Researches On Cell Culture And Experimental Infection And Development Of Indirect Sandwich ELISA To Detect Porcine Rotavirus

Porcine rotavirus is one of the main pathogens of viral diarrhea which does harm to swine industry. The key technique of cell culture for porcine rotavirus was studied using rotavirus OSU strain and experimental infection of porcine rotavirus in piglets was investigated. Indirect sandwich enzyme-linked inmunosorbent assay(ELISA) was developed for detecting porcine rotavirus. The study contributed to the further application study on porcine rotavirus.Porcine rotavirus was cultured successfully on MA-104 cells. The virus was treated for 1h by trypsin on final concentration of 20μg/mL before inoculating and then inoculated in maintain medium with 4μg/mL trypsin. The cytopathic effect such as kytoplasm connecting, botryoidalis accretion and net-balloon was clearly observed post 10 passage. Double capsid virus was detected by Electronmicroscope. Healthy 3-day-old colostrums-deprived piglets were inoculated orally with 1×10(6.8) TCID₅₀ of porcine rotavirus. Piglet developed watery diarrhea post 10h infection, and died post 42h infection. During necropsy, milk curd in stomach, fluid in thinwalled small intestines, ectocolon were observed. During histopathological examination, slight to moderate villous stunting were observed. Bulging and sloughing of villous epithelial cells were prominent. Neutrophils were clustered in the lamina propria at villous tips subjacent to disrupted epithelium.In severity segment of villous destruction, necrosis and sloughing of exposed lamina propria resulted in complete loss of villous strcture.Indirect sandwich ELISA was developed for detecting porcine rotavirus. Rotavirus was extracted from cell culture by ultracentifigation, which was used as antigen for immunization of pig and rabbit respectively. Porcine anti RV polyclone IgG and rabbit anti RV IgG were both extracted, and indirect sandwich ELISA was developed by these two kind of IgG. Through the optimization test, the results were as follows: the coating porcine anti RV IgG concentration was 4μg/mL, and the coating tempetature and time was at 4℃incubating for a night. The blocking buffer was 1% bovine serum albumin(BSA) and reaction for 60min at 37℃. Antigen was incubated for 90min at 37℃. The concentration of rabbit anti RV IgG was 3.5μg/mL, and incubated for 90min at 37℃. The optimation dilution of horseradish peroxidase(HRP)-labelled goat anti-rabbbit IgG was 1:8000, and incubated for 90min at 37°C. Substrate reacted for 15min.The cutoff of optical density(OD)at 450 nanometers(nm) wavelength was 0.161. The indirect sandwich ELISA was confirmed to have good reproducibility by the repeating test, and was showed specificity because there were no cross reaction with Transmissible gastro-enteritis virus(TGEV), Porcine reproductive and respiratory Syndrome virus(PRRSV),Porcine Parvovirus(PPV),Japanese encephalitis virus(JEV), Escherichia coli, Actinobacillus pleuropenumoniae.The indirect sandwich ELISA was proved to have high sensitivity and the least detectable antigen was 1.25μg/mL.The coated polystyrene microtiter plates can preserved at ordinary temperature,-20℃and 4℃for at least 4 weeks after washed.