Molecular Epidemiological Investigation And Genomic Analysis Of Bovine Kobuvirus In Some Provinces In China

Bovine kobuvirus(BKV),which belongs to the genus kobuvirus,family Picornaviridae.The virus are spherical and non-enveloped,single-stranded RNA viruses with a positive sense polarity and the diameter is between 30 nm and 40 nm.There are currently three BKV genomes in Gen Bank database,from Japan,the UK and Egypt,respectively.It had been suggested that BKV may be associated with diarrhea in calves,but the pathogenicity of BKV still needs to be determined.Calves within 5 months of age are most susceptible to infection.Since BKV was first identified in Japan in 2003,this virus has been detected in bovine diarrhea feces and healthy feces in 10 countries,indicating that BKV is widely distributed geographically.The highest infection rates of BKV reach 77.8% in dairy cattle and 14.0% in beef cattle.In 2003,BKV was found in the diarrhea feces of dairy cattle for the first time in China,and the detected infection rate was as high as 34.9%.The popularity of BKV is a potential threat to the health development of domestic cattle industry.In addition,yak,as one of the main livestock products in the Qinghai-Tibet Plateau,has not been reported about yak-sourced BKV.As a new virus,BKV has few research data in China.In this study,molecular detection was conducted on samples of dairy cattle feces from some provinces in China to understand the correlation between the virus and diarrhea in dairy cattle and the molecular characteristics of BKV and to investigate the infection of BKV in yaks in Qinghai-Tibet plateau.The results achieved are shown as follows: 1.Molecular detection and phylogenetic analysis of bovine kobuvirus from dairy calves from four provinces in ChinaIn order to understand the infection status and phylogenetic analysis of BKV in cattle in some provinces of China,a total of 173 calf fecal samples(96 diarrheic and 77 non-diarrheic)were collected from 14 dairy farms across four Chinese provinces of Liaoning(three farms),Henan(three farms),Shandong(three farms)and Shanxi(five farms)from January 2018 to April 2018.These provinces are the main dairy cattle-producing areas in China.The ages of the tested calves ranged from 2 days old to 4 months old.All samples were detected by a BKV RT-PCR assay according to a previous report by Jeoung H Y et al.Moreover,37 partial 3D gene(631 bp),15 complete VP1 gene(801 bp),and 12 partial VP0 gene(660 bp)were successfully obtained from the nucleotide sequences of positive sample of BKV.The results showed that the average positive rate of 173 samples was 24.86% and the dairy farm positive rate was 64.29%,indicated BKV was highly distributed in China.Among the 96 diarrheic samples,34 were detected as BKV positive(35.42%),and 9 out of 77 non-diarrheic samples were detected as BKV positive(11.69%).The BKV detection rate for the diarrheic fecal samples was significantly higher than that for the non-diarrheic samples(P<0.001).Of the 34 BKV positive diarrheic samples,28 were confirmed to be co-infection positive for other viruses.Analysis of these sequences from 37 partial 3D gene fragments obtained in this study revealed that the strains share 92.6%–100% nucleotide sequence identity(97.0%–100% amino acid sequence identity)with each other,and 88.6%–96.4% nucleotide sequence identity(92.4%–100% amino acid identity)with other 3D sequences in the Gen Bank database.Further analysis reveals that these BKV 3D sequences are divided into 2 large branches,and all the 37 strains from this study are located on the class 1 branch which is further distinguished by 10 small branches.These 15 complete BKV VP1 sequences in this study share 74.2%–100.0% nucleotide sequence identity(80.1%–100.0% amino acid identity)with each other,and share 72.9%–94.9% nucleotide sequence identity(80.5%–97.8% amino acid identity)with the other BKV VP1 sequences in the Gen Bank database.Further analysis reveals that all the VP1 sequences fall into 8 distinct branches.7/15 VP1 sequences from this study were clustered into the known lineage 2.Interestingly,8/15 VP1 sequences from our study were clustered into 3 independent branches distinct from the 5 known lineages.The 12 BKV VP0 sequences from this study were found to share 78.5%–100% nucleotide and 84.1%–100% amino acid sequence identity with each other,and 80.2%–84.7% nucleotide and 85.0%–91.8% amino acid sequence identity with the only 3 BKV VP0 sequences in the Gen Bank database.11 of the BKV strains from the this study clustered on 1 independent branch,1 strain from this study and 3 known strains from Gen Bank clustered on an independent branch.Further analysis found that the 11 sequences in independent branch shared a unique amino acid insertion in two forms(S or N).The results showed that BKV was widely distributed in China and may be associated with diarrhea in calves and the co-infection was severe in the BKV diarrhea-positive samples.These BKV 3D sequence from this study enriched the genetic diversity.Moreover,8/15 BKV VP1 sequences were independently divided into 3 new branches,indicating that these VP1 may represent 3 novel lineages.Compared with the only 3 BKV VP0 sequences in the Gen Bank database and the 1/12 sequences obtained in this study,the 11/12 VP0 sequences independently showed a unique amino acid insertion trend.The above information provides references for further study of the infection and molecular characteristics of BKV.2.Successfully obtained the first BKV genome in ChinaIn order to further study the characteristics of the BKV genome,13 pairs of primers were used to amplify the full genome sequences of the BKV genome and sequences were assembled using Seq Man software.A nearly complete BKV genome of 7907 nucleotides in length which contains the 7395 bp complete ORF,named CHZ/CHINA,was successfully obtained from a positive BKV diarrhea sample of calve from Henan in this study(Gen Bank accession No.MK080265).This genomic sequence comprises a G + C content of 56.14%,and the sequence encodes a putative polyprotein precursor of 2465 amino acids in length.The ORF genome of strain CHZ/China shares 87.4%–88.3% nucleotide and 93.7%–96.4% amino acid identity with the 3 known ORFs from BKV strains.A phylogenetic analysis based on genomic sequences revealed that strain CHZ/CHINA clusters on an independent branch,with the five L,VP0,VP3 and VP1 protein sequences generating the same result as for the genomes.The results showed that CHZ/CHINA displays a larger genetic distance from the other three genomes and CHZ/CHINA may represent a novel BKV strain.In this study,the first Chinese BKV genome was successfully obtained and the findings contribute to better understanding the molecular characteristics and phylogenetic analysis of BKV.3.Successfully established a real-time RT-PCR assay for detecting bovine kobuvirusIt had been suggested that BKV may be associated with diarrhea in calves.As one of the main breeding animals in Qinghai-Tibet plateau,yak has a high death rate of calves caused by diarrhea in April and May every year,which brings major economic losses to the local herdsmen.However,there is no epidemiological investigation report on yak-sourced BKV.In the previous work,our group used the only existing 3 reports on BKV RT-PCR method to detect BKV in fecal samples of yak calf diarrhea,but the result did not detect BKV,which may be because the sensitivity was not high enough.In order to establish a more sensitive method for detecting BKV,by designing primers targeted to 3D fragment of BKV strains and optimizing reaction conditions and system in this study,a TB Green real-time RT-PCR assay was successfully developed.The results demonstrated that the assay had a good linear relationship between 4.86×108 and 4.86×102 copies/?L of virus concentration,the linear correlation coefficient was 0.9995 and the amplification efficiency was 92.75%.This assay only was specific for detection of BKV,no cross reaction were observed with other species of diarrhea-causing pathogens;the minimum detection limit was 4.86×102 copies/?L;both the intra-and inter-group coefficients of variation were <2%.Comparing to other three reported RT-PCR assays,this assay had a significant high detection rate of BKV for clinical feces samples from both diarrheal dairy cattle and yak.Total 131 yak diarrhea samples collected from Qinghai,Xizang and Sichuan Tibetan areas,the BKV detection rate was 31.30%(The positive rates in Qinghai,Tibet,and Sichuan Tibetan areas were 44.44%,41.18%,and 13.46%,respectively and the field positive rates were 50.00%,100.00%,and 37.50%,respectively).The real-time RT-PCR assay for detecting BKV developed in this study has a good specificity and stability,which provide a reliable method for detection and epidemiological investigation of BKV,and the existence of BKV on yaks was also proved.