Research On Cultivation And Post-treatment Process Of Vero Cell-derived H5N1 Influenza Virus

Outbreaks of avian influenza virus caused social panic and economic losses have been seen many times in China in recent years.Vaccination is the most economic and ef-fective measure for influenza prevention and control.Chicken embryos is the only virus culture matrix in all Influenza vaccine approved in China.But the supply of chicken em-bryos needs a long time,it can't provide enough vaccine in response to influenza pandemic that can happen at uncertain times.Vero cell is WHO resigned cell strain to produce vaccine and can be cultured at large scale by CellFactory and Microcarrier.Vero cell-derived influenza virus is closer to the epidemic strains.Therefore,it is necessary to develop Vero cell-derived pandemic influenza vaccine.Cellfactory used to culture cells at large scale is mature and widespread in the do-mestic and overseas.It has convenient and flexible security operation,less occupying space,good controllability.This paper mainly studies the growth of Vero cells in Cellfactories and optimize the culture conditions of influenza virus.The optimum culture conditions for H5N1 influenza virus in cellfactory:cellage for 60?72 h?2 ?g/ml TPCK-Trypsin?0.1 MOI?the harvest time for 72 h?culture temperature for 33±0.5 ??pH 7.6 and the viral titer is 1:256?1:1024.Immunofluorescence test found H5N1 Va vi-rus can well replicate in Vero cells.Hemagglutination inhibition Assay of H5N1 Va for phenotype identification shows Vero cell-derived virus is H5N1 Va.BLAST pairwise alignments confirms the surface antigen genes of H5N1 Va,HA and NA,come from A/Anhui/1/2005(H5N1).There are some problems in vaccine production with a continuous cell line,Such as residual DNA has potential tumorigenicity.Vaccine production need to control Vero cell within 150 generation,and need purification in order to reduce residual Vero cell DNA and proteins.In this paper,we study Vero cell-derived pandemic influenza vaccine post-treatment process.Virus harvest fluid is clarified using 0.65?0.45?0.22 ?m filter car-tridges,concentrated with 300 KD UF membrane,Benzonase treatment at 37 ? for 4 h,concentrated with 1000 KD UF membrane below 5 psi,Chromatographic purification with Sepharose 4 FF and DEAE Sepharose FF.Finally,Vero cell-derived influenza vac-cine reaches Chinese pharmacopoeia requirements.The study will lay the foundation for development of Vero cell-derived pandemic influenza vaccine.