Viral Metagenomic Study On Calf Fecal In Daqing City And Establishment Of The Triplex RT-PCR Method Of BNoV,BKV And BToV

At present,the etiology of calf diarrhea in China is complex,and new viral infectious diseases and virus mutation continue to occur,which seriously affects the health of calves in China.Traditional pathogen detection technology can no longer meet the needs of rapid diagnosis of animal infectious diseases.With the continuous development of gene sequencing technology Viral Metagenomics(VM)based on Next-generation DNA sequencing technology has been used to analyze calf enterovirus community.There are relatively few studies on intestinal VM detection of calves in Daqing dairy population.Therefore,this experiment systematically investigated the composition of calf enterovirus community in Daqing area,analyzed the genetic variation of local epidemic strains,and then investigated the ecological changes of calf enterovirus,which had early warning significance for bovine viral infectious diseases.This study was based on the VM technology of Illumina Nova Seq6000 sequencing platform.VM was applied to analyze the viral community composition of 155 calf fecal samples from 9large-scale dairy farms in Daqing,and to compare the differences between the viral communities in the intestine of calves with diarrhea and calves without clinical diarrhea symptoms;the triplex RT-PCR method was established to detect Bovine Norovirus(BNoV),Bovine Kobuvirus(BKV)and Bovine Torovirus(BToV),which were detected by VM and newly caused diarrhea of calves in China.The epidemiological situation of these viruses was investigated,and the g enetic evolution characteristics and epidemic trend of virus genes were systematically analyzed.The main research results are as follows:(1)9 fecal mixed sample pools were sequenced with high-throughput sequencing,with a total data of 193.13G.There were 6 517 147 virus-liked reads from 21 families were successfully annotated in the calf intestine after quality control.According to the De Brujin algorithm,a total of 53 571 contigs were obtained through de novo assembly.Vertebrate viruses were annotated to a total of 202 197 reads,representing 3.10%of the viral communities in the intestine of calves,including Parvoviridae,Circoviridae,Smacoviridae,Reoviridae,Poxviridae,Hepadnaviridae,Herpesviridae,Polyomaviridae,Coronaviridae,Picornaviridae,Caliciviridae,Astroviridae,Orthomyxoviridae,and Retroviridae.A total of 47 069 reads were annotated in the Coronaviridae,with the highest percentage of 0.72%.By comparing the viral communities in the intestine of calves without clinical symptoms of diarrhea with those of calves with diarrhea,we found that the relative abundance of Parvoviridae,Picornaviridae and Reoviridae in the diarrhea group libraries were on average 3.05,1.54 and5.61 times higher than that of the libraries of the group without clinical symptoms of diarrhea.The contigs of 98 Bovine Rotavirus,26 Bovine Parvovirus,28 Bovine Enterovirus,145 BKV and 15 Bovine Hungarovirus were assembled,which may be the main viruses causing diarrhea in calves.Multiple viral nucleic acids were detected in the clinical diarrhea free group library which mainly included Circoviridae,Caliciviridae,Coronaviridae,Astroviridae,Parvoviridae,Picornaviridae,Reoviridae.The nearly complete genome sequences of Bovine Rotavirus,Bovine Coronavirus,and Bovine Astrovirus were obtained by assembly.The genetic evolution analysis showed that the strains obtained by VM assembly were closely related to the prevalent strains in China,exhibiting significant geographical clustering and genetic diversity.Successfully assembled domestic emerging bovine-derived viruses,such as BNoV,BKV,BToV,Bovine Nebovirus.The positive rates of inter libraries assembly were 66.66%,77.77%,77.77%,44.44%,respectively.The positive rate of assembly results of BNoV,BKV and BToV among sequencing libraries were more than 50%,indicating that these three viruses had become prevalent in the Daqing and require pathogen testing to prevent and control the outbreak of infectious diseases.(2)The triplex RT-PCR detection method for rapid identification of BNoV,BKV,and BToV was successfully established by targeting the Rd Rp gene of BNoV,the 3D gene of BKV,and the N gene of BToV.It showed the minimum detection limits for BNoV,BKV,and BToV plasmid standards using triplex RT-PCR were 2.73×10~4 copies/μL,2.87×10~4 copies/μL and 3.18×10~3copies/μL,respectively.The triplex RT-PCR method was used to detect 153 fecal samples of calves,and it was found that the positive rate of mixed infection of the three viruses reached7.18%.The positive detection rates of BNoV,BKV,and BToV were 11.11%,25.49%,and38.56%,respectively.Compared with the single RT-PCR detection method established by other scholars,the coincidence rates for the three viruses mentioned above are 94.77%,93.46%,and91.50%,respectively.The highly pathogenic genotype GⅢ.1 of BNoV and the predominantly prevalent genotype GⅢ.2 were both detected in Daqing;the positive BKV samples detected in Daqing were all classified as Aichivirus Type B,with nucleotide similarity between strains higher than 90%,and were more closely related to the prevalent strain in China and more distantly related to the Aichivirus Type D strain;the positive BToV samples detected had the nucleotide similarity between strains was higher than 97%and the genetic distance from the original classical s train Breda 1 was far.This study preliminarily explored the viral spectrum composition of calf feces from scale farms in Daqing area,providing a theoretical basis for the pathogenic diagnosis of calf viral diarrhea.The triplex RT-PCR assay for BNoV,BKV,and BToV was developed to provide technical support for the detection of the above three viruses and the molecular epidemiological investigation.