| Yak diarrhea is a gastrointestinal disease caused by a variety of factors,and the main clinical features are elevated body temperature,mucosal erosion and shedding,and weight loss.Common viruses that can cause diarrhea in yaks include: bovine viral diarrhea virus(BVDV),bovine coronavirus(BCoV)and bovine rotavirus(BRV).The clinical symptoms of yaks caused by the three pathogens are similar,and mixed infection is likely to occur,which on the one hand causes greater economic losses in the yak breeding industry in China,and on the other hand also brings great difficulties to clinical control.The aim of this study was to understand the viral pathogens of diarrhea in yaks in Ganzi County,Ganzi Tibetan Autonomous Prefecture,Sichuan Province,and to perform molecular detection of BVDV,BCoV and BRV and related genome analysis in fecal samples collected from clinically healthy yaks,and the following results were obtained:1.From March 2021 to September 2021,a total of 185 yak fecal samples were collected from three yak pastures in Ganzi County,Ganzi Tibetan Autonomous Prefecture,Sichuan Province.BVDV,BCoV and BRV were detected by RT-PCR,respectively.The results showed that the detection rate of BVDV was 41.62%(77/185);the detection rate of BCoV was 7.03%(13/185);and the detection rate of BRV was 0%(0/185).The co-infection rate of BVDV and BCoV was 7.03%(13/185).2.According to the published reference sequences of BVDV Npro gene and BCoV S gene in Gen Bank,gene amplification primers were designed by software Primer 5.0 software to amplify the BVDV Npro gene and BCoV S gene of positive samples,respectively.A total of 12 BVDV Npro gene and 4 BCoV S complete gene sequences as well as 9 BCoV S1 gene sequences were obtained.Nucleotide and amino acid homology analysis was performed by Meg Align between the Npro gene sequences of 12 BVDV strains and the sequences of 20 reference strains.The results showed that the nucleotide and amino acid identities of Npro gene of BVDV strains obtained in this study were 97.4% ～ 99.7% and 95.7% ～100%,respectively,with those of the reference strains.The homology with strain SWU-Z6 was the highest,with nucleotide homology of 97.4% ～ 99.7% and amino acid homology of 96.9% ～ 100%;12 BVDV epidemic strains obtained by the neighboring method were selected by MEGA X software to construct a phylogenetic tree based on the Npro gene with the reference strain.The results showed that the 12 BVDV strains obtained in this study had a close genetic relationship with the Chinese strain SWU-Z6,all of which belonged to the BVDV-1a subtype.The S1 fragment of S gene of 9 BCoV strains was analyzed by Meg Align.The results showed that the nucleotide identities were 96.4% ～ 98.5% and the amino acids were 94.8% ～ 98.2%.The phylogenetic tree was constructed by MEGA X software,and it was found that the S1 gene of BCoV in yaks in Ganzi County was closely related to the Canadian strain Quebec and the American strain 4-17-08,but distributed on different branches with the Chinese strains BCV-AKS-01 and HLJ-1-CHN.The above results indicated that there was a possibility of different genetic evolution of the S1 gene of BCoV in China.The phylogenetic tree was constructed by MEGA X software using the whole S gene of 4 strains of BCoV and 22 reference strains.It was found that the S gene of 4 strains of BCoV obtained in this experiment was similar to that of the Chinese strain BCV-AKS-01.In summary,BVDV and BCoV were detected in the healthy yak population in Ganzi County,and BVDV type 1a was predominant in healthy yaks.The results of this study will help to gain insight into the genetic variation of BVDV and BCoV in yak sources in Ganzi County and provide a reference basis for the control of yak diarrhea in this area. |
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