Studies On Some Biological Characteristics And P32 Gene Encoding For The Main Structural Protein Of Goat Pox Virus

Goat pox, which is listed in Group A diseases of the OIE and in Group one diseases of China, is a highly contagious viral disease of goats, characterized by fever, ocular and nasal discharges. Pox lesions appear on the skin, the respiratory and gastro-intestinal mucosae. The disease is caused mainly by goat pox virus (GPV) which is one of pox viruses, classified in the genus Capripoxvirus of the family Poxviridae. Since October 2002, the disease firstly outbroke in Guizhou province and spreaded quickly. The disease inflicts the production of goat cultivation in Guizhou province. We have isolated and verified the pathogen of the doubtful goat pox disease in Guizhou province. Then we studied the major structural protein gene P32 of GPV and the pathomorphology of clinical goat pox disease cases and artificial infection cases were observed. The mainly study content are followed.The Vero-E6, BHK-21 cells were infected by GPV-LD and GPV-QL strains , then they showed some cytopathic effects after 3⁷ days, such as getting to round, gathering to clustering. The fluorescent antibody test showed some specific flavovirens fluorescence in the cytoplasm. There were lots of mature and immature particles in the cytoplasm of infected cells under the transmission electron microscope(TEM). The genus special fragments P32 gene(969bp) and ITR gene(289) were amplified from the DNA samples which were extracted from the infected cell cultures by PCR, the results of sequencing showed the fragments were the special fragments in the GPV genome. The 2000TCID₅₀ GPV-LD strain infected cell cultures were used to infect the 3 months old healthy goats through subcutaneous inoculation. The infected goats showed the same clinical symptom and pathological changes as the clinical cases of goat pox and the goat pox viruses were retrieved from the infected goats.Two specific primers were designed according to the P32 gene and ITR gene sequences of goat poxvirus, the sensitivity and specificity were compared with each other. The results showed the primers have the specificity of Capripoxvirus and do not have consensual reaction with the Orf virus of parapoxvirus, Fowl pox virus of Avipoxvirus and the skin samples of healthy goats. The homologies of nucleotide were above 99.5% and 100% between P32 gene or ITR gene sequences of goat pox viruses. The minimum DNA detection amount of P32 gene primers and ITR gene primers were 19.06ng and 24.40pg. On the whole, the ITR primers are more fit used to diagnose the clinical goat pox disease, and the P32 primers are more fit used to research the virus membrane protein.According to the goat pox virus complete gene sequence, a size of 64 bp gene fragment which locates in gp064 region of goat pox virus (GPV) genome was selected and a pair of primers and a TaqMan-MGB probe against the gene fragment were designed with Primer Express 2.0 software. Then, the fluorescence quantitative PCR (FQ-PCR) assay was developed for quantitative detection of goat pox cases. The FQ-PCR can detect the goat pox virus from the nose swabs, blood samples, skin samples, lung samples, stomach samples, et al., which were selected from GPV infected goats, while the common PCR can detect the GPV only from the skin samples and mucous membrane samples with exanthema variolosum. The minimum DNA detection amount of the FQ-PCR is 0.1 TCID₅₀ according to the standard curve of this test. And the FQ-PCR can detect the exist of GPV before 3～5 days of the skin exanthema variolosum appearance. So the FQ-PCR is a useful technology for the technology of goat pox.The pathomorphology of clinical goat pox cases and artificial infection cases in Guizhou province were observed. The major pathological changes were pox lesions on the skin, respiratory tract and gastro-intestinal tract. And there were hyperplasia and degeneration in epithelial cell and inflammatory cell in diseased regions, such as macrophages, leukomonocyte, leucocyte neutrophils, et al. The cytopathic effects such as cytochondriome tumentia, endocytoplasmic reticulum expansion and Golgi's complex expansion were also observed under the TEM. A lots of mature and immature particles in the cytoplasm of affected cells were observed under TEM, including initial stage particles, intermediate stage particles and mature particles. The phenomena that GPV could damage the vascular endothelial cells and medullated nerve fibers were finded under TEM. The test results remain that mature particles got the peplos through the cell organ membrane perhaps was one of the way for particles getting the peplos.The P32 genes of GPV-QL, GPV-LD, GPV-Y and GPV-B were inserted into the pMD18-T vector. The sequence analysis of recombinant plasmids showed that the P32 gene of GPV strains isolated from Guizhou shared 99.9% nucleotides with each other, 99.5%～100% with the GPV strains of Chinese and 99.5%-99.6% with the GPV strains of foreign country. Amino acids homology is similar to the nucleotide homology. The Phylogenetic analysis showed the relationship of Guizhou strains were nearer with Chinese other strains than that of foreign strains. The research indicated the P32 gene of capripoxvirus is very conservative and the heredity of capripoxvirus has some relation with the geographic setting. The bioinformatics analysis showed that P32 protein was a kind of membrane protein which had a larvaceous dominant epitope in the 227-251 segment of the amino acid sequence.The different P32 gene fragments were amplified by PCR method from pMD18-T-P32/LD recombinant plasmids. The different P32 gene fragments were cloned into prokaryotic expression vector and eukaryotic expression vector. The proteins were induced to express in their host bacterium and their antigenicity were researched. Then the ELISA for detecting the serum antibody of goat pox were established against the pure P32 protein. The results showed the P32/N_1/3 and P32/M_1/3 gene fragments did not express in both eukaryotic expression system and prokaryotic expression system, while the complete P32 gene expressed well in both eukaryotic expression system and prokaryotic expression system. The results of SDS-PAGE and Western-Blotting showed a specific protein band about 35.5KD in eukaryotic expression products and a specific protein band about 64KD in prokaryotic expression products were detected. The expression protein can be purified by Ni⁺ affinity chromatograph and G-100 chromotography techniques. The agar diffusion reaction and animal experiment identified that the pure P32 protein had good antigenicity. The clinical experiment verified the indirect ELISA assay which made use of the pure P32 protein was useful for detecting the goat pox serum antibody.For detecting the BHK-21 cell apoptosis caused by goat pox virus, the H.E staining method, apoptosis detection kit, flow cytometry and DNA Ladder test were used to detect the BHK-21 cell cultures which infected by the goat pox virus. The results showed that the goat pox virus could cause the BHK-21 cell apoptosis. The research consequences provide some evidences to explain some pathological changes of goat pox.