Generation Of Vero Cell Adapted H1N1 Influenza Virus By Reverse Genetics

Outbreaks of H1N1 influenza are caused by influenza virus (SIV) belonging to the family Orthomyxoviridae. Since its first case report in Mexico in March 2009, a novel swineorigin H1N1 influenza virus (S-OIV) has now spread worldwide, infecting more than 621719 people, claiming more than 18001 lives thus far. On June 11,2009, the World Health Organization (WHO) raised the H1N1 global pandemic to level 6. Now the main method to prevent influenza is to inoculate vaccines.In the market the influenza vaccines are mainly the inactive vaccines made by chicken embryos. There are many aspects should be promoted of the influenza vaccine production by egg embryos.For example, the vaccine produced cope with influenza pandemic is not enough.Due to the disadvantages of chicken embryos,it is important to improve the influenza based on mammal cells. Vero cell is WHO resigned cell strain to produce vaccine and can be cultured at large scale by the bioreactor which has great foreground to produce influenza vaccine. But Vero cell is not the influenza virus susceptible cell naturally.So it is important to obtain high-growth H1N1 influenza virus in Vero cells for the influenza vaccine production by Vero cells.All eight cDNA segments of Vero cell adapted influenza virus A/yunnan/1/2005Va (H3N2) were individually cloned into vector pHW2000. All of the plasmids were sequenced to ensure that additional mutations were not introduced during PCR.A gene library about Vero cell adapted influenza virus A/yunnan/1/2005Va (H3N2) was created.Development of vaccines against this pathogen has been underway in a number of laboratories and a few vaccines candidates have been reported and being tested for their efficacy.The plasmid-based system has been shown to be a simple and reliable method for generation of vaccine virus strains. The construction of reassortant virus harboring the HA and NA glycoproteins of A/California/reassortant/NYMC X-179A, the NS and M of Vero cell adapted influenza virus A/yunnan/1/2005Va (H3N2) while PB2,PB1,PA and NP genes are those derived from laboratory-adapted strains such as A/PR/8/34 can be done through transfection of 8 individual plasmids into mammalian cells. we cloned HA and NA genes of A/California/reassortant/NYMC X-179A. NS and M of Vero cell adapted influenza virus A/yunnan/1/2005Va (H3N2) into a bidirectional plasmid, pHW2000, and subsequently used them for transfection according to previously described protocols. The rescued viruses were continuously passaged for nine times. The subtype was determined by HI test,Immunoflourescence Stain Assay,SRID. The ICR mice were immunized with the inactivated influenza vaccine produced in Vero cells using the reassortant strain. The results showed reassortanted influenza virus(H1N1),which are adapted to Vero cells was prepared and there was no significant difference in immunogenicity of this strain while compared with parental strain A/California/reassortant/NYMC X-179A (HIN1). The conclusion is that the Vero cell adapted influenza virus A/yunnan/1/2005Va (H3N2) can be used as donor strain to prepare reassortmented vaccine strains adapted to Vero cells.