Identification Of An Enterovirus E HY12 And Establishment Of Sandwich ELISA For Viral Antigen Detection

Bovine enterovirus infection is an emerging infectious disease contributing to a significant economic loss to cattle industry. As the causative agent, BEV belongs to the genus enterovirus within the family of Picornaviridae. Based on the latest virus taxonomy, the genus of enterovirus consists of 9 species of enterovirus(A, B, C, D, E, F, G, H, J) and 3species of rhinovirus(A, B, C), where the enterovirus speciesE and F(EV-E and EV-F) cause infections to cattle. Like other species within the genus, BEVs are small, non-enveloped viruses with an icosahedral virion and a positive-stranded RNA genome. BEV has been often isolated from cattle with a clinical signs varying from respiratory diseases to enteric, reproductive diseases and infertility, even from feces of the presumably healthy cattle. As BEV infection is a newly discovered disease in China, its diagnosis, prevention and pathogenesis still remains largely unknown.In this study, the virus with diameter of 20-30 nm was isolated from cattle manifesting pyrexia, cough, dyspnea, diarrhea with high morbidity(33/70, 47 %) and mortality(17/33, 51 %) on a farm in Jilin province. Physiochemical properties showed the virus to be either enterovirus within Picornaviridae or parvovirus within Parvoviridae. Degenerated primers were designed and used to amplify the potential virus sequences using PCR for parvovirus, enterovirus, and foot and mouth disease virus, respectively. Sequencing the PCR-amplified fragment revealed it contained the sequence that shared a high sequence identity with enterovirus, and isolated virus was named as HY12 strain. Phylogenetic analysis demonstrated that HY12 was closely related to an Australia bovine enterovirus strain of SL305(90%), while it only has 75% sequence identity with the newly reported bovine enterovirus stains in China. The HY12 strain is clustered to the enterovirus E.The VP1 and VP2 genesencoded by HY12 were PCR-amplified and cloned to prokaryotic expression vector pGEX-4T to generate pGEX-4T-1-VP1 and pGEX-4T-1-VP2 constructs. The recombinant proteins VP1 and VP2 were expressed, purified and used as antigen to generate either monoclonal or polyclonal antibodies. A sandwich ELISA for detection of bovine enterovirus antigens was developed using antibodies raised againstthe recombinant proteins VP1 and VP2 derived from enterovirus E HY12, and used to investigate the BEV infections in cattle population in Jilin Province. The concentrations of anti-VP1 for antibody coating and anti-VP2 conjugated with HRP were optimized using square matrix titrimetry. The criterion for sandwich ELISA was determined. Sample was assessed as positive when its OD490 is larger than or equal to(≥) 0.145. The established ELISA had a high sensitivity and specificity with a well repeatability and simplicity. Epidemiological survey indicated that BEV infection rates in cattle populations of Jilin province were 8.16 %~58.7 %. The results in this study will provide a sensitive, specific, simple and rapid antigen detection method for diagnosis and epidemiologic survey of BEV infection.