Gene Analysis And Preliminary Study On Infectious Clone Construction Of Bovine Viral Diarrhea Virus NM Strain

Bovine viral diarrhea-mucosal disease(BVD/MD),also known as bovine mucosal disease,is a cow reproductive disorder caused by Bovine viral diarrhea-mucosal disease virus(BVDV).Cattle has severe diarrhea,nasal endoscopy,esophageal ulcer erosion,fetal cattle immunosuppression,continuous detoxification,and a disease of the bovine respiratory and digestive tract that is prevalent in the world.BVDV has achieved cross-host transmission through genetic recombination and mutation at the same time that sick cattle are in contact with other animals.In recent years,there have been reports that deer,pigs,and sheep have been infected with BVDV,which also makes my country face more severe challenges in the prevention and treatment of BVDV.Vaccination is an effective means to prevent and control BVDV.At present,there are commercial inactivated vaccines on the market in China,which have a certain protective effect.However,inactivated vaccines have a short protection period and require long-term uninterrupted immunization.Therefore,the development of new and safe live vaccines is still the focus of vaccine research.In this study,the full-length sequence of the BVDV NM strain isolated and preserved in the laboratory was amplified by the PCR method,and the total length of the BVDV NM strain was12307 bp after splicing.The coding region of the genome is position 387-12080,and the lengths of Npro,Core,Erns,E1,E2,P7,NS2-3,NS4 A,NS4B,NS5 A,and NS5 B are 504,306,681,585,1122,210,3408,192,1041,1488,2157 bp.Compared with the non-cytopathic JL strain isolated and preserved in the laboratory,the homology between the two is 79.9%.The homology and evolution analysis of the full length of the NM strain showed that the BVDV NM strain had the highest homology with the BVDV Singer Arg isolate from Shandong,China,at 99.98%,indicating that the BVDV NM strain may be a mutation of the Singer Arg strain from Shandong,China.After RNA secondary structure prediction,it was found that both the 5’and 3’ends of the NM strain contained a Y-shaped hairpin structure,while the JL strain had no hairpin structure.The E2 protein of JL strain and NM strain contains a large number of phosphorylation modification sites and glycosylation sites,but their modification sites are quite different.In order to verify the correctness of the regulatory elements required by the virus rescue system,a minigenome system was constructed in this experiment.The system is based on the p CI-neo vector and uses the overlap PCR method to fuse hammerhead ribozyme,BVDV NM strain 5’UTR,EGFP reporter gene,BVDV NM strain 3’UTR and hepatitis D ribozyme,respectively.After sending it to the biological company to verify that the sequence is correct,the cells are transfected to verify whether the regulatory regions and starter elements of the terminal sequence can function normally.After 48 hours of transfection of the minigenome,green fluorescence can be observed through an inverted fluorescence microscope,indicating that the regulatory elements required for the virus rescue system have been successfully constructed.In summary,this study carried out PCR amplification,sequence determination and biological information analysis on the BVDV NM strain,and constructed and verified a microgenome containing the promoter elements of the strain,which provided a basis for the establishment of a reverse genetic operating system for the BVDV NM strain.