Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.

Human infection with avian influenza A(H7N9) virus is associated mainly with the exposure to infected poultry. The factors that allow interspecies transmission but limit human-to-human transmission are unknown. Here we show that A/Anhui/1/2013(H7N9) influenza virus infection of chickens (natural hosts) is asymptomatic and that it generates a high genetic diversity. In contrast, diversity is tightly restricted in infected ferrets, limiting further adaptation to a fully transmissible form. Airborne transmission in ferrets is accompanied by the mutations in PB1, NP and NA genes that reduce viral polymerase and neuraminidase activity. Therefore, while A(H7N9) virus can infect mammals, further adaptation appears to incur a fitness cost. Our results reveal that a tight genetic bottleneck during avian-to-mammalian transmission is a limiting factor in A(H7N9) influenza virus adaptation to mammals. This previously unrecognized biological mechanism limiting species jumps provides a measure of adaptive potential and may serve as a risk assessment tool for pandemic preparedness.

A pandemic-capable influenza virus requires a hemagglutinin (HA) surface glycoprotein that is immunologically unseen by most people and is capable of supporting replication and transmission in humans. HA stabilization has been linked to 2009 pH1N1 pandemic potential in humans and H5N1 airborne transmissibility in the ferret model. Swine have served as an intermediate host for zoonotic influenza viruses, yet the evolutionary pressure exerted by this host on HA stability was unknown. For over 70 contemporary swine H1 and H3 isolates, we measured HA activation pH to range from pH 5.1 to 5.9 for H1 viruses and pH 5.3 to 5.8 for H3 viruses. Thus, contemporary swine isolates vary widely in HA stability, having values favored by both avian (pH >5.5) and human and ferret (pH ≤5.5) species. Using an early 2009 pandemic H1N1 (pH1N1) virus backbone, we generated three viruses differing by one HA residue that only altered HA stability: WT (pH 5.5), HA1-Y17H (pH 6.0), and HA2-R106K (pH 5.3). All three replicated in pigs and transmitted from pig-to-pig and pig-to-ferret. WT and R106 viruses maintained HA genotype and phenotype after transmission. Y17H (pH 6.0) acquired HA mutations that stabilized the HA protein to pH 5.8 after transmission to pigs and 5.5 after transmission to ferrets. Overall, we found swine support a broad range of HA activation pH for contact transmission and many recent swine H1N1 and H3N2 isolates have stabilized (human-like) HA proteins. This constitutes a heightened pandemic risk and underscores the importance of ongoing surveillance and control efforts for swine viruses.

Highly pathogenic H5 influenza viruses have been introduced into North America from Asia, causing extensive morbidity and mortality in domestic poultry. The introduced viruses have reassorted with North American avian influenza viruses, generating viral genotypes not seen on other continents. The experiments and analyses presented here were designed to assess the impact of this genetic diversification on viral phenotypes, particularly as regards mammalian hosts, by comparing the North American viruses with their Eurasian precursor viruses.

Highly pathogenic H5N1 influenza viruses have been isolated from a number of avian and mammalian species. Despite intensive control measures the number of human and animal cases continues to increase. A more complete understanding of susceptible species and of contributing environmental and molecular factors is crucial if we are to slow the rate of new cases. H5N1 is currently endemic in domestic poultry in only a handful of countries with sporadic and unpredictable spread to other countries. Close contact of terrestrial bird or mammalian species with infected poultry/waterfowl or their biological products is the major route for interspecies transmission. Intra-species transmission of H5N1 in mammals, including humans, has taken place on a limited scale though it remains to be seen if this will change; recent laboratory studies suggest that it is indeed possible. Here we review the avian and mammalian species that are naturally susceptible to H5N1 infection and the molecular factors associated with its expanded host range.