Using the guinea pig as a model host, we show that aerosol spread of influenza virus is dependent upon both ambient relative humidity and temperature. Twenty experiments performed at relative humidities from 20% to 80% and 5 °C, 20 °C, or 30 °C indicated that both cold and dry conditions favor transmission. The relationship between transmission via aerosols and relative humidity at 20 °C is similar to that previously reported for the stability of influenza viruses (except at high relative humidity, 80%), implying that the effects of humidity act largely at the level of the virus particle. For infected guinea pigs housed at 5 °C, the duration of peak shedding was approximately 40 h longer than that of animals housed at 20 °C; this increased shedding likely accounts for the enhanced transmission seen at 5 °C. To investigate the mechanism permitting prolonged viral growth, expression levels in the upper respiratory tract of several innate immune mediators were determined. Innate responses proved to be comparable between animals housed at 5 °C and 20 °C, suggesting that cold temperature (5 °C) does not impair the innate immune response in this system. Although the seasonal epidemiology of influenza is well characterized, the underlying reasons for predominant wintertime spread are not clear. We provide direct, experimental evidence to support the role of weather conditions in the dynamics of influenza and thereby address a long-standing question fundamental to the understanding of influenza epidemiology and evolution.

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While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

Neutralizing antibodies against influenza viruses have traditionally been thought to provide protection exclusively through their variable region; the contributions of mechanisms conferred by the Fc domain remain controversial. We investigated the in vivo contributions of Fc interactions with their cognate receptors for a collection of neutralizing anti-influenza antibodies. Whereas five broadly neutralizing monoclonal antibodies (bNAbs) targeting the conserved stalk region of hemagglutinin (HA) required interactions between the antibody Fc and Fc receptors for IgG (FcγRs) to confer protection from lethal H1N1 challenge, three strain-specific monoclonal Abs (mAbs) against the variable head domain of HA were equally protective in the presence or absence of FcγR interactions. Although all antibodies blocked infection, only anti-stalk bNAbs were capable of mediating cytotoxicity of infected cells, which accounts for their FcγR dependence. Immune complexes generated with anti–HA stalk mAb efficiently interacted with FcγRs, but anti–HA head immune complexes did not. These results suggest that FcγR binding capacity by anti-HA antibodies was dependent on the interaction of the cognate Fab with antigen. We exploited these disparate mechanisms of mAb-mediated protection to reengineer an anti-stalk bNAb to selectively enhance FcγR engagement to augment its protective activity. These findings reveal a previously uncharacterized property of bNAbs and guide an approach toward enhancing mAb-mediated antiviral therapeutics.

The NS1 protein is the only nonstructural protein encoded by influenza A virus. It has been proposed that the NS1 performs several regulatory functions during the viral replication cycle, including the regulation of synthesis, transport, splicing, and translation of mRNAs. Through the use of reverse genetics, a viable transfectant influenza A virus (delNS1) which lacks the NS1 gene has been generated. Our results indicate that the NS1 of influenza A virus is an auxiliary (virulence) factor which plays a crucial role in inhibiting interferon-mediated antiviral responses of the host.