Detection of Gammaherpesvirus Signal in Dogs and Recombinant Protein Expression

• Main Authors: Chen Hsiang Huang, 黄貞翔
• Other Authors: 邱繡河
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/51759555618834945847

碩士 === 國立中興大學 === 獸醫微生物學研究所 === 96 === Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is associated with Kaposi’s sarcoma (KS), multicentric Castleman’s disease, and primary effusion lymphoma (PEL). Previously, our preliminary polymerase chain reaction (PCR) data showed that KSHV DNA may exist in dogs. To confirm this discovery, we designed nested PCR primers specific for the gene encoding KSHV DNA polymerase and performed an extensive investigation. Among 173 dog genomic DNA samples that had been examined by nested PCR, 2 (1.15%) were positive for KSHV DNA sequence. After culturing the bone marrow and peripheral leukocytes of both positive canines, we observed small clumps of cells, which revealed the possibility of cell immortalization. Notably, we also detected the gene signal of KSHV DNA polymerase in the cultured cells by nested PCR. However, by ISH and RT-PCR, we did not detect any expression nor existence of KSHV latent and lytic genes. On the other hand, six KSHV gene fragments, which encode the N-terminal and C-terminal regions of ORF65, ORF73 and K8.1A, were cloned from KSHV infected cell line BC-2. Prokaryotic expression plasmids were constructed to express respective recombinant proteins. Using purified recombinant N-terminal and C-terminal ORF73 (ORF73-N and ORF73-C) proteins as detecting antigens, we screened dog serum samples to investigate any exposure to the virus. The results showed that 17.5% (7 out of 40) of dog sera reacted strongly with the antigens. Apart from the serological assay, we further immunized mice with recombinant ORF73-N and ORF73-C proteins to develop polyclonal antibody against ORF73 protein, which could be used as a reagent for the detection of latent KSHV infection. In conclusion, we report for the first time the detection of KSHV DNA sequence in leukocyte DNA as well as in cultured leukocytes of dogs. Additionally, based on our serological data, some dogs may have encountered or were infected with KSHV-related virus.