Pathogenicity Analysis Of Bvdv And Immune Effect Evaluation Of E2 Protein In New Zealand White Rabbits

Bovine viral diarrhea is a disease caused by bovine viral diarrhea virus.Mixed infection of one or more pathogens can cause bovine respiratory disease syndrome(BRDC).Infectious bovine rhinotracheitis virus(IBRV),bovine parainfluenza virus type3(BPIV-3)and bovine viral diarrhea virus(BVDV)are the main viruses causing bovine respiratory diseases.The main clinical symptoms after infection with bovine viral diarrhea virus(BVDV)are shortness of breath and shortness of breath,reduced or eliminated feeding,mucosal congestion and ulcer spots in the oral cavity,massive salivation,watery or loose stool-like diarrhea,odor and occasional blood.In addition to the above clinical symptoms,BVDV infection usually causes immunosuppression,so it is easy to be mixed with mycoplasma,influenza virus,bovine pasteurella and other respiratory diseases,which aggravates the symptoms of bovine respiratory tract to syndrome together with other respiratory diseases.A series of epidemiological investigations have shown that the incidence of the disease is high,but the mortality rate is low,but in the acute infection type,the mortality rate is high.The widespread spread of the disease has attracted the attention of the global cattle industry,causing huge economic losses to the global cattle industry.BVDV belongs to Flaviviridae and Pestivirus.The International Committee on Taxonomy of Viruses currently divides BVDV into three genotypes : BVDV-1,BVDV-2 and BVDV-3(Ho Bi-like).Among them,BVDV-1 has the widest range of transmission and the greatest impact in China.At present,the main method to prevent bovine viral diarrhea virus in China is to inoculate inactivated vaccine.However,BVDV belongs to RNA virus,and its mutation rate is faster than that of DNA virus,which leads to the decrease of vaccine protection.The E2 protein of BVDV plays an extremely important role in the immune response and is the preferred protective antigen for genetically engineered vaccines.At present,there are few studies on BVDV-1d subtype in China,and the infection mechanism of the disease is not clear.Therefore,this study selected economical,convenient and easy to operate alternative animals to carry out pathogenicity tests.The protection of E2 subunit vaccine against BVDV strain was evaluated in experimental animals.First,from 2021 to 2022,the epidemiological investigation of bovine viral diarrhea virus in Inner Mongolia showed that in a cattle farm with BVDV eradication plan,we identified 33 BVDV nucleic acid positive from 103 samples,the positive rate was 32.04 %,we isolated 5 strains of NCP BVDV,the separation rate was 15.15 %,and 1 strain of CP BVDV,the separation rate was 3.03 %.The CP strain was successfully isolated from nasal swabs of calves with severe clinical symptoms in Hohhot,Inner Mongolia.In this study,we named the strain HH839,and determined the biological characteristics of the strain by virus titer determination,electron microscopy,serum neutralization test,q PCR,replication kinetics determination,whole genome sequencing and genetic evolution analysis.Second,this study investigated the pathogenicity of different biotypes of BVDV in New Zealand White rabbits,including CP-type BVDV infection group,NCP-type BVDV infection group and CP-type NCP-type BVDV mixed infection group.The suitability of New Zealand White rabbits as a model of BVDV infection was investigated by clinical signs observation,autopsy observation,RT-PCR identification of nasal and anal swabs and each organ,and histopathological observation.The results showed that different biotypes of BVDV could cause different degrees of pathogenicity in New Zealand White rabbits after artificial infection by intravenous injection at the ear margins.Third,this study evaluated the effectiveness of BVDV E2 recombinant protein as a genetic engineering vaccine on a New Zealand white rabbit model.The subunit vaccine was prepared with Freund ’s adjuvant.The 2 to 3-month-old New Zealand white rabbits were immunized with E2 recombinant protein 1mg / head,and the serum was collected regularly.After immunization,the mice were challenged according to the method of pathogenicity test,and the effectiveness of E2 subunit vaccine was evaluated by indirect Elisa,RT-PCR,histopathological observation and immunohistochemistry.The results of indirect Elisa test showed that E2 subunit vaccine could induce BVDV specific antibody in New Zealand white rabbits.BVDV specific antibody could be detected 7 days after the first immunization,and the antibody level reached the highest value 28 days after the first immunization.The nasal swabs of New Zealand white rabbits in the immune challenge group were negative by RT-PCR.Histopathological observation showed that the trachea and lungs of the immune challenge group showed slight histopathological changes;the results of immunohistochemistry showed that the immune group was negative and the control group was positive.In conclusion,a single CP-type BVDV strain was successfully isolated.New Zealand white rabbits can be used as an experimental animal infection model for BVDV.Recombinant E2 protein has good immunogenicity,and the results provide a reference for the development of subunit vaccine for bovine viral diarrhea virus.