Generation of influenza A viruses entirely from cloned cDNAs

Abstract

We describe a new reverse-genetics system that allows one to efficiently generate inf luenza A viruses entirely from cloned cDNAs.

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

“…The A/WSN/1933 H1N1 (here referred to as WSN33) 12-plasmid reverse genetics system (38) was kindly provided by Yoshihiro Kawaoka (Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI). The expression plasmid for T7T7NS1 was developed by reverse transcription-PCR (RT-PCR) cloning using our laboratory strain of PR8 and the general methodology previously described by Hoffmann et al (39).…”

“…To map the SUMOylation site, specific mutations in the sequence of NS1 were subsequently inserted by using the same site-directed mutagenesis approach, resulting in the expression constructs for T7T7NS1K70A, T7T7NS1K219A, and the double mutant T7T7K70AK219A. To generate the pPolI/WSN/T7T7NS1 construct, the pPolI/WSN/NS construct derived from the WSN33 12-plasmid reverse genetics system (38) was PCR amplified by using primers complementary to the 5= untranslated region (UTR) and the 3= UTR of the WSN33 NS gene segment, producing a PCR product comprising the backbone of the original plasmid and the 5= and 3= UTRs of the NS gene segment but missing the totality of the ORF for NS1 and NS2. This PCR product was ligated with the PCR-amplified coding region of the NS gene segment from our PR8 strain, therefore generating a recombinant carrying the 5= and 3= UTRs of the WSN33 NS gene segment and the ORF from the PR8 NS gene segment.…”

Heterologous HA DNA vaccine prime—inactivated influenza vaccine boost is more effective than using DNA or inactivated vaccine alone in eliciting antibody responses against H1 or H3 serotype influenza viruses

“…Another type of influenza vaccine is the cold-adapted live influenza virus vaccine (CAIV) which has been shown to be more immunogenic than TIV in inducing protective immunity and may be associated with a longer-lasting and more cross-protective immune response than is elicited by TIV [5]. A new technology termed 'reverse genetics' has been developed to generate high growth reassortants [6][7][8][9][10][11] and combines viral genes from the high growth yield laboratory strain of influenza A virus A/PR/8/34 (H1N1) with genes encoding protective antigens of the target viral strains [12]. However, this technology does not change the subsequent manufacturing process needed to produce large stocks of vaccine viruses to make the final TIV or CAIV formulations.…”

Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses

“…This reassortant virus is also attenuated by removal of the polybasic cleavage site of the HA which is associated with high pathogenicity [4][5][6]. These reverse genetics(RG)-derived reassortants [7,8] are then subjected to extensive safety testing before distribution to the influenza vaccine manufacturers. This procedure is essential to allow use of the virus under the biosafety level 2 enhanced, which is the highest safety level available in egg-based manufacturing facilities, and to generate the potential high growth phenotype required for adequate vaccine antigen yield.…”