Development and evaluation of molecular tests for investigation of tissue tropism of adenovirus in tissues of brushtail possum (Trichosurus vulpecula) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies at Massey University
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Date
2006
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Massey University
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Abstract
(Trichosurus vulpecula) is considered to be a vertebrate pest, which causes severe damage to the native ecosystem. Biological control, such as immunocontraception, is considered to be the only foreseeable long-term solution to the control of the possum population. Possum adenovirus has been investigated as a possible candidate for use in the biological control of this pest in New Zealand. Attempts to isolate the virus by cell culture have been unsuccessful and could be due to inappropriate culture systems. The localization of adenovirus in possums may be indicative of the tissue tropism of the virus and be helpful in finding the appropriate tissue samples for cell culture. However, this information is not available to date. In this study, the main aim was to establish a sensitive detection method to detect the presence of adenovirus in possum tissues and allow an investigation of tissue tropism. Direct and indirect in situ polymerase chain reaction (in situ PCR) and in situ hybridization (ISH) were established using canine adenovirus type 1 (CAdV-1) as a parallel model for optimizing the experimental conditions. The result showed that both in situ PCR and ISH detection systems were able to detect canine adenoviruses in cultured MDCK cells at a low level of infection. In situ PCR methods were able to detect CAdV-1 in MDCK cells at 8 hours after infection with strong staining in the nuclei, while ISH was able to detect CAdV-1 at 14 hours after infection. The same approaches were applied to detect possum adenovirus in formalin-fixed, paraffin-embedded sections of possum intestinal tissues using a probe from possum adenovirus hexon gene. However, no possum adenovirus was detected in these tissues. This result indicates that further investigation using the same approach should be applied to other possum tissues. In order to investigate the presence of antibody to possum adenovirus, an agar gel immunodiffusion (AGID) test was established using canine adenovirus type 1 (CAdV-1) as antigen. Possum sera from various regions of New Zealand were obtained from the possum serum bank of this laboratory. Of the 268 sera tested, none of them were antibody positive to CAV-1. This result could be due to the insensitivity of AGID test per se and the absence of the shared cross-reaction antigen between these two viruses, suggesting that further investigation using viruses from the group of atadenovirus as antigen is needed.
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Adenovirus diseases, Trichosurus vulpecula, Diseases