Molecular Epidemiological Investigation Of Bovine Diarrhea Virus In Ningxia And Establishment Of BVDV RPA Visualization Detection Method

In recent years,emerging infectious diseases have become common,the types have gradually increased,and the epidemic background has become more complex,and the effective prevention of sudden infectious diseases has become one of the major scientific problems of human public health.In this study,the virus metagenomics method was used to analyze the virus community structure of dairy and beef cattle in Ningxia.RT-PCR was used to conduct epidemiological investigation and genetic evolution analysis of bovine diarrhea-related viruses.The virus was isolated and identified in samples with positive Bovine viral diarrhea virus(BVDV)tests.In addition,the study established a visual detection method for BVDV based on Recombinase polymerase amplification(RPA).Through experiments,we obtained the following results:(1)The number of contigs annotated to the virus sequence after high-throughput sequencing of cattle samples in Ningxia accounted for 16.89%of the total reads number,and beef cattle accounted for 39.40%.We annotated 4 Realms,6 Kingdoms,11 Phylums,23 Classes,33 Orders,55 Families.There are 22 animal viridae,and Bovine diarrhea virus families include:flaviviridae,coronavirus,caliciviridae,astroviridae,and reoviridae.(2)According to the viruses found in the virus metagenomics of the Ningxia region,pathogenetic investigation of BVDV,Bovine rotavirus(BRV),Bovine coronavirus(BCoV),Bovine norovirus(BNoV)and Bovine astrovirus(BoAstV)were protected by RT-PCR.The results showed that the positive rates of dairy cows BVDV,BCoV,BRV,BNoV and BoAstV were 36.48%,40.88%,4.40%,30.82%and 26.42%,respectively.The positive rates of beef cattle BVDV,BCoV,BRV,BNoV and BAstV were 47.48%,30.94%,53.96%,62.59%and 33.09%.(3)The positive samples of BVDV detected by RT-PCR were inoculated in MDBK cells for virus isolation,and the biotype,morphology,indirect fluorescence,cytotoxicity,RT-PCR identification and genome-wide evolutionary sequence analysis proved that the B VDV-NX strain was successfully isolated,which was 50～60 nm round capsuled particles,the titer of TCID50 was 10-9.17,and the isolated strain was BVDV-1d subtype.(4)The study established the BVDV visual RPA detection methods,and the optimal reaction temperature for BVDV RPA detection was 37℃,the optimal reaction time was 25 min,the minimum detection threshold of gel electrophoresis RPA was 1×101 copies/pL,and the minimum detection threshold of BVDV SYBR Green I visualization was 1×109 copies/μL in sunlight.Under UV,the minimum detection threshold of BVDV SYBR Green I was 1×105 copies/μL.The established RPA detection method has good specificity,and the RPA detection threshold of clinical samples was equal to that of PCR detection,and the visualization was obvious.The results of these studies show that dairy cattle and beef cattle in Ningxia carry significant differences in virus bearing.There are differences between BVDV,BRV,BCoV,BoAstV,and BNoV among different species,different regions and different breeding modes.The BVDV-NX strain was successfully isolated.The established BVDV RPA visualization detection method has strong specificity,high sensitivity and obvious visualization.