Molecular Prevalence And Genomic Characterization Of Bovine Norovirus

Bovine Norovirus(BNoV)is a pathogen causing calves diarrhea and has been detected in 19 countries,including Italy,SouthKorea and China etc.,.The BNoV cluster into genogroup III(GIII),and can be further divided into two distinct genotypes: genotype 1(GIII.1)and genotype 2(GIII.2).Recently,our team confirmed the presence of BNoV in China,but the prevalence and genomic characteristics of BNoV in China are still unclear.The study aimed to investigate the molecular prevalence and genomic characteristics of BNoV in China,and the results as shown as follows;1.The prevalence of BNoV was first determined in dairy calve in China,and the virus exhibited a unique evolution characteristic.BNoV is a pathogen causing calf diarrhea,the aims of this study was to investigate the prevalence and molecular characteristics of BNoV in several regions of China.A total of 265 fecal samples,including 211 diarrheic fecal samples and 54 healthy fecal samples,were collected from 25 farms in six provinces.BNoV were detected by RT-PCR,and the results showed that the detection rate of BNoV in diarrhea samples(43/211,20.4%)was significantly higher than the healthy samples(9.26%)(P<0.01),which proved that the virus was associated with calves diarrhea;the detection rate of Sichuan province,Liaoning province,Henan province,Xinjiang province and Shandong province was 34.8%(8/23),53.2%(25/47),11.1%(3/27),6.9%(6/87)and 9.1%(1/11),respectively.But,no samples were detected as BNoV positive in Shanxi Province.Phylogenetic analysis of 43 RdRp fragments from five provinces showed that 5 strains clustered into GIII.1 and the remaining 38 strains clustered into GIII.2.Phylogenetic analysis of 26 VP1 fragments from five provinces showed that 1 strain clustered into GIII.1,the 23 strains clustered into GIII.2 and the remaining 2 strains clustered on a unique branch.Phylogenetic analysis of 17 VP2 fragments from four provinces showed that all 17 strains clustered on a branch in GIII.2.These results indicated that the VP1 and VP2 fragments of Chinese BNoV strains have rich genetic diversity in China.Moreover,as an emerging diarrhea pathogen with unique evolutionary characteristics BNoV has been widely circulated among dairy calves,providing an important reference for control of calf diarrhea in China.2.First detection of Bovine Norovirus from Yak(Bos grunniens)in China The yak(Bos grunniens)is a unique domestic bovine species and an iconic symbol in the high altitude region of the Qinghai-Tibetan Plateau.The purpose of this study was to investigate the presence of BNoV in yak in China.During May 2018 and July 2018,77 diarrheic were collected fecal samples of young yaks from Sichuan province.2 samples were detected as BNoV-positive by RT-PCR.Phylogenetic analyses based on RdRp revealed these BNoV strains are close to the Chinese GIII.2 strain from dairy calves.Moreover,we obtained two 1311 bp RdRp sequences which have 15 identical nucleotide mutations and 3 identical amino acid mutations with available BNoV I RdRp sequences.The result first confirmed the presence of BNoV in yak,and awareness for BNoV surveillance and transmission in yak should be raised.3.A novel VP1 genotype of BNoV was identified,and the complete genomes were successfully obtained.In the previous work,the VP1 fragments of 2 strains showed significant differences compared from all other GIII VP1 genesstrain Bo/BET-17/18/CH was selected for genomic amplification.The complete genome of strain Bo/BET-17/18/CH was successfully sequenced.It was 7321 nucleotides(nt)in length,shared 79.4%-80.9% nt identity with all five BNoV genomes,clustered on a separate branch of the phylogenetic tree,suggesting that strain Bo/BET-17/18/CH could represent a novel BNoV strain.Two interesting characteristics were found in the genome: i)the VP1 sequence differed greatly from known BNoV VP1 sequences,suggesting that the VP1 sequence could represent a novel BNoV genotype;ii)a recombination event is predicted within the ORF1-ORF2 overlap.Moreover 16.3%(7/43)of the BNoV were identified as the novel VP1 genotype,which were distributed on four farms across two provinces,indicating that the novel VP1 genotype strain has spread.These findings extend our understanding of the genetic evolution and epidemics of BNoV.4.A Real-time RT-PCR assay for detecting BNoV was established.The 3 'end of RdRp sequence of BNoV is often used for molecular detection targets.Our previous results showed that the Chinese BNoV RdRp had unique evolutionary characteristics and nucleotide mutations in 3'end,which may affect the accuracy of BNoV detection rate in China.The aims of the present study were to establish a sensitive and specific real-time RT-PCR assay for detection of BNoV.The degenerate primers were designed based on the BNoV RdRp sequences in China,a Real-time RT-PCR assay for detecting BNoV was successfully established,after optimizing.The minimum detection limit was 3.7copies/?L.This assay only specifically detected BNoV,do not detected other common bovine diarrhea pathogens;the intra-and intercoefficients of variation were <2%.71.1% of the 45 diarrheic samples were detected as BNoV-positive by this assay,which was higher than other 2 assay from previously reported.Therefore,this assay in this study has the characteristics of high sensitivity,good specificity and stability,which provides a reliable method for the detection and epidemiological investigation of BNoV.Therefore,the Real-time RT-PCR assay for detecting BNoV established in this study has a good specificity,stability and sensitivity,which will contribute the molecular detection of BNoV in China and provide a basis for scientific prevention and control of BNoV.