Research On H7N9 Influenza Virus Cultured In Vero Cells And Its Purification Process

The influenza outbreak and cause epidemic every year on a global scale,which is a serious threat to human health.Since 2013,there have been five outbreaks of H7N9 epidemic in China.As of April 2019,there were 1,642 cases of H7N9 infection,of which 612 died and the mortality rate was as high as 39%.Vaccination is the most cost-effective preventive measure to aganist influenza.However,current influenza vaccines are based on chicken embryo,which have many defects that resulting in allergies,and long periodic time of supplyment.The World Health Organization recommends the use of Vero cells as an alternative to the production of influenza vaccines.The virus strain used in this study is the Vero cell adaptation strain A/Shanghai/02/2013(H7N9)Va that reassorted by reverse genetics in the laboratory,and we further optimize its culture conditions on Vero cells.The virus production rate on Vero cells was increased,the haemagluttinin titer was up to 1:512 and LgTCID50/ml was 8.5,both remained stable after passage for 20 generations.Then we establish a virus bank.The production process of large-scale cultivation of influenza virus in cell factories was optimi2ed,and the haemagluttinin titer of virus was stable at 1:1024,which realized large-scale production.The H7N9Va virus solution was purified by filtration clarification,formaldehyde inactivation,300KD ultrafiltration,and then purified by a two-stage chromatography including anion exchange chromatography Unigel-30Q and complex mode chromatography Capto core 700.The salt concentration in the loading buffer of the Unigel-30Q chromatography was 0.5M NaCl+20mM PB,pH 6.8-7.4,and the salt concentration in the loading buffer of Capto core 700 chromatography was 0.15M NaCl+20 mMPB,pH 7.4.More importantly,and the anion exchange Unigel-30Q can achieve a residual DNA removal rate of more than 90%.The entire purification process avoids the virus capture step,greatly reduces the virus loss rate,and can quickly and efficiently purify the virus.The final virus recovery rate was 58.4%,the final content of haemagluttinin was 132.12μg/ml.the residual DNA removal rate of Vero cells was 99.95%,and the final content of residual DNA was 28.69 pg/dose,the protein removal rate was 98.87%,and the final Vero cell host protein content was 28.28 ng/dose,The albumin content was 11.36 ng/dose,both of which met the current Chinese Pharmacopoeia and WHO standards.In summary,this research determined the appropriate conditions for the culture of influenza virus A/Shanghai/02/2013(H7N9)Va in Vero cells,and established a virus bank.The large-scale cultivation influenza virus H7N9Va in Vero cells has been achieved,and the fast and efficient purification process of the influenza virus whole virus inactivated vaccine has been obtained,which is of great significance for the future emergency response to pandemic influenza outbreaks.