Adaptability On Vero Cells And Inactivation Research Of Rotavirus P[2]G3 Strain And P[8]G1 Strain

Rotavirus is the most common cause of severe infantile diarrhea worldwild and also the important reason for the children deaths in developing countries, responsible for 50% of intestinal infection. Rotavirus, a member of the family Reoviridae, is divided into seven groups(A-G) according to the antigenicity of the structure protein VP6. RV vaccines at present aim directly at group A rotaviruses, the major cause of severe dehydrating gastroenteritis among the infants and children. RV diarrhea has already become a severe public health problem and brung heavy disease burden and economic burden to society. There is no specifics to RV diarrhea now. And the improvement of water quality and public health condition can not cut down the attack rate of RV diarrhea obviously. Therefore, to develop RV vaccine in high performance, safety and lower price is the only approach to prevent and control RV infection. WHO has been thinking highly of the research and development of RV vaccine. The RV vaccine utilized clinically are all attenuated live vaccine for oral use, which has gained fine immunization and protection effect when taken by susceptible population. However, because of the danger of seroconversion and potential severe side effect (intu-ssusception), the attenuated live vaccine is not considered as the main aspect of the research of RV vaccine yet. Some extra-intestinal RV vaccines, e.g. subunit vaccine, virus-like particle, inactivated vaccine, DNA vaccine and transgenic plant vaccine can result in some protection effect in animal model and show potential application value, although they have not reached the clinical observation stage yet. Among these new vaccines, inactivated vaccine is becoming a hot spot of the research of RV vaccine gradually in advantage of its safety and stability.We cultured two serotype RV P[2]G3 strain (SA11 strain) and P[8]G1 strain (Wa strain) purified by plaque adapting to Vero cells to reach the demand of scale virus culture by microcarrier. We passaged RV serially on Vero cells and determined the best MOI to be 0.05. RV growth curve is drawn showing that the virus titer reached the peak 3-4 d after virus inoculation and droped afterward. The maximum titer of SA11 strain (9th generation) is 7.0 CCID₅₀/ml, Wa strain (9th generation) is 6.25 CCID₅₀/ml. Both strains achieved higher titer through serial passage: SA11 strain (10th generation) is about 7.25 CCID₅₀/ml, Wa strain (10th generation) is about 6.5 CCID₅₀/ml, and both strains keeped the same genome banding pattern showing fine genetic stability. Higher viral titer can be gained when cultured on Vero cells by microcarrier: SA11 strain (9th generation) is 7.25 CCID₅₀/ml, Wa strain (9th generation) is 6.75 CCID₅₀/ml. Harvest virus and inactivate byβ-Propiolactone, formaldehyde and heating. The inactivation effect was detected by blind passage for 3 generations on MA104 cells. Only the SA11 strain cultured on MA104 cells and by microcarrier inactivated under the condition of heating 30 min at 56℃and then addition of 1:4000 (v/v)β-Propiolactone sturred at 4℃for 8 days was inactivated thoroughly. The complete virosome can be seen by electron microscope. The concentrated venom of Wa strain was purified by Sepharose 4FF solvent resistant column. The first peak at 280nm is the purified virus peak determined by SDS-PAGE and Western blot. The protein density is 0.788mg/ml working out in the way of Bradford.This research cultured two serotype RV P[2]G3 strain (SA11 strain) and P[8]G1 strain (Wa strain) adapting to Vero cells successfully. Higher viral titer can be gained when cultured on Vero cells by microcarrier. We have also researched for the technology of viral inactivation and purification that will promote the research and development of the RV inactivated vaccine.