Isolation And Identification Of Bovine Rotavirus In Shaanxi Province And Analysis Of Genetic Variation Of VP7 Gene

Bovine rotavirus, which also called calf diarrhea virus, and belonged to Reoviridae family, Rotavirus genus, double strands RNA, containing eleven segments. Less than one month calves usually infect rotavirus. The clinical symptom are depressed, lose appetite, diarrhea, severe dehydration and acid poisoning. About half of calves'diarrhea is related to rotavirus infection.VP7 is a kind of glucoprotein N linked oligomerization carubinose, which is coded by the 7th, 8th or 9th segment, is the main protective antigen, composes outer capsid and determines G serological type. VP7 is also a kind of Ca2+-link protein, which is related to formation and stabilization. VP7 recombined protein and VP7 synthesis peptides (275~295 aa) can produce neutralization antibody protecting animals from RV infection. A strain of bovine rotavirus was isolated from Shaanxi province, and its structure gene VP7 was cloned and genetic variation was analysed. The results as follows:1. According to different serological types of RV VP7 gene, three pair of primers were designed, RT-PCR was used to amplify calf diarrhea feces which these calves maybe infect BRV. 500 bp was gained which confirmed calf diarrhea was produced by G6 serological type of bovine rotavirus.2. Bovine rotavirus was isolated by using MA104 cells from diarrhea feces which were identified by PCR, CPE appeared when diarrhea feces was blindly passaged for four generations, CPE showed that cells fell off, appeared net-balloon and parts of cells died. TCID50 of virus was 105.20/20μL when virus was passaged to eight generation. PAGE assay was used to determine the distribution of nucleic acid. The distribution appeared 4∶2∶3∶2, which belonged to group A rotavirus.VP7 gene of strain isolated was amplified and 1062 bp was obtained. It is confirmed bovine rotavirus was isolated from diarrhea feces.3. DNAStar software was used to analyze the VP7 gene homology between the RV strain isolated and the NCDV reference strain, WC3 strain, CHLY strain, which were received in GenBank. It is showeded that the homology of nucleotide is 99.1% compared with NCDV reference strain, is 95.6% compared with WC3 strain, and is 97.7% compared with CHLY strain. It is also showeded that the homology is fairly low compared with other serological types. VP7 gene variation of RV strain isolated appeared that basic groups were replaced at 58, 97, 302, 458, 486, 699,742,882 and930.. These results indicated that nucleotide diversity is low in the same serological types, but high in different serological types.A strain of bovine rotavirus was isolated from Shaanxi province, and diagnosis method of BRV was established. Genetic variation of VP7 gene of Shaanxi strain was analysed, which provided new information for BRV molecular epidemiology in China.