Anice C. Lowen

Influenza Virus Transmission Is Dependent on Relative Humidity and Temperature

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.

Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N.

Since 2003, more than 380 cases of H5N1 influenza virus infection of humans have been reported. Although the resultant disease in these cases was often severe or fatal, transmission of avian influenza viruses between humans is rare. The precise nature of the barrier blocking human-to-human spread is unknown. It is clear, however, that efficient human-to-human transmission of an antigenically novel influenza virus would result in a pandemic. Influenza viruses with changes at amino acids 627 or 701 of the PB2 protein have been isolated from human cases of highly pathogenic H5 and H7 avian influenza. Herein, we have used the guinea pig model to test the contributions of PB2 627 and 701 to mammalian transmission. To this end, viruses carrying mutations at these positions were generated in the A/Panama/2007/99 (H3N2) and A/Viet Nam/1203/04 (H5N1) backgrounds. In the context of either rPan99 or rVN1203, mutation of lysine 627 to the avian consensus residue glutamic acid was found to decrease transmission. Introduction of an asparagine at position 701, in conjunction with the K627E mutation, resulted in a phenotype more similar to that of the parental strains, suggesting that this residue can compensate for the lack of 627K in terms of increasing transmission in mammals. Thus, our data show that PB2 amino acids 627 and 701 are determinants of mammalian inter-host transmission in diverse virus backgrounds.

Animal Models for Influenza Virus Pathogenesis and Transmission

Influenza virus infection of humans results in a respiratory disease that ranges in severity from sub-clinical infection to primary viral pneumonia that can result in death. The clinical effects of infection vary with the exposure history, age and immune status of the host, and also the virulence of the influenza strain. In humans, the virus is transmitted through either aerosol or contact-based transfer of infectious respiratory secretions. As is evidenced by most zoonotic influenza virus infections, not all strains that can infect humans are able to transmit from person-to-person. Animal models of influenza are essential to research efforts aimed at understanding the viral and host factors that contribute to the disease and transmission outcomes of influenza virus infection in humans. These models furthermore allow the pre-clinical testing of antiviral drugs and vaccines aimed at reducing morbidity and mortality in the population through amelioration of the virulence or transmissibility of influenza viruses. Mice, ferrets, guinea pigs, cotton rats, hamsters and macaques have all been used to study influenza viruses and therapeutics targeting them. Each model presents unique advantages and disadvantages, which will be discussed herein.

Transmission of a 2009 Pandemic Influenza Virus Shows a Sensitivity to Temperature and Humidity Similar to That of an H3N2 Seasonal Strain

ABSTRACT In temperate regions of the world, influenza epidemics follow a highly regular seasonal pattern, in which activity peaks in midwinter. Consistently with this epidemiology, we have shown previously that the aerosol transmission of a seasonal H3N2 influenza virus is most efficient under cold, dry conditions. With the 2009 H1N1 pandemic, an exception to the standard seasonality of influenza developed: during 2009 in the Northern Hemisphere, an unusually high level of influenza virus activity over the spring and summer months was followed by a widespread epidemic which peaked in late October, approximately 2.5 months earlier than usual. Herein we show that aerosol transmission of a 2009 pandemic strain shows a dependence on relative humidity and temperature very similar to that of a seasonal H3N2 influenza virus. Our data indicate that the observed differences in the timings of outbreaks with regard to the seasons are most likely not due to intrinsic differences in transmission between the pandemic H1N1 and seasonal H3N2 influenza viruses.

Mutations in the NS1 C-terminal tail do not enhance replication or virulence of the 2009 pandemic H1N1 influenza A virus.

The ‘classical’ swine H1N1 influenza A virus lineage was established after the devastating 1918 human pandemic virus entered domestic pig herds. A descendent of this lineage recently re-emerged in humans as the 2009 pandemic H1N1 virus. Adaptation in pigs has led to several changes in the multifunctional viral NS1 protein as compared with the parental 1918 virus, most notably a K217E substitution that abolishes binding to host Crk/CrkL signalling adapters, and an 11 aa C-terminal truncation. Using reverse genetics, we reintroduced both these features into a prototype 2009 H1N1 strain, A/California/04/09. Restoration of Crk/CrkL binding or extension of NS1 to 230 aa had no impact on virus replication in human or swine cells. In addition, minimal effects on replication, pathogenicity and transmission were observed in mouse and ferret models. Our data suggest that the currently circulating 2009 H1N1 virus is optimized to replicate efficiently without requiring certain NS1 functions.

Effects of Receptor Binding Specificity of Avian Influenza Virus on the Human Innate Immune Response

ABSTRACT Humans infected by the highly pathogenic H5N1 avian influenza viruses (HPAIV) present unusually high concentrations in serum of proinflammatory cytokines and chemokines, which are believed to contribute to the high pathogenicity of these viruses. The hemagglutinins (HAs) of avian influenza viruses preferentially bind to sialic acids attached through α2,3 linkages (SAα2,3) to the terminal galactose of carbohydrates on the host cell surface, while the HAs from human strains bind to α2,6-linked SA (SAα2,6). To evaluate the role of the viral receptor specificity in promoting innate immune responses in humans, we generated recombinant influenza viruses, one bearing the HA and neuraminidase (NA) genes from the A/Vietnam/1203/2004 H5N1 HPAIV in an influenza A/Puerto Rico/8/1934 (A/PR/8/34) backbone with specificity for SAα2,3 and the other a mutant virus (with Q226L and G228S in the HA) with preferential receptor specificity for SAα2,6. Viruses with preferential affinity for SAα2,3 induced higher levels of proinflammatory cytokines and interferon (IFN)-inducible genes in primary human dendritic cells (DCs) than viruses with SAα2,6 binding specificity, and these differences were independent of viral replication, as shown by infections with UV-inactivated viruses. Moreover, human primary macrophages and respiratory epithelial cells showed higher expression of proinflammatory genes after infection with the virus with SAα2,3 affinity than after infection with the virus with SAα2,6 affinity. These data indicate that binding to SAα2,3 by H5N1 HPAIV may be sensed by human cells differently than binding to SAα2,6, inducing an exacerbated innate proinflammatory response in infected individuals.

The DBA.2 Mouse Is Susceptible to Disease following Infection with a Broad, but Limited, Range of Influenza A and B Viruses

ABSTRACT We assessed the relative susceptibilities to disease of the DBA.2 and C57BL/6 mouse models upon infection with a range of influenza A and B viruses. DBA.2 mice were more susceptible to disease upon inoculation with human H1N1 influenza A virus strains, several swine influenza viruses, and influenza B viruses but were not overtly susceptible to infection with human seasonal H3N2 strains. Hemagglutination inhibition and immunoglobulin isotype profiling indicated that DBA.2 and C57BL/6 mice generate comparable humoral responses upon equivalent 50% mouse lethal dose (MLD50) challenges with influenza virus. Our data demonstrate the utility of DBA.2 mice for the elucidation of influenza virus pathogenicity determinants and the testing of influenza vaccines.

2009 H1N1 swine flu: the 2010 perspective

In May 2009, as the H1N1 swine flu outbreak was in the early stages, a conference was held at the New York Academy of Sciences to discuss what was known about the virus and what was being done to stop the outbreak. In May 2010, a follow‐up conference was again held at the New York Academy of Sciences, but now to discuss the H1N1 outbreak retrospectively. The report presented here summarizes the 2010 conference proceedings.