The rescue of H1N1 subtype swine influenza virus.
- Author:
Yaping PENG
1
;
Hongbo ZHOU
;
Chun LI
;
Meilin JIN
Author Information
1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
- Publication Type:Journal Article
- MeSH:
Animals;
COS Cells;
Cercopithecus aethiops;
Chick Embryo;
Chickens;
Influenza A Virus, H1N1 Subtype;
genetics;
physiology;
Plasmids;
genetics;
RNA Polymerase I;
genetics;
RNA Polymerase II;
genetics;
Recombination, Genetic;
genetics;
Swine;
Transfection;
Virus Replication
- From:
Chinese Journal of Biotechnology
2008;24(5):857-861
- CountryChina
- Language:Chinese
-
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.
