Molecular Detection And Genomic Characterization Of Bovine Coronavirus In Yak

Bovine coronavirus?BCoV?is a single-stranded positive-strand RNA virus,and the virus belongs to group2a of genus Betacoronavirus in family Coronaviridae.BCoV can cause diarrhea in neonatal calves,winter dysentery in adult cattle,and respiratory tract illness in cattle of all ages,leading to serious economic losses to the global cattle industry.Yak plays an important role for the local people in production and living on the Qinghai-Tibet Plateau.The sero-epidemiological investigation showed high infection rates of BCoV in yak,but the molecular prevalence of yak BCoV has not been reported.The aim of this study was to investigate the prevalence and molecular characteristics of BCoV from yak in the Qinghai-Tibet Plateau,and the results were as follows:1.The RT-PCR assay for detecting bovine coronavirus was successfully establishedAs one BCoV positive sample identified by virus Metagenomics was not been detected by RT-PCR assays previous reported,which was suspected that the reasons are sequence mutation or low sensitivity of the assays.Thus,it was a need to establish a RT-PCR assay for detecting BCoV.The BCoV polymerase gene Nsp11conserved region was selected to design primers sequence based on analysis of all23 BCoV genome sequences in the GenBank database.A RT-PCR assay for detecting BCoV was successfully established by optimizing the reaction conditions and reaction system.The method has good specificity and stability,and high sensitivity with detection limit is 3.2×10³ copies·?L^-1.To evaluate the effect of this assay in clinically fecal samples,the detection rate of this assay in clinically fecal samples were compared to that of other two RT-PCR assays.The RT-PCR assay established in this study showed that the detection rate of BCoV was significantly higher than that of the other two RT-PCR assays,providing a more accurate test for the diagnosis and epidemiological investigation of BCoV.2.Molecular prevalence and phylogenetic analysis of BCoV from yak in four Tibetan regionsIn order to investigate the prevalence and molecular characteristics of yak BCoV,336 diarrhea samples of yak from 29 herds were collected from the Qinghai-Tibet Plateau from May 2015 to August 2018,including 60 samples in Tibet,60 samples in Qinghai,156 samples in Sichuan,and 60 samlpes in Yunnan,and 98 yak nasal samples from yak with respiratory diseases were collected in Sichuan.All samples were detected for BCoV by RT-PCR assay established in this study.Representative samples were selected to clone S,HE and N genes for understanding the molecular characteristics of BCoV in yak.The results showed that the detection rate of BCoV was 69.05%,and the farm positive rate was 100%.The positive rate of BCoV in 98yak nasal scorpions with respiratory diseases was 72.45%.It indicated that BCoV was circulated widely in yak in the Qinghai-Tibet Plateau.S,HE,and N genes fragments from 40 diarrhea samples from four regions were successfully cloned.Phylogenetic analysis based on S,HE and N genes fragments showed that most of the BCoV strains from yak have a unique evolutionary trend.12 complete S gene from diarrhea samples from four regions were successfully cloned,and they shared96.9%¹00.0%nt similarity and 95.5%¹00.0%aa similarity between each other,shared 96.9%⁹8.8%nt similarity and 95.1%⁹8.8%aa similarity with other BCoV strains available in the GenBank database.Phylogenetic analysis showed that the 12complete S gens cloned in this study had a unique genetic evolution characterization.This is the first molecular prevalence of BCoV in yak.The results showed that BCoV is highly prevalent in yak and has unique evolutionary characteristics,which contribute to prevent and control of yak diarrhea.3.A BCoV strain from yak was successfully isolated and its complete genome was amplifiedBCoV-positive diarrhea fecal samples of yak were used to isolate in HRT-18 cell line,and a BCoV strain,resulting in cytopathic effect?CPE?in HRT-18 cell,was successfully isolated,named YAK/HY24/CH/2017.The virus was identified by RT-PCR assay,indirect immunofluorescence and electron microscopic.The virus titer of the strain after plaque purification was 10^8.17TCID₅₀/ml.43 pairs of primers were designed using Primer 6.0 software,and the complete genome sequences of the strain was successfully amplified.The genome of the strain was 31032 bp in length.Phylogenetic tree based on the complete sequence showed that the YAK/HY24/CH/2017 strain was closest to the US strain U00735.2.Compared with all BCoV S gene sequences in GenBank,the S gene of strain has 26 unique amino acid variations?17 unique amino acid variations in the S1 subunit and 9 unique amino acid variations in the S2 subunit?,and the recombination event was predicted in the S1B receptor binding domain produced by the antibody.The YAK/HY24/CH/2017 strain HE gene receptor binding domain?141²83?has four unique amino acid variations,and its receptor binding domain underwent recombination events.The recombinant region was located at 182⁷02 bp,which may affect the function of HE gene.In this study,the BCoV of yak was first isolated,and the first complete of BCoV from yak was first amplified,which contribute to further understand the biological characteristics and genetic evolution of BCoV from yak.