Host innate immunity is the first line of defense against invading pathogens, including influenza viruses. Ferrets are well recognized as the best model of influenza virus pathogenesis and transmission, but little is known about the innate immune response of ferrets after infection with this virus. The goal of this study was to investigate the contribution of localized host responses to influenza virus pathogenicity and transmissibility in this model by measuring the level of messenger RNA expression of 12 cytokines and chemokines in the upper and lower respiratory tracts of ferrets infected with H5N1, H1N1, or H3N2 influenza viruses that exhibit diverse virulence and transmissibility in ferrets. We found a strong temporal correlation between inflammatory mediators and the kinetics and frequency of transmission, clinical signs associated with transmission, peak virus shedding, and virulence. Our findings point to a link between localized innate immunity and influenza virus transmission and disease progression.

Continued H5N1 virus infection in humans highlights the need for vaccine strategies that provide cross-clade protection against this rapidly evolving virus. We report a comparative evaluation in ferrets of the immunogenicity and cross-protective efficacy of isogenic mammalian cell-grown, live attenuated influenza vaccine (LAIV) and adjuvanted, whole-virus, inactivated influenza vaccine (IIV), produced from a clade 1 H5N1 6:2 reassortant vaccine candidate (caVN1203-Len17rg) based on the cold-adapted A/Leningrad/134/17/57 (H2N2) master donor virus. Two doses of LAIV or IIV provided complete protection against lethal homologous H5N1 virus challenge and a reduction in virus shedding and disease severity after heterologous clade 2.2.1 H5N1 virus challenge and increased virus-specific serum and nasal wash antibody levels. Although both vaccines demonstrated cross-protective efficacy, LAIV induced higher levels of nasal wash IgA and reduction of heterologous virus shedding, compared with IIV. Thus, enhanced respiratory tract antibody responses elicited by LAIV were associated with improved cross-clade protection.

Human infections with highly pathogenic H5N1 avian influenza viruses continue to occur in many parts of the world and pose a considerable public health threat. With the use of animal models, the identification of virulence determinants has been instrumental in improving our understanding of how these viruses cause severe disease in humans. Two genetically similar H5N1 viruses (A/Thailand/16/2004 and A/Thailand/SP83/2004) exhibit high or low virulence phenotypes, respectively, in multiple animal models. Reassortant viruses were generated from this virus pair and evaluated in ferrets. Each of the polymerase genes of A/Thailand/16/2004 virus individually conferred increased virulence to A/Thailand/SP83/2004 virus while the neuraminidase of the low virulence virus reduced virulence and replication efficiency of the virulent virus in ferrets unless the homologous HA was present. Our results demonstrate that H5N1 virus virulence determinants are polygenic and that there is an important correlation between polymerase adaptation, efficient replication in the host, and virulence.

Although H5N1 influenza viruses have been responsible for hundreds of human infections, these avian influenza viruses have not fully adapted to the human host. The lack of sustained transmission in humans may be due, in part, to their avian-like receptor preference. Here, we have introduced receptor binding domain mutations within the hemagglutinin (HA) gene of two H5N1 viruses and evaluated changes in receptor binding specificity by glycan microarray analysis. The impact of these mutations on replication efficiency was assessed in vitro and in vivo. Although certain mutations switched the receptor binding preference of the H5 HA, the rescued mutant viruses displayed reduced replication in vitro and delayed peak virus shedding in ferrets. An improvement in transmission efficiency was not observed with any of the mutants compared to the parental viruses, indicating that alternative molecular changes are required for H5N1 viruses to fully adapt to humans and to acquire pandemic capability.

This study investigated whether transmissible H5 subtype human-avian reassortant viruses could be generated in vivo. To this end, ferrets were coinfected with recent avian H5N1 (A/Thailand/16/04) and human H3N2 (A/Wyoming/3/03) viruses. Genotype analyses of plaque-purified viruses from nasal secretions of coinfected ferrets revealed that approximately 9% of recovered viruses contained genes from both progenitor viruses. H5 and H3 subtype viruses, including reassortants, were found in airways extending toward and in the upper respiratory tract of ferrets. However, only parental H5N1 genotype viruses were found in lung tissue. Approximately 34% of the recovered reassortant viruses possessed the H5 hemagglutinin (HA) gene, with five unique H5 subtypes recovered. These H5 reassortants were selected for further studies to examine their growth and transmissibility characteristics. Five H5 viruses with representative reassortant genotypes showed reduced titers in nasal secretions of infected ferrets compared to the parental H5N1 virus. No transmission by direct contact between infected and naive ferrets was observed. These studies indicate that reassortment between H5N1 avian influenza and H3N2 human viruses occurred readily in vivo and furthermore that reassortment between these two viral subtypes is likely to occur in ferret upper airways. Given the relatively high incidence of reassortant viruses from tissues of the ferret upper airway, it is reasonable to conclude that continued exposure of humans and animals to H5N1 alongside seasonal influenza viruses increases the risk of generating H5 subtype reassortant viruses that may be shed from upper airway secretions.