| Background Enteroviruses,a family of small RNAs,are positive strand RNA viruses which exist widely in nature.At present,more than 70 serum-types enteroviruses have been found.Normally,enteroviruses cause mild cases such as low fever,fatigue,burnout,etc.However,some serious cases may result in systemic infection or damages to brain,spinal cord,heart and other important organs with poor prognosis,sequelae or even death.In recent years,the aggression of infectious diseases caused by enteroviruses has spread widely,and the epidemic areas have gradually spread in Europe,Asia and the Pacific region.The epidemic scale also gradually changed from sporadic distribution to collective,large-area outbreak without obvious regularity among the severe cases.As the epidemic continues to spread in the Asia Pacific region,there is a similar epidemic in mainland China and the number of infected people has been on a rapid rise.In many domestic areas with underdeveloped public health conditions,enterovirus-related infectious diseases,for which there is no effective treatment at present,have posed a great threat to the healthy population.Therefore,the research of targeted prophylactic vaccine has become an urgent task in the pharmaceutical field.Objective In order to study the production process and and preliminarilyexplore the candidate strains for enterovirus vaccine,this experiment was carried out to verify the applicability of existing production and purificationprocesses for the production of different enteroviruses,and to determine preliminarily whether the selected strains had high stable genetic traits required for the production of experimental vaccines.Methods The 4 standard strains,Coxsackievirus group B type 2(CoxB2),group B type 6(CoxB6),group A type 7(CoxA7)and Echo virus type 14(Echo 14),were inoculated on Vero cells and were cultured,harvested,concentrated by PEG10000,extracted by chloroform,purified by OptiPrepTM Density Gradient Medium.After The purification liquidswere made,RNA of the 4 strains were extracted to obtain VP1 fragments,the major antigenic epitope gene,which were amplified by the RT-PCR method.Through sequencing the 4 VP1 fragments and comparing the results respectively with the standard sequences of original strains,the genetic stability of the 4 strains were analyzed.Subsequently,the purified CoxB6 virus was inactivated by the existing production processes to prepare for experimental vaccine,which was injected into baby rhesus monkeys to evaluate preliminarily the immunogenicity by testing the level of neutralizing antibody.Results These 4 different strains of enterovirus all adapted to Vero cells and grew in Vero cells,which caused cytopathic effect(CPE)48 hours after inoculation.And after 72 hours’ culture,the proliferated viruses of the 4 strains could be harvested for concentrating and purifying.High purity of virus in the purification liquids was detected by SDS-PAGE(polyacrylamide gel electrophoresis)and the virus titer of the 4 strains were all more than 9.5 lgCCID50/ml detected by micro hole method.Comparisons between the VP1 gene as well as protein sequences from the 4 adapted strainsand the standard sequences from their corresponding original strains shew that the mutation rate was close to the natural mutation average level of enterovirus.The CoxB6 virus could be inactivated completely by formaldehyde,and the expeirmental inactivated vaccine of CoxB6 successfuly induced neutralizing antibodies in rhesus monkeys.Conclusion Existing production and purification processes is applicable for different enteroviruses;Under the condition of the existing production processes,the VP1 gene and protein sequence of the 4 selected strains did not changed significantly.They all maintained a good stability in genetic traits,providing the reference for the next step of vaccine development.At the same time,the experimental inactivated vaccine prepared by the existing production processes can induce the immune response of the experimental animals. |
PDF Full Text Request