Molecular Detection And Genomic Characteristics Of Group A Rotavirus In Yak

Bovine group A Rotavirus?BRVA?is an important diarrhea-causing pathogen in calf,which brings huge economic losses to the cattle industry worldwide.Moreover,the public health significance of BRVA cross-species transmission is receiving more and more attention.The previous investigation of our laboratory had shown that the infection of BRVA is an important cause of diarrhea in yak in the Qinghai-Tibet Plateau.However,the molecular and genomic characteristics of BRVA in yak are still large unknown,which hamper the prevention and control of BRVA in yak.The purpose of this study was to investigate the prevalent genotype and genomic characteristics of BRVA in yak.The results were as follows:1.Detection of group A rotavirus in yak in parts of Qinghai-Tibet Plateau from2017 to 2018In order to understand the current prevalence of BRVA in yak,a total of 219 fecal samples from yak calves??3 months?with diarrhea were collected from 29 farms in northwest Sichuan and Tibet during June 2017 to July 2018.Specific RT-PCR assay was used for detecting BRVA from these samples,and positive samples were further used to isolate this virus.The results showed that the detection rate of group A rotavirus in yak in northwest Sichuan was 61.62%?114/185?,and the detection rate in Tibet was 52.94%?18/34?,which showed that BRVA infection is still an important cause of yak diarrhea in these regions currently.Among them,the co-infection of BRVA and BCoV was 70.45%?93/132?,the co-infection of BRVA and BVDV was18.94%?25/132?.The result provide a reference for the diagnosis and prevention of yak diarrhea.2.Molecular characteristics of VP4 and VP7 genes of BRVA in yakThe VP4 and VP7 proteins are rotavirus outer capsid proteins which induce the production of neutralizing antibodies.The P-type was determined by VP4 and the G-type was determined by VP7,respectively.In order to investigate the molecular characteristics of VP4 and VP7 genes of BRVA in yak,86 BRVA positive samples from Sichuan,Tibet,Qinghai and Yunnan were used to inoculate into MA-104 cells and undergone 3 generations blind passage.The positive samples of MA-104 cells detected by RT-PCR were used to amplify the VP4 and VP7 sequences.RotaC was used to identify G and P genotype.The results showed that 65 MA-104 cells samples were detected as BRVA positive,among which 34 VP4 sequences with 664-667 bp in length were obtained.32/34 of VP4 sequences were identified as P[1]type,the remain 2 VP4 sequences were identified as P[11]type.26 VP7 sequences with 885bp in length were obtained,and 23/26 VP7 sequences were identified as G6 type,the remain 3 were identified as G10 type.Among the 17 samples simultaneously amplified the VP4 and VP7 genes,G6P[1]type was 16/17,G6P[11]type was 1/17,indicting that G6P[1]type was the prevalent genotype in yak.The phylogenetic analysis indicated that the VP7 genes of the G6 strains and the VP4 genes of the P[1]strains from four different provinces were all clustered into an independently branch respectively,no geographical difference was found in phylogenetic trees.The results showed that BRVA in yak undergo a unique evolutionary,contributing to the prevention and control of BRVA in yak.3.Isolation and Genomic characteristics of BRVA from yakGenome of rotavirus contains 11 segments,and gene reassortment is a common phenomenon in rotavirus genetic evolution.In order to better understand the genetic diversity of rotavirus,whole genome based genotyping system has been established.Currently,there is sole BRVA genome in GenBank from China,and there is no genome information of BRVA from yak.In order to better know the molecular characteristics and genetic evolution of BRVA in yak,Two strains which could cause cytopathic effect were isolated,and they were confirmed to be BRVA by RT-PCR,indirect immunofluorescence and electron microscopy,named HY-1,and QH-1respectively.After plaque purification,the virus titers were 10^8.39 TCID₅₀/ml?HY-1?and 10^5.84 TCID₅₀/ml?QH-1?,respectively,which laid a foundation for the biological characteristics and vaccine research of BRVA in yak.Primers were designed to the amplify the genome sequences of HY-1 and QH-1 strains.The full-length of HY-1strain is 18474 bp,the whole genome genotype constellation was obtained as G6-P[11]-I2-R2-C2-M2-A3-N2-T6-E1-H1.The full-length of QH-1 strain is 18492bp,the whole genome genotype constellation was obtained as G6-P[1]-I2-R2-C2-M2-A3-N3-T6-E2-H3.Sequence alignment and phylogenetic tree indicated that among the11 genes of HY-1 strain,VP1 and VP3 genes were assumed to be of sheep origin,VP2,VP4,NSP1 and NSP3 genes were assumed to be of bovine origin,VP6 and VP7 genes were assumed to be of ruminant origin,NSP2,NSP4 and NSP5 genes were assumed to be of human origin.Among the 11 genes of QH-1 strain,VP1 and VP3genes were assumed to be of sheep origin,VP2,VP4,NSP1,NSP3 and NSP5 genes were assumed to be of bovine origin,VP6 and VP7 genes were assumed to be of ruminant origin,NSP2 and NSP4 genes were assumed to be of human origin.The result showed that the two genomes of HY-1 and QH-1 strains experienced a complex genes reassortment in the course of evolution,and both of them have 2-3 segments were assumed to be of human origin.Amino acid analysis showed that,compared with Chinese same P genotype strains,there were 7 and 20 amino acid changes in the VP8 region in VP4 gene of HY-1 and QH-1 strains,respectively.Moreover,4 amino acid changes in QH-1 strain occurred in 8-1and 8-3 regions.Compared with Chinese same G genotype strains,9 and 8 amino acid changes occurred in the 7-1 and 7-2regions of the VP7 gene of HY-1 and QH-1 strains,respectively.These important amino acid changes of VP8 and VP7 may affect on the pathogenicity and immunity of the strains,which need to be further experimental verified.This is the first report about the genomes of BRVA strains in yak,which provided an important reference for understanding its molecular characteristics and genetic evolution.At the same time,as the two genomes of HY-1 and QH-1 strains showed a complex reassortment,it was suggested that the strains may have zoonotic potential which is important for public health.