Isolation And Identification Of Bovine Rotavirus And Establishment Of Its Detection Methods

Bovine Rotavirus(BRV),belonging to the Rotavirus genus with Reoviridae family,is one of the main pathogens that cause diarrhea in calves.Since BRV was first reported in 1969,it has occurred and spread in various countries which causing serious economic losses to the industry.The main symptoms of the disease are diarrhea and dehydration in calves within 1-month age.And the disease is difficult to distinguish other pathogens caused by the clinical symptoms of bovine diarrhea.Therefore,the establishment of a rapid diagnostic method is of great significance for the prevention and control of BRV.In this study,the clinical samples were collected from large cattle farms in different regions of Hebei province,and a cytotoxic strain was successfully isolated.Genetic and phylogenetic analysis of VP6 gene had been analysis and expressed in vitro,and ELISA method had been established based on VP6 protein of BRV.In addition,a double TaqMan fluorescent quantitative PCR detection method was established based on the conservative sequences of BRV VP6 and BCV N genes.This study provides a rapid and effective diagnostic method for BRV in China and lays the foundation for future vaccine research and development.In order to understand the infection of BRV in Hebei province,RT-PCR was used to detect 72 diarrhea samples collected from different cattle farms in Hebei province from March to June 2019.The diarrhea stool samples were processed and infected with Marc-145 cells to isolate the virus.Then,the cytopathology of isolation was observed and detected by RT-PCR,TCID50 and physical and chemical identification.The results of the cells showed shrinkage,rounding,and unobvious border lesions after 5 generations,and RT-PCR was identified as positive.According to the Reed-Muench method,the TCIDso of 15th isolation was 10-5.24/0.1mL.It is tolerant to chloroform,ether and strong acid,but is more sensitive to heat at 60? for 10 min to inactivate the virus completely,indicating that the CH-HB-BD strain was successfully isolated.In order to understand the genetic variation of the isolation,this study successfully amplified and sequenced the VP6 gene,and carried out sequence and amino acid homology with the domestic and foreign rotavirus A(RVA)VP6 gene.The results showed that the isolation was closely related to strains RVA G1P8-3-50 from the United States(accession number:KU956011).RVA WC3(accession number:AF411322)and German RVA 88977(accession number:KJ940164),which shared the highest homology(96.4%)with strain WC3(accession number:AF411322).The results of nucleotide identities were consistent with amino acid homology,indicating that the VP6 gene can be used as a target gene for the establishment of diagnostic methods.In addition,the isolation is closely related to the vaccine strain RVA NCDV(accession number:DQ870496)with the amino acid homology of 95.8%,and only three changes in amino acid positions No.122(I?L),211(V?I),349(I?V).In this study,VP6 protein of the isolation was expressed,and the reactogenicity of the protein was identified by Western blot.An indirect ELISA method for detecting IgG antibodies in bovine serum was established using recombinant VP6 protein.Through the optimization of reaction conditions,it was determined that the optimal coating condition for protein was 6?g/mL at 4? overnight,and 5%skim milk was the best blocking solution.At the same time,the concentrations of primary and secondary antibodies were 1:500 and 1:10000 among different determined concentrations in this study.In addition,the results show that the ELISA method established in this study have good repeatability and specificity;and the coefficient of variation between groups is less than 7%,which has no cross-reactivity with BCV,BVDV and BNoV.The cut-off value is 0.290 calculated by 30 BRV-negative sera,and the positive rate of 285 clinical samples showed 50.1%(144/285).In order to establish a rapid and quantitative detection method for bovine rotavirus(BRV)and bovine coronavirus(BCV),primers and probes were designed based on the VP6 gene of BRV and the N gene of BCV in GenBank.After optimizing the system by the square matrix method,a duplex TaqMan real-time PCR method for simultaneous detection of BRV and BCV was successfully established.This method has no cross-reaction with BVDV,BPV,BEV,and IBRV indicated that the method established in this study has strong specificity.The sensitivity results showed that the minimum detection limit of BCV and BRV were 41.8 copies/?L and 3.55 copies/?L,respectively.The intra-and inter-group coefficients of variation were less than 2%,indicating that the method was reproducible.The duplex TaqMan real-time PCR assay was used to detect 72 bovine diarrhea stool samples,the positive rate of BRV was 50.0%(36/72),the positive BCV was 75.0%(54/72)and the co-infection rate was 33.3%(24/72).However,the detection results of conventional PCR method for BRV was 47.2%(34/72)and BCV was 70.8%(51/72),and the co-infection rate was 29.1%(22/72).The coincidence rates of BRV and BCV was 97.2%and 96.8%respectively,which showed that this method could be used to detect clinical samples.The established duplex TaqMan real-time PCR assay has important implications for clinical pathogen detection and epidemiological investigation of BRV and BCV.