We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator—together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 × 10 ³ plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 × 10 ⁴ –5 × 10 ⁷ pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.

The virulence of influenza virus is a multigenic trait. One determinant of virulence is the multifunctional NS1 protein that functions in several ways to defeat the cellular innate immune response. Recent large-scale genome sequence analysis of avian influenza virus isolates indicated that four C-terminal residues of the NS1 protein is a PDZ ligand domain of the X-S/T-X-V type and it was speculated that it may represent a virulence determinant. To test this hypothesis, by using mice as a model system, the four C-terminal amino acid residues of a number of influenza virus strains were engineered into the A/WSN/33 virus NS1 protein by reverse genetics and the pathogenicity of the viruses determined. Viruses containing NS1 sequences from the 1918 H1N1 and H5N1 highly pathogenic avian influenza (HPAI) viruses demonstrated increased virulence in infected mice compared with wt A/WSN/33 virus, as characterized by rapid loss of body weight, decreased survival time, and decreased mean lethal dose. Histopathological analysis of infected mouse lung tissues demonstrated severe alveolitis, hemorrhaging, and spread of the virus throughout the entire lung. The increase in pathogenicity was not caused by the overproduction of IFN, suggesting the NS1 protein C terminus may interact with PDZ-binding protein(s) and modulate pathogenicity through alternative mechanisms.

Vaccination represents the most effective form of protection against influenza infection. While neutralizing antibodies are typically measured as a correlate of vaccine-induced protective immunity against influenza, nonneutralizing antibodies may contribute to protection or amelioration of disease. The goal of this study was to dissect the individual contributions of the immunoglobulin G1 (IgG1) and IgG2a antibody isotypes to vaccine-induced immunity against influenza virus. To accomplish this, we utilized an influenza vaccine regimen that selectively enhanced IgG1 or IgG2a antibodies by using either DNA or viral replicon particle (VRP) vectors expressing influenza virus hemagglutinin (HA) (HA-DNA or HA-VRP, respectively). After HA-DNA vaccination, neutralizing antibodies were detected by both in vitro (microneutralization) and in vivo (lung viral titer) methods and were associated with increased IgG1 expression by enzyme-linked immunosorbent assay (ELISA). Vaccination with HA-VRP did not strongly stimulate either neutralizing or IgG1 antibodies but did induce IgG2a antibodies. Expression of IgG2a antibodies in this context correlated with clearance of virus and increased protection against lethal influenza challenge. Increased induction of both antibody isotypes as measured by ELISA was a better correlate for vaccine efficacy than neutralization alone. This study details separate but important roles for both IgG1 and IgG2a expression in vaccination against influenza and argues for the development of vaccine regimens that stimulate and measure expression of both antibody isotypes.

H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT) H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu) residue at amino acid position 226 in the hemagglutinin (HA) receptor-binding site (RBS), responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.