Characterization of a Feline Influenza A(H7N2) Virus.

During December 2016-February 2017, influenza A viruses of the H7N2 subtype infected ≈500 cats in animal shelters in New York, NY, USA, indicating virus transmission among cats. A veterinarian who treated the animals also became infected with feline influenza A(H7N2) virus and experienced respiratory symptoms. To understand the pathogenicity and transmissibility of these feline H7N2 viruses in mammals, we characterized them in vitro and in vivo. Feline H7N2 subtype viruses replicated in the respiratory organs of mice, ferrets, and cats without causing severe lesions. Direct contact transmission of feline H7N2 subtype viruses was detected in ferrets and cats; in cats, exposed animals were also infected via respiratory droplet transmission. These results suggest that the feline H7N2 subtype viruses could spread among cats and also infect humans. Outbreaks of the feline H7N2 viruses could, therefore, pose a risk to public health.

A Phylogeny-Based Global Nomenclature System and Automated Annotation Tool for H1 Hemagglutinin Genes from Swine Influenza A Viruses.

The H1 subtype of influenza A viruses (IAVs) has been circulating in swine since the 1918 human influenza pandemic. Over time, and aided by further introductions from nonswine hosts, swine H1 viruses have diversified into three genetic lineages. Due to limited global data, these H1 lineages were named based on colloquial context, leading to a proliferation of inconsistent regional naming conventions. In this study, we propose rigorous phylogenetic criteria to establish a globally consistent nomenclature of swine H1 virus hemagglutinin (HA) evolution. These criteria applied to a data set of 7,070 H1 HA sequences led to 28 distinct clades as the basis for the nomenclature. We developed and implemented a web-accessible annotation tool that can assign these biologically informative categories to new sequence data. The annotation tool assigned the combined data set of 7,070 H1 sequences to the correct clade more than 99% of the time. Our analyses indicated that 87% of the swine H1 viruses from 2010 to the present had HAs that belonged to 7 contemporary cocirculating clades. Our nomenclature and web-accessible classification tool provide an accurate method for researchers, diagnosticians, and health officials to assign clade designations to HA sequences. The tool can be updated readily to track evolving nomenclature as new clades emerge, ensuring continued relevance. A common global nomenclature facilitates comparisons of IAVs infecting humans and pigs, within and between regions, and can provide insight into the diversity of swine H1 influenza virus and its impact on vaccine strain selection, diagnostic reagents, and test performance, thereby simplifying communication of such data. IMPORTANCE A fundamental goal in the biological sciences is the definition of groups of organisms based on evolutionary history and the naming of those groups. For influenza A viruses (IAVs) in swine, understanding the hemagglutinin (HA) genetic lineage of a circulating strain aids in vaccine antigen selection and allows for inferences about vaccine efficacy. Previous reporting of H1 virus HA in swine relied on colloquial names, frequently with incriminating and stigmatizing geographic toponyms, making comparisons between studies challenging. To overcome this, we developed an adaptable nomenclature using measurable criteria for historical and contemporary evolutionary patterns of H1 global swine IAVs. We also developed a web-accessible tool that classifies viruses according to this nomenclature. This classification system will aid agricultural production and pandemic preparedness through the identification of important changes in swine IAVs and provides terminology enabling discussion of swine IAVs in a common context among animal and human health initiatives.

Development of high-yield influenza A virus vaccine viruses.

Vaccination is one of the most cost-effective ways to prevent infection. Influenza vaccines propagated in cultured cells are approved for use in humans, but their yields are often suboptimal. Here, we screened A/Puerto Rico/8/34 (PR8) virus mutant libraries to develop vaccine backbones (defined here as the six viral RNA segments not encoding haemagglutinin and neuraminidase) that support high yield in cell culture. We also tested mutations in the coding and regulatory regions of the virus, and chimeric haemagglutinin and neuraminidase genes. A combination of high-yield mutations from these screens led to a PR8 backbone that improved the titres of H1N1, H3N2, H5N1 and H7N9 vaccine viruses in African green monkey kidney and Madin-Darby canine kidney cells. This PR8 backbone also improves titres in embryonated chicken eggs, a common propagation system for influenza viruses. This PR8 vaccine backbone thus represents an advance in seasonal and pandemic influenza vaccine development.

Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus.

Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.

Novel residues in avian influenza virus PB2 protein affect virulence in mammalian hosts.

Highly pathogenic avian H5N1 influenza viruses have sporadically transmitted to humans causing high mortality. The mechanistic basis for adaptation is still poorly understood, although several residues in viral protein PB2 are known to be important for this event. Here, we demonstrate that three residues, 147T, 339T and 588T, in PB2 play critical roles in the virulence of avian H5N1 influenza viruses in a mammalian host in vitro and in vivo and, together, result in a phenotype comparable to that conferred by the previously known PB2-627K mutation with respect to virus polymerase activity. A virus with the three residues and 627K in PB2, as has been isolated from a lethal human case, is more pathogenic than viruses with only the three residues or 627K in PB2. Importantly, H5N1 viruses bearing the former three PB2 residues have circulated widely in recent years in avian species in nature.

Improving pandemic influenza risk assessment.

Assessing the pandemic risk posed by specific non-human influenza A viruses is an important goal in public health research. As influenza virus genome sequencing becomes cheaper, faster, and more readily available, the ability to predict pandemic potential from sequence data could transform pandemic influenza risk assessment capabilities. However, the complexities of the relationships between virus genotype and phenotype make such predictions extremely difficult. The integration of experimental work, computational tool development, and analysis of evolutionary pathways, together with refinements to influenza surveillance, has the potential to transform our ability to assess the risks posed to humans by non-human influenza viruses and lead to improved pandemic preparedness and response.

Identification of amino acid changes that may have been critical for the genesis of A(H7N9) influenza viruses.

UNLABELLED: Novel influenza A viruses of the H7N9 subtype [A(H7N9)] emerged in the spring of 2013 in China and had infected 163 people as of 10 January 2014; 50 of them died of the severe respiratory infection caused by these viruses. Phylogenetic studies have indicated that the novel A(H7N9) viruses emerged from reassortment of H7, N9, and H9N2 viruses. Inspections of protein sequences from A(H7N9) viruses and their immediate predecessors revealed several amino acid changes in A(H7N9) viruses that may have facilitated transmission and replication in the novel host. Since mutations that occurred more ancestrally may also have contributed to the genesis of A(H7N9) viruses, we inferred historical evolutionary events leading to the novel viruses. We identified a number of amino acid changes on the evolutionary path to A(H7N9) viruses, including substitutions that may be associated with host range, replicative ability, and/or host responses to infection. The biological significance of these amino acid changes can be tested in future studies. IMPORTANCE: The novel influenza A viruses of the H7N9 subtype [A(H7N9)], which first emerged in the spring of 2013, cause severe respiratory infections in humans. Here, we performed a comprehensive evolutionary analysis of the progenitors of A(H7N9) viruses to identify amino acid changes that may have been critical for the emergence of A(H7N9) viruses and their ability to infect humans. We provide a list of potentially important amino acid changes that can be tested for their significance for the influenza virus host range, replicative ability, and/or host responses to infection.

Synergistic effect of the PDZ and p85ß-binding domains of the NS1 protein on virulence of an avian H5N1 influenza A virus.

The influenza A virus NS1 protein affects virulence through several mechanisms, including the host's innate immune response and various signaling pathways. Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype continue to evolve through reassortment and mutations. Our recent phylogenetic analysis identified a group of HPAI H5N1 viruses with two characteristic mutations in NS1: the avian virus-type PDZ domain-binding motif ESEV (which affects virulence) was replaced with ESKV, and NS1-138F (which is highly conserved among all influenza A viruses and may affect the activation of the phosphatidylinositol 3-kinase [PI3K]/Akt signaling pathway) was replaced with NS1-138Y. Here, we show that an HPAI H5N1 virus (A/duck/Hunan/69/2004) encoding NS1-ESKV and NS1-138Y was confined to the respiratory tract of infected mice, whereas a mutant encoding NS1-ESEV and NS1-138F caused systemic infection and killed mice more efficiently. Mutation of either one of these sites had small effects on virulence. In addition, we found that the amino acid at NS1-138 affected not only the induction of the PI3K/Akt pathway but also the interaction of NS1 with cellular PDZ domain proteins. Similarly, the mutation in the PDZ domain-binding motif of NS1 altered its binding to cellular PDZ domain proteins and affected Akt phosphorylation. These findings suggest a functional interplay between the mutations at NS1-138 and NS1-229 that results in a synergistic effect on influenza virulence.

Evolution of highly pathogenic avian H5N1 influenza viruses and the emergence of dominant variants.

Highly pathogenic avian H5N1 viruses have circulated in South-east Asia for more than a decade and have now spread to more than 60 countries. The evolution of these viruses is characterized by frequent reassortment of the so-called 'internal' genes, creating novel genotypes. Additionally, over time, the surface glycoprotein, haemagglutinin (HA), which is the primary target of the adaptive immune response, has evolved by point mutation into 20 genetically and potentially antigenically distinct clades. To investigate the evolution of avian H5N1 influenza viruses, we undertook a high-resolution analysis of the reassortment of internal genes and evolution of HA of 651 avian H5N1 viruses from 2000 to 2008. Our analysis suggested: (i) all current H5N1 genotypes were derived from a single, clearly defined sequence of initial reassortment events; (ii) reassortment of just three of the internal genes had the most importance in avian H5N1 virus evolution; (iii) HA and the constellation of internal genes may be jointly important in the emergence of dominant variants. Further, our analysis led to the identification of evolutionarily significant molecular changes in the internal genes that may be significant for the emergence of these dominant variants.

Modeling targeted layered containment of an influenza pandemic in the United States.

Planning a response to an outbreak of a pandemic strain of influenza is a high public health priority. Three research groups using different individual-based, stochastic simulation models have examined the consequences of intervention strategies chosen in consultation with U.S. public health workers. The first goal is to simulate the effectiveness of a set of potentially feasible intervention strategies. Combinations called targeted layered containment (TLC) of influenza antiviral treatment and prophylaxis and nonpharmaceutical interventions of quarantine, isolation, school closure, community social distancing, and workplace social distancing are considered. The second goal is to examine the robustness of the results to model assumptions. The comparisons focus on a pandemic outbreak in a population similar to that of Chicago, with approximately 8.6 million people. The simulations suggest that at the expected transmissibility of a pandemic strain, timely implementation of a combination of targeted household antiviral prophylaxis, and social distancing measures could substantially lower the illness attack rate before a highly efficacious vaccine could become available. Timely initiation of measures and school closure play important roles. Because of the current lack of data on which to base such models, further field research is recommended to learn more about the sources of transmission and the effectiveness of social distancing measures in reducing influenza transmission.

Genotype turnover by reassortment of replication complex genes from avian influenza A virus.

Reassortment among the RNA segments of Influenza A virus caused the two most recent human influenza pandemics; recently, reassortment has generated viral genotypes associated with outbreaks of avian H5N1 influenza in Asia and Europe. A statistical analysis has been developed for the systematic identification and characterization of reassortant viruses. The analysis was applied to the genes of the replication complex of 152 avian influenza A viruses isolated between 1966 and 2004 from predominantly terrestrial and domestic aquatic avian species. The results indicated that reassortment among these genes was pervasive throughout this period and throughout both the Eurasian and North American lineages of the virus. Evidence is presented that the circulating genotypes of the replication complex are being replaced continually by novel genotypes created by reassortment. No constraints for coordinated reassortment among genes of the replication complex were evident; rather, reassortment almost always proceeded one segment at a time. A maximum-likelihood estimate of the rate of reassortment was derived. For significantly diverged Asian avian influenza A viruses from the period 1991-2004, it was estimated that the median duration between creation of a new genotype and its next segment reassortment was 3 years. Reassortments that introduced previously unobserved influenza genetic material were detected. These findings point to substantial potential for rapid generation of novel avian influenza A viruses, emphasizing the importance of intensive surveillance of these host species in preparation for a possible pandemic.

Kinetics of influenza A virus infection in humans.

Currently, little is known about the viral kinetics of influenza A during infection within an individual. We utilize a series of mathematical models of increasing complexity, which incorporate target cell limitation and the innate interferon response, to examine influenza A virus kinetics in the upper respiratory tracts of experimentally infected adults. The models were fit to data from an experimental H1N1 influenza A/Hong Kong/123/77 infection and suggest that it is important to include the eclipse phase of the viral life cycle in viral dynamic models. Doing so, we estimate that after a delay of approximately 6 h, infected cells begin producing influenza virus and continue to do so for approximately 5 h. The average lifetime of infected cells is approximately 11 h, and the half-life of free infectious virus is approximately 3 h. We calculated the basic reproductive number, R(0), which indicated that a single infected cell could produce approximately 22 new productive infections. This suggests that antiviral treatments have a large hurdle to overcome in moderating symptoms and limiting infectiousness and that treatment has to be initiated as early as possible. For about 50% of patients, the curve of viral titer versus time has two peaks. This bimodal behavior can be explained by incorporating the antiviral effects of interferon into the model. Our model also compared well to an additional data set on viral titer after experimental infection and treatment with the neuraminidase inhibitor zanamivir, which suggests that such models may prove useful in estimating the efficacies of different antiviral therapies for influenza A infection.

Mitigation strategies for pandemic influenza in the United States.

Recent human deaths due to infection by highly pathogenic (H5N1) avian influenza A virus have raised the specter of a devastating pandemic like that of 1917-1918, should this avian virus evolve to become readily transmissible among humans. We introduce and use a large-scale stochastic simulation model to investigate the spread of a pandemic strain of influenza virus through the U.S. population of 281 million individuals for R(0) (the basic reproductive number) from 1.6 to 2.4. We model the impact that a variety of levels and combinations of influenza antiviral agents, vaccines, and modified social mobility (including school closure and travel restrictions) have on the timing and magnitude of this spread. Our simulations demonstrate that, in a highly mobile population, restricting travel after an outbreak is detected is likely to delay slightly the time course of the outbreak without impacting the eventual number ill. For R(0) < 1.9, our model suggests that the rapid production and distribution of vaccines, even if poorly matched to circulating strains, could significantly slow disease spread and limit the number ill to <10% of the population, particularly if children are preferentially vaccinated. Alternatively, the aggressive deployment of several million courses of influenza antiviral agents in a targeted prophylaxis strategy may contain a nascent outbreak with low R(0), provided adequate contact tracing and distribution capacities exist. For higher R(0), we predict that multiple strategies in combination (involving both social and medical interventions) will be required to achieve similar limits on illness rates.

Homology model of the structure of influenza B virus HA1.

Influenza B virus is one of two types of influenza virus that cause substantial morbidity and mortality in humans, the other being influenza A virus. The inability to provide lasting protection to humans against influenza B virus infection is due, in part, to antigenic drift of the viral surface glycoprotein, haemagglutinin (HA). Studies of the antigenicity of the HA of influenza B virus have been hampered by lack of knowledge of its structure. To address this gap, two possible models have been inferred for this structure, based on two known structures of the homologous HA of the influenza A virus (subtypes H3 and H9). Statistical, structural and functional analyses of these models suggested that they matched important details of experimental observations and did not differ from each other in any substantive way. These models were used to investigate two HA sites at which viral variants appeared to carry a selective advantage. It was found that each of these sites coevolved with nearby sites to compensate for either size or charge changes.