Abstract
Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high- growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6+2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/ MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics.
Highlights
Influenza viruses are members of orthomyxoviridae family and composed of 8 segmented negative-sense, single-stranded RNAs
Beside well-known human threats posed by H5N1 avian influenza, the potential for newly emerging influenza virus has been elevated with recent outbreak of Swine influenza viruses into the human (Novel Swine-Origin Influenza A Virus Investigation Team, 2009)
The eight plasmids set of X-31 and X-31ca were transfected into 293T cells, and after 3 days of incubation, the supernatants were collected for plaque assay on Madin-Darby Canine kidney (MDCK) cells
Summary
Influenza viruses are members of orthomyxoviridae family and composed of 8 segmented negative-sense, single-stranded RNAs. A total of 16 haemagglutinin subtype and 9 neuraminidase subtype were identified in influenza A type (Webby and Webster, 2001; Nicholson et al, 2003). Due to segmented nature of RNA genes, the virus can exchange their RNA segments genes with other subtypes of influenza viruses causing major changes of antigenicity. The replication of the RNA genes is error-prone, allowing frequent nucleotide substitutions at genetic level (Webby and Webster, 2001). Frequent antigenic drift and shift provides the diversity of influenza viruses that causes the emergence and re-emergence of epidemic and pandemic viruses. Beside well-known human threats posed by H5N1 avian influenza, the potential for newly emerging influenza virus has been elevated with recent outbreak of Swine influenza viruses into the human (Novel Swine-Origin Influenza A Virus Investigation Team, 2009). The development of novel antiviral therapy and efficient vaccine is urgently needed
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