterilise by autoclaving at ] 0 Ibs. AMMONIUM ACETATE, pH 5.2 (5 M) CHlCOONH4 Distil led H20 Steri lised by fi ltration through 0.2 !Am filter. 3 .85 g up to 1 0.0 ml ANTIBIOTIC / TRYPSIN / VERSENE. (ATV) Trypsin Difco, 1 :250 Verse ne (EDT A) tetrasodium salt NaCI KCI Dextrose Pen ic i l l in Streptomycin Phenol red 0.5 g 0.2 g 8 .0 g 0.4 g 1 .0 g 0.58 g 2 x 1 05 units 0. ] g 0.02 g Adjuste to pH 7.2, filter through 0.2 !Am fi lter. d ispense i nto 10 ml aliquots and store at -20 0e. BLOCKING SOLUTION (FOR DIG DETECTION) 1 % w/v Blocking reagent ( Roche) d issolved i n Maleic acid buffer, pH 7 .5 . BOUIN'S FIXATIVE Picric acid, saturated aqueous solution 75 ml Formali n (40% formaldehyde) 25 ml Glacial acetic acid 5 ml BUFFER EB (ELUTION BUFFER) 1 0 mM Tris.HCl, pH 8.5 Appendices CITRIC ACID - PHOSPHATE BUFFER ( 0.1 M), pH 5.0 Citric acid . H20 Na2HPO� Distil led H20 7 .30 g 1 1 . 86 g up to 1 000 ml DENA TURA TION SOLUTION (FOR COLONY HYBRIDISATION) 0.5 M NaOH, 1 .5 M NaCI. 0. 1 % SDS DEPC-TREATED H20 234 Add diethy lpyrocarbonate (DEPC) to a final volume 0.0 I % ( v/v) . Stir at 37 QC for an hour. Leave at 37 QC overnight, followed by autoc!aving. DETECTION BUFFER (FOR DIG DETECTION) 1 00 mM Tris-HCI , 1 00 mM NaCI, pH 9.5 EAGLE MINIMUM ESSENTIAL MEDIA + NON-ESSENTIAL AMINOACIDS, WITH L­ GLUTAMINE (MEM+N) MEM + n (Sigma) NaHCO} Distil led H20 Steril ise by filtration through 0.2-ftm filter. 3 X EE BUFFER 30 mM EPPS (Sigma), pH 8.0, 3 mM EDT A ELISA COA TING BUFFER 9.7 g 2.2 g up to 1 000 ml (0.05 M CARBONATE-BICARBONATE BUFFER, PH 9.6) Na2CO} NaHCO, Distil led H20 ELISA BLOCKING BUFFER 1 .59 g 2.93 g up to 1 000 ml 3% bovine serum albumin (Sigma) (w/v) in ELISA Washing Buffer ELISA WASHING BUFFER 0.05% Tween 20 in PBS Appelldices GEL LOADING BUFFER I x TBE 50Q'o glyccrol 0.005% Bromophenol Blue 0.025% Xylene Cyanol GLUTAMAX™- l SUPPLEMENT (GIBCO BRL) 200 mM in 0.85% NaCI GTNE BUFFER, pH 7.5 Glyc ine Tris-Cl NaCI EDTA MALEIC ACID BUFFER, PH 7.5 1 00 mM maleic acid, pH 7.5 1 50 mM NaCI 200 mM 50 mM l OO mM I mM NE'tJTRALISA TION SOLUTION (FOR COLONY AND PLAQUE HYBRIDISATION) 1 .0 M Tris-HCI , pH 7.5 1 .5 M NaCI OPD CHROMOGEN SOLUTION 235 A tablet contai n i ng 5 mg of orthophenylenediamine d ihydrochloride (GPD, Pierce) was dissolved in 1 0 m l Citric Acid Phosphate Buffer (0. 1 M , pH 5 .0) and 3 5 II I H202 added. The solution was prepared immediately before use. PENICILLIN STREPTOMYCIN KANAMYCIN (PSK) Streptomyci n Kanamyci n Penic i l l in 1 .0 g 1 .0 g I X 1 06 units Make up i n 1 00 m] with PBS, filter through 0.2-11111 filter, d ispense i nto 20-ml aliquots and store at -20 °C unti l required. PHOSPHATE BUFFERED SALINE, pH 7.0 (PBS) NaCI KCl Na2HP04 KH2P04 8.0 g 0.2 g 1 . 1 5 g 0.2 g Make up to 1 000 ml with dHzO, adjust to pH 7.0 and steri l ise by autoclaving. Appendices PHOSPHATE BUFFER KH2P04 K2HP04 Distilled H20 PHOSPHOTUNGSTIC ACID STAIN Potassium phosphotungstate Distil led H20 2.3 g 1 2.5 g up to 1 00 ml 2 g up to l OO ml Adjust to pH 7.2 with KOH and filter through 0.2-�Lm filter. PROBE STRIPPING SOLUTION (FOR ALKALI-LABILE dUTP) 0.2 M NaOH, 0. 1 % (w/v) SDS PROTEINASE K STOCK SOLUTION (10 ,.,gI ..... ) Protei nase K Distil led H20 SDS STOCK SOLUUTION 2.6 mg 260.0 !-LI 1 0% SDS i n H20 filtered through 0.2-!-Lm filter. SOC MEDIUM Tryptone (Gibco BRL) Yeast extract (Gibco BRL) 1 M NaCl I M KCI 2 g 0.55 g I ml 1 ml 236 Stir to dissolve, autoclave, and store at RT. Immediately before use, add 2 M MgClz ( 1 ml) and 2 M glucose ( 1 ml), which had been filtered through 0.2 !-Ll filter. 20 X SSC STOCK SOLUTION NaCl Tri-sodium citrate 1 75 .32 g 88.23 g Distil led H20 up to \ 000 ml Check that pH is 7 - 8 . Adjust i f required. Store at RT. 2 X SSC WASH SOLUTION 2 x SSC, 0. 1 % SDS 0.5 X SSC WASH SOLUTION 0.5 x SCc, O. I % SDS Appendices T AE ELECTROPHORESIS BUFFER STOCK SOLUTION (50x) Trizma base EDT A, sodium salt Distil led H20 242 g 1 8.6 g up to 1 .0 l itre 237 Dissolve in approximately 800 ml dH20. Adjust to pH 8 .0 with glacial acetic acid (57 mill ) . Make up to a final volume of 1 000 m1. Store at RT. TB MEDIUM Tryptone (Gibco BRL) Yeast extract (Gibeo BRL) Glycerol 1 2 g 24 g 4 ml Distil led H20 up to 900 ml Stir to dissolve, autoclave and cool to RT. Add l OO ml of Phosphate Buffer and mix well . TBE ELECTROPHORESIS BUFFER: STOCK SOLUTION (lOx) Trizma base Boric acid EDTA, sodium salt Disti l led H20 TE BUFFER (10 mM) Trizma base 1 .2 1 g 1 08 g 55 g 9.3g up to 1 000 ml EDT A 0.372 g EDT A, sodium salt Add approximately 800 ml of dH20. Adjust to pH 8 .0 with concentrated HCI. Make up to a final volume of 1 000 ml . Store at RT. TISSUE CULTURE MATERIALS Nunclon® Nunclon® Falcon® Nunclon® 25-cm.1, 75-cm} and I SO-em} polystyrene tissue culture flasks 96-well and 6-well multiplates 24-wel l , /lat bottomed multi plates 8-well chamber sl ides. TNE BUFFER, pH 8.0 Tris.HCL 1 0 mM l OO mM I mM NaCI EDTA Appendices WASHING BUFFER (FOR DIG DETECTION) 1 00 mM maleic acid, pH 7.5 1 50 mM NaCI 0.3(}t v/v Tween-20 VERONAL-BUFFERED SALINE (Sx) NaCl 85.0 g Barbital 5 .75 g Sodium barbital 3.75 g MgCI2 .6H2O 1 .0 1 5 g CaCI2·2H1O 0.22 g Distilled H2O up to 2000 ml 238 Bibliography Anon.a "BCM Search Launcher: Sequence Uti l i ties. " Web page. 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The hlue 'race l ine' i n the tille of each chapter has heen copied from a weh site located at: http://horseracing.miningco.com/sports/horserac ing/lihrary/h1clip.htm#clip Erratum Page number / line number 85 / 10 88 / 1 7 97 / 27 124 / 2 128 / 9 13 1 / 29 1 40 / 25 173 / 6 1 86 / 4, 29 188 / 14,24 1 89 / 4 1 89 / 22 197 / 8 199 / 25 204 / 17 206 / 4 250 263 / 3 1 267 Alterations to text in bold font 13 isolates (Gleeson and Studdert, 1977) washed as above, incubated for 30 minutes with 0.1 mlof Streptavidin Horse Radish Peroxidase (pearce) diluted 1:500 in washing butTer, washed again Of35 yearlings tested, 30 (85.7%) had three of the remaining nine horses as opposed to 76.7% Approximately 61% of all the horses lOO �g/ml (Frazer et al. 1997) not (Shi et al. 1997) (Fodor. 1997) not (Varga et al. 1997) (foal F4 in table 3.3) as described in chapter 3. 100 I'l:iml Approximately 5 J.lg (Tekstra et al. 1999) not (Van Der Voom et al. 1999) (Frazer et al. 1997) not (Sbi et al. 1997) Additional reference: Fodor SPA. 1997. Massively parallel gcoomics. Science 277: 393-395. Price DA, Klencrman P, Booth BL, Phillips RE, & Sewell AK. 1 999. Cytotoxic T Iymphocytes, chemokines and antiviral immunity. lmmullology Today 20 (5): 212-216. Additional reference: Tek.<;tra J, Beekhuizen H, Van De Gevel JS, Van Benten JJ, Tuk CW, & Heelen RH. 1999. Infection of human endothelial cells with staphylococcus aureus induces the production of monocyte chemotactic protein-I (Mep-I) and monocyte chemotaxis. Clillical and E.'Cperimentallmmullology 117 (3): 489-495.