Isolation And Identification Of CPV Strains From Tibetan Mastiff And Nucleotide Sequence Analysis Of Virus’ VP2Genes

To identify the gene type of canine parvovirus(CPV) in Tibetan mastiff, seven fecal sample were collected by cotton swab from Tibetan Mastiff whose CPV test results were positive according to the Immune Colloidal Gold kit. Six strains were isolated by cultivation in F81cells. These strains were identified by Hemagglutination(HA) test,50%tissue-culture infecttive dose(TCIDso), physicochemical assay. The results showed that the virus can be cultivated in feline kidney81(F81) cells. And the typical cytopathogenic effect(CPE) was spotted in F81cells from6to13days after infection, presented with cytomixis, shapes change. The isolates were able to agglutinate the porcine erythrocytes effectively, the HA titer were27to211,also were able to agglutinate the feline erythrocytes, the HA titer were21to24, but can not agglutinate the erythrocytes of cow, goat, mouse, rabbik. These strains were all anti-acid(PH3), anti-heat(60℃), and insensitive to200mL/L aether and480mL/L chloroform, and was inhibited by5-IDUR. The isolates’VP2complete gene and a825bp fragment on it were amplified by PCR with Primer3,4and Primer1,2. Combine the isolates’VP2genes with pMD19-T in order to sequence the nucleotide residues in VP2. The isolates’VP2contain1775bp nucleotide residues and code for584amino acids, same as other CPV VP2gene on GenBank. Compare the isolates with CPV-5.us.79(GenBank accession number:EU659116.1), the isolates’mutations are on the base of CPV-2a, parts of altered nucleotide residues belong to samesense mutation, and some altered amino acid residues were very close to some key point on the CPV surface, but the biological significances were not clear. compared with ten reference strains’(HQ883267.1、GQ865519.1, FJ222823.1, FJ005214.1et al), home and abroad, from GenBank, the most similar one was HQ883267.1, the homology was99.0%to99.6%. Put the six isolates and the reference strains, from GenBank, home and abroad, together to establish the phylogenetic tree. According to the tree, the isolates had a pretty close relationships with reference strains from home and had a far relationships with reference strain from abroad, got a close relationships with CPV strains but a far relaionships with FPV or MEV. Six isolates and CPV reference strains had an common ancestor. All isolates were proved to be CPV-2a.In this research, a new CPV isolation method based on collecting samples by cotton swab had been established. This was the first time to investigate the epidemiology and variation of CPV in Tibetan mastiff, and could afford us some extraordinary helpful experiment status in improving the effectiveness of prophylaxis for CPV.