The rescue of H1N1 subtype swine influenza virus / 生物工程学报

ABSTRACT

The swine influenza virus (SIV) strain A/Swine/TianJin/01/2004(H1N1) (A/S/TJ/04) was rescued successfully by an eight-plasmid rescue system. The cDNAs of SIV 8 gene segments were synthesized by RT-PCR and cloned into the RNA polymerase I/II bidirection expression vector PHW2000 independently, resulting in 8 recombinant plasmids. The 8 recombinant plasmids were cotransfected into COS-1 cell, 30 h later TPCK-trypsin was added to 0.5 microg/mL. The COS-1 cell and supernatant were harvested 48 h after cotransfection and were inoculated into the allantoic cavity of 9-day-old specific-pathogen free (SPF) chicken eggs. The allantoic fluid of dead eggs was harvested and passaged 3 generations in SPF chicken eggs to get infective virus. The successful rescue of A/S/TJ/04 SIV was identified by hemagglutination assay, hemagglutination inhibition assay, sequence analysis and electron microscope observation. The successful rescue of SIV built a platform for the research of the relationship between genome structure and function of SIV, the mechanisms of trans-species transmission of influenza virus and for the generation of new SIV as vaccine.