Investigation Of BVD Infection And Isolation And Identification Of Epidemic Strains In An Area Of Henan

Bovine viral diarrhea(BVD)is a highly contagious infectious disease transmitted between cattle.The cause of the disease is Bovine viral diarrhea virus(BVDV).There are many genotypes and subtypes of BVDV.The clinical symptoms of different genotypes or subtypes of infected cattle are different.The common symptoms are diarrhea,fever and respiratory symptoms.In recent years,BVD has been widely popular in China and even around the world,causing huge economic losses.Due to the particularity of the BVDV genome,mutations are likely to occur during transmission.Among them,the E2 protein has the highest coefficient of variation.At the same time,this protein is a major breakthrough in vaccine development due to its good immunogenicity and antigenicity.This nature of the E2 protein has brought great challenges to the successful development of vaccines.The disease has hindered the sustained and healthy development of large-scale cattle breeding,so mastering the entire gene sequence of BVDV is of great significance for vaccine development and BVD prevention and control.The experiment collected 200 bovine sera from a cattle farm in a certain area in Henan,and obtained the prevalence of BVD in the area by seroantigen positive rate investigation;and isolated a BVDV strain from the spleen of sick and dead cows for genotype identification and whole gene Sequence determination and biotype,TCID50 identification and other tests.Based on the data obtained from the serological positive rate identification results,reasonable prevention and control measures are formulated,and the whole genome sequence of the isolate is determined to provide basic biological information for vaccine research and reverse genetic work.The biological information analysis software is used to obtain the isolate nucleotide Sequence homology with the primary amino acid sequence of the E2 protein,and compare the primary amino acid sequence of the isolated strain E2 protein with the amino acid sequence of the E2 protein of the SD1303 strain with the highest homology and the SD15 strain with the lowest homology.In 2019,200 samples of bovine serum from 4 large-scale cattle farms in Henan were tested for BVDV by RT-PCR,and the positive rate was 17.5%;the spleens of dead cows infected with BVDV were collected.The genotype of the strain was identified as BVDV Ⅱ.The strain was named BVDV HEN01 strain;compared with the SM strain of the control group,the MDBK cells inoculated with the BVDV-HEN01 strain did not undergo cytopathy through cell culture;Cells inoculated with SM strain in the control group showed cytopathic phenomena such as straining and accumulation;cells in the blank group grew normally.According to the test results,the strain can be determined to be non-cytopathic(NCPE);the entire gene of the strain was designed Amplify the primers,recover and purify the amplified PCR product gel to obtain the target fragment,connect the target fragment to the pMD-18 T vector,transform into the DH5α E.coli competent state,identify and extract the plasmid after culturing,and sequence the sequencing results.After splicing,the whole genome sequence was obtained.The TCID50 of the virus was determined to be 10-4.1 by indirect immunofluorescence.Observing the virus particles in the cell culture using an electron microscope showed that the diameter of the virus particles was about 50 nm.Based on the basic biological information of the measured whole gene sequence,the structure of E2 protein was successfully predicted.