Mouse Model For Enterovirus E HY12 Infection And Antibody Change In Experimentally Infected Mice

Bovine enterovirus(BEV) is the causative agent of an emerging infectious disease in China that has a significant economic impact to the cattle industry. As the etiological agent, BEV causes a disease of cattle with clinical signs characterized by fever, cough, dyspnea, diarrhea, dehydration, and even death. According to the latest classification by the International Committee on Taxonomy of Viruses(ICTV), BEV were classified to two species EV-E(formerly BEV-A) and EV-F(formerly BEV-B). As BEV infection is an emerging infectious disease in China, the pathogenesis, diagnosis and prevention remains largely unknown.In this study, a previously isolated enterovirus E HY12 was used to experimentally infect mice in an attempt to establish a mouse model. Inoculation of HY12 viruses to different mice strains showed that ICR suckling mice aged at 3 days were easily infected. PCR detection of viral genome sequences and virus isolation showed that viruses in infected ICR mice last at least 18 days. Minimal infective dose of HY12 to mice was determined to be 2×106 TCID50. Infection routes including intranasal, gastrointestinal gavages, intraperitoneal injection, subcutaneous injection and intramuscular injection of HY12 to mice were also determined, and showed that all routes were suitable for inoculation. Histopathological observations revealed an obvious pathological changes and inflammatory cell infiltration in the intestine, lung, liver, brain and kidney. Simultaneously, immunohistochemistry detection demonstrated that HY12 can be detected in most tissues especially in the intestine, lung, and brain, thus laying a solid basis for future investigation related to the pathogenesis of HY12.An indirect ELISA for detecting enterovirus antibody was established using the VP2 recombinant proteins as coating antigens and employed to determine the antibody change in mice experimentally infected with bovine enterovirus HY12. The conditions of ELISA method were optimized. Threshold defining positive and negative samples was determined statistically based on BEV-negative mice serum samples. Criteria for serum with OD490≥ 0.10 is considered as BEV-positive sample. Compared with the virus neutralization test, indirect ELISA method had a high sensitivity and specificity. Statistical analysis also showed that the coefficients of variation for positive serum and negative serum within the sample plate were 3.8% and 5.2%, respectively, indicating a stability of the ELISA assay. Mice were chosen as animal model and infected with HY12 enteroviruses to determine viral immunogenicity. Antibody titers were assayed using indirect ELISA. The antibody was detected as early as in the first week, then gradually increased and reached peak level in the sixth week before it decreased. The indirect ELISA established in this study for detecting BEV antibody will contribute to the diagnosis and epidemiological survey to this newly-emerging infection.In summary, we successfully established HY12 suckling mouse model and the indirect ELISA method for detection of the antibody for enterovirus, thus laying the basis for future researches related to viral pathogenesis, diagnosis and seroepidemiology, and vaccine development.