Isolation And Identification Of The Local Strain Of Porcine Epidemic Diarrhea Virus LJB/03

A porcine epidemic diarrhea virus (PEDV) designated LJB/03 was isolated from the feces of diarrheaed piglets and cultured in Vero cell propagated in maintenance medium(MM) containing 3μg/ml trypsin.The isolated virus was identified by stable cytopathic effects (CPE), TCID50, RT-PCR, immunofluorescence, ELISA ect. The experimental results indicated that the bland passages of 20 genrations were undertaken untill the CPE could be seen on 21 genrations in 48 h and were harvested in 72h. CPE was shorten and intensified with increased passages.On passage 40 in Vero cell culture, CPE characterized by cellular swelling , refracted reinforcement and particulate matter increaser was detected microscopically after 24h; A great quantity of cells defluxioed and the airspace amang cells was enlarged after 36h; the cells having defluxioed reached 80% and Vero cells reticulated after 48h.But the cell control still retained a pyknomonolayer and there was a small quantity of cells having defluxioed . At passage levels 1, 10, 20, 30,and 40, PEDVs were identified through RT-PCR for every 10 passage on the M gene in order to certificated that the CPE caused by PEDV and the virus propagated in Vero cells. The results were masculine.Indirect immunofluorescence test indicated that viral antigen could be detected and could be seen green fluorescence in Vero cells. No green fluorescence could be seen with the negative control of mock-infected Vero cell.Indirect ELISA detected that P/N was 7.6.It was justified that the virus in Vero cell culture was masculine. Titer of the virus was10-5.2TCID50/0.1 ml at 40 passage.Above experiments certified that it was PEDV from the feces and it propagated in Vero cell culture in medium containing trypsin.