Detection And Genomic Characteristics Of Bovine Torovirus In Yak

Bovine torovirus(BToV)is a single-stranded positive-strand RNA virus,and the virus belongs to genus Torovirus in family Tobaniviridae.BToV is an important pathogen of bovine diarrhea,which can cause bovine diarrhea at all ages and great economic losses to the world cattle industry.BToV also has respiratory tissue tropism,which may also be a respiratory pathogenic factor.Yak is the most important living material for herdsmen in Qinghai-Tibet Plateau.Diarrhea is common in newborn yak,resulting in major economic losses in the yak industry.BToV has been detected in domestic dairy cows in the early stage of our laboratory,but the molecular prevalence of yak BToV has not been reported.The purpose of this study was to develop an insulated isothermal RT-PCR(RT-iiPCR)assay to investigate the presence of BToV in yak and to analyze the characteristics of BToV genome.1.The RT-iiPCR for detecting BToV was successfully established,and the existence of BToV in yak was confirmedIn the early stage of this study,the nested RT-PCR method,which is the most cited abroad,was used to re-examine the stool samples of yak diarrhea that were detected as BToV positive by macro virus group technology.The detection rate of BToV was low,and the analysis found that the detection target sequence had changed.Thus,it is necessary to re-establish the RT-PCR method for detecting BToV.The 13complete M gene sequences in GenBank and 2 yak-derived BToV M gene fragments(obtained by nested RT-PCR method)were selected for comparison and analysis,and Beacon Designer 8 was used to design primers and probes.By optimizing the reaction conditions and system,a RT-iiPCR method for detecting BToV was established.This assay has good specificity and stability.It only specially amplifies the target fragment of BToV and does not amplify BVDV,BRV,BEV,BCoV,BAstV,Escherichia coli K99,etc.The sensitivity of this assay was high with a minimum detection limit of23.2 copies/?L^-1.The detection ration for clinical samples was same as that of a real-time quantitative PCR reported abroad and superior to the nested RT-PCR assay.Among 180 yak diarrheic fecal samples collected from 22 farms in 3 provinces of Qinghai-Tibet Plateau from May 2018 to September 2020,7.8%(14/180)samples tested as BToV-positive using this assay.Among them,the positive rate in Tibet,Qinghai,and Sichuan was 11.8%(4/34),8.9%(4/45)and 5.9%(6/101)respectively and the farm positive rate was 31.8%(7/22).In this study,we developed a new RT-iiPCR assay,providing a reliable tool for the rapid detection of BToV.This research firstly revealed that BToV could infect yak.Although the positivity rate of BToV is low,the results indicated that BToV may already have a wide geographical distribution.The results of this study provide a reference for the diagnosis and control of diarrhea in yak.2.Successfully obtained the first BToV genome in yakTo further study the characteristics of the BToV genome in yak,27 pairs of primers were designed according to the all 5 complete BToV genomic sequences in GenBank were used to amplify the full genome sequences of the BToV genome in positive fecal samples.Sequences were assembled using Seq Man software.A complete BToV genomic sequence was obtained from a Tibetan calf yak fecal,which was 28314 nucleotides in length with a GC content of 36.31%,named BToV yak-XZ01,belonged to genotype?strain.BToV yak-XZ01 shared 82%?97%nt sequence identity with the 5 BToV genomic sequences in the GenBank database.It shared the highest sequence identity with the native BToV genome SC2(97%).A phylogenetic tree was constructed based on the genome,ORF1a/1b,S,HE gene sequences showed that the BToV yak-XZ01 was most closely related to the native BToV,indicating that yak BToV may have been transmitted from native BToV,but it also has a certain genetic distance.Compared with all 5 complete BToV pp1ab protein sequences in the GenBank database,the pp1ab protein of the BToV yak-XZ01 had 21unique amino acid variations,mainly concentrated in the Papain-like proteinase domain(9/21)and Rd Rp polymerase domain(5/21).Papain-like proteinase and Rd Rp polymerase play a key role in viral replication,and these mutations may affect viral replication.Compared with all complete BToV HE gene sequences in the GenBank database,the HE gene of the BToV yak-XZ01 shared a unique identical aa variant(P44S)in the esterase domain.In addition,an identical recombination event was identified in the S1 subunit of the S protein.Both the S1 subunit and the HE protein esterase domain are involved in the process of virus binding to the receptor,and these mutations may affect the receptor-binding function of the virus.In this study,a genotype?BToV genome was obtained,and a recombinant event was predicted in its S gene.The results of this study enriched the genome data of BToV,which will be helpful for better understanding of BToV's molecular characteristics and genetic evolution.