Adaptation Of Enterovirus71 H6 Strain In Vero Cells

Enterovirus71 (EV71) infection has been associated with hand-foot-and-mouth disease (HFMD), herpangina, aseptic meningitis, polio-like paralysis, brain- stem encephalitis, pulmonary edema. Several EV71 outbreaks associated with severe neurological diseases were recently reported in Asia-Pacific region, including Malaysia in 1997, Australia in 1999, Singapore in 2000, Japan in 1997 and 2000, Taiwan in 1998, and China in 2008 and 2009. The cases with EV71-associated neurological syndrome continually increased in many countries during past ten years. With the spread of EV71 in the world, people are focus on how to control and therapy virus. There is no specific medicine and vaccine to therapy and prevention of the EV71. So it is important to research and develop one vaccine which can control the epidemic of EV71. To provide a large number of antigens for production of vaccines, adaptation and passages of EV71 H6 strain in Vero calls was studied in present thesis.In this study, EV71 H6 strain isolates were investigated systematically. To obtain the adaptive strain of EV71 H6 in Vero cells, EV71 H6 strain was isolated from RD calls, and was inoculated to Vero cells for plaque screening and further subculture. Then biological, Immunological, and physical-chemical Properties of EV71 H6 adaptive strain were studied.The faeces sample which from HFMD infants clinically diagnosed was transacted by routine method. The sample was cultured in RD cell, and the pronounced cytopathic effect revealed after 3 times blind passages. Then EV71 H6 was inoculated to Vero cells for plaque screening and further subculture. In the process of adaptation, the size of plagues of all passages had no change. The adaptation of virus to Vero cells was observed from the 5th passage expressed in the cytopathic effect (CPE) more significant and the titers of virus increased from 4.75 LogCCID50/ml to 8.13 LogCCID50/ml. Further cultured, the virus titer was stable and beyond 8.00 LogCCID50/ml. The optimal propagation conditions of EV71 were tested. The optimal MOI of adapted strain was 0.01, the optimal harvest time was 96h. The virus strain was stable under storage at -80℃. Physic-chemically, EV71 was a RNA virus, and sensitive to heat, resistant to aether, acid. The adaptive strain of EV71 H6 can be neutralized by specific anti-EV71 serum. The test of neutralization was done on RD cells, and the neutralization antibody titer of rabbits were gradually increase, and the maximum titer was as high as 1:1024. It suggests that this adaptive strain of EV71 H6 on Vero cells have good immunogenicity.The results of the preliminary experiments show that EV71 H6 can be well adapted in Vero cells culture, and keep stable biological character, and it offers valuable information for further research of EV71 vaccine.