Epidemiological Investigation And Identification Of BVDV In Four Western Provinces(Regions)

Bovine viral diarrhea virus（BVDV）is the pathogen of bovine viral diarrhea-mucosal disease（BVD/MD）,it mainly causes bovine diarrhea,reproductive disorders and respiratory diseases,which seriously hinder the development of the cattle breeding industry.BVDV has been widely spread around the world,it also has varying degrees of prevalence in China.At present,the research about the prevalence of BVDV in western China is limited.In order to understand the prevalence of BVDV in the western region,this study first conducted nucleic acid amplification testing on cattle serum samples collected from four western provinces（regions）through RT-PCR,the results determine the positive rate and subtype distribution of BVDV.Then,BVDV wild strains were isolated and identified by cell culture,indirect immunofluorescence staining and transmission electron microscope observation,finally purified through plaque assay.The results of the study are as follows:1.The epidemiological investigation of BVDV in the four western provinces（regions）A total of 1234 clinical serum samples were collected from 17 herds of diary cattle、beef cattle and yaks in western China（Shaanxi,Ningxia,Xinjiang,Tibet）from 2019 to2020.Design primers based on the 5’UTR of the conserved region of BVDV,and all samples were tested by RT-PCR.The results showed that the average positive rate of BVDV in the four western provinces（regions）was 7.2%（89/1234）,and the positive rate of herds was 82.4%（14/17）,the positive rate of dairy cattle,beef cattle and yak were 42.70%（38/89）,33.71%（30/89）and 10.77%（21/195）.The positive rates of BVDV in Shaanxi,Ningxia,Xinjiang and Tibet were 4.57%（44/963）,30%（18/60）,37.5%（6/16）and 10.77%（21/195）.The serum samples that were positive by RT-PCR were sequenced for 5’UTR gene,265’UTR sequences were successfully obtained,these sequences were compared with the reference sequences in Gen Bank,and phylogenetic tree was constructed based on the5’UTR gene for analysis,the result showed that all these sequences were distributed in the BVDV-1 genotype,including three subtypes,BVDV-1a（n=15）,BVDV-1c（n=9）and BVDV-1q（n=2）.BVDV-1a subtypes are distributed in Shaanxi（n=9）,Xinjiang（n=3）,Ningxia（n=1）and Tibet（n=2）;BVDV-1c is distributed in Shaanxi（n=1）,Ningxia（n=5）and Tibet（n=3）;BVDV-1q is distributed in Ningxia（n=2）.Some samples were selected for N^pro gene sequencing,and the genotyping results of the 4 N^pro sequences successfully obtained were consistent with the 5’UTR.2.Isolation and identification of wild BVDV strainsSerum samples that were positive by RT-PCR were inoculated with MDBK cells for virus isolation,and then identified by RT-PCR test,indirect immunofluorescence staining and transmission electron microscope observation.The results showed that a total of 19BVDV wild virus strains were obtained,of which 15 were non-cytopathic biotype（NCP）and 4 were cytopathic biotype（CP）.4 CP biotype isolates were further purified by plaque assay,and finally 2 were purified,named SX-1-2/1a/2019 and XZ-N1/1c/2020,and according to the Reed-Muench method,the results showed that the values of TCID₅₀/100μL were 10^3.8and 10^5.2.This study clarified that BVDV was prevalent in four western provinces（regions）.The prevalent genotype was BVDV-1,and the prevalent subtype was BVDV-1a,BVDV-1c and BVDV-1q.The obtained two CP biotype isolates provide a theoretical basis and practical significance for further research on the pathogenicity and immunogenicity of the BVDV.