Islam T. M. Hussein

Connecting the study of wild influenza with the potential for pandemic disease.

Continuing outbreaks of pathogenic (H5N1) and pandemic (SOIVH1N1) influenza have underscored the need to understand the origin, characteristics, and evolution of novel influenza A virus (IAV) variants that pose a threat to human health. In the last 4-5years, focus has been placed on the organization of large-scale surveillance programs to examine the phylogenetics of avian influenza virus (AIV) and host-virus relationships in domestic and wild animals. Here we review the current gaps in wild animal and environmental surveillance and the current understanding of genetic signatures in potentially pandemic strains.

Reassortment of Influenza A Viruses in Wild Birds in Alaska before H5 Clade 2.3.4.4 Outbreaks

Sampling of mallards in Alaska during September 2014–April 2015 identified low pathogenic avian influenza A virus (subtypes H5N2 and H1N1) that shared ancestry with highly pathogenic reassortant H5N2 and H5N1 viruses. Molecular dating indicated reassortment soon after interhemispheric movement of H5N8 clade 2.3.4.4, suggesting genetic exchange in Alaska or surrounds before outbreaks.

Ecosystem Interactions Underlie the Spread of Avian Influenza A Viruses with Pandemic Potential.

Despite evidence for avian influenza A virus (AIV) transmission between wild and domestic ecosystems, the roles of bird migration and poultry trade in the spread of viruses remain enigmatic. In this study, we integrate ecosystem interactions into a phylogeographic model to assess the contribution of wild and domestic hosts to AIV distribution and persistence. Analysis of globally sampled AIV datasets shows frequent two-way transmission between wild and domestic ecosystems. In general, viral flow from domestic to wild bird populations was restricted to within a geographic region. In contrast, spillover from wild to domestic populations occurred both within and between regions. Wild birds mediated long-distance dispersal at intercontinental scales whereas viral spread among poultry populations was a major driver of regional spread. Viral spread between poultry flocks frequently originated from persistent lineages circulating in regions of intensive poultry production. Our analysis of long-term surveillance data demonstrates that meaningful insights can be inferred from integrating ecosystem into phylogeographic reconstructions that may be consequential for pandemic preparedness and livestock protection.

New England harbor seal H3N8 influenza virus retains avian-like receptor specificity.

An influenza H3N8 virus, carrying mammalian adaptation mutations, was isolated from New England harbor seals in 2011. We sought to assess the risk of its human transmissibility using two complementary approaches. First, we tested the binding of recombinant hemagglutinin (HA) proteins of seal H3N8 and human-adapted H3N2 viruses to respiratory tissues of humans and ferrets. For human tissues, we observed strong tendency of the seal H3 to bind to lung alveoli, which was in direct contrast to the human-adapted H3 that bound mainly to the trachea. This staining pattern was also consistent in ferrets, the primary animal model for human influenza pathogenesis. Second, we compared the binding of the recombinant HAs to a library of 610 glycans. In contrast to the human H3, which bound almost exclusively to α-2,6 sialylated glycans, the seal H3 bound preferentially to α-2,3 sialylated glycans. Additionally, the seal H3N8 virus replicated in human lung carcinoma cells. Our data suggest that the seal H3N8 virus has retained its avian-like receptor binding specificity, but could potentially establish infection in human lungs.

A point mutation in the polymerase protein PB2 allows a reassortant H9N2 influenza isolate of wild-bird origin to replicate in human cells.

H9N2 influenza A viruses are on the list of potentially pandemic subtypes. Therefore, it is important to understand how genomic reassortment and genetic polymorphisms affect phenotypes of H9N2 viruses circulating in the wild bird reservoir. A comparative genetic analysis of North American H9N2 isolates of wild bird origin identified a naturally occurring reassortant virus containing gene segments derived from both North American and Eurasian lineage ancestors. The PB2 segment of this virus encodes 10 amino acid changes that distinguish it from other H9 strains circulating in North America. G590S, one of the 10 amino acid substitutions observed, was present in ~12% of H9 viruses worldwide. This mutation combined with R591 has been reported as a marker of pathogenicity for human pandemic 2009 H1N1 viruses. Screening by polymerase reporter assay of all the natural polymorphisms at these two positions identified G590/K591 and S590/K591 as the most active, with the highest polymerase activity recorded for the SK polymorphism. Rescued viruses containing these two polymorphic combinations replicated more efficiently in MDCK cells and they were the only ones tested that were capable of establishing productive infection in NHBE cells. A global analysis of all PB2 sequences identified the K591 signature in six viral HA/NA subtypes isolated from several hosts in seven geographic locations. Interestingly, introducing the K591 mutation into the PB2 of a human-adapted H3N2 virus did not affect its polymerase activity. Our findings demonstrate that a single point mutation in the PB2 of a low pathogenic H9N2 isolate could have a significant effect on viral phenotype and increase its propensity to infect mammals. However, this effect is not universal, warranting caution in interpreting point mutations without considering protein sequence context.

Genetic characterization of H5N2 influenza viruses isolated from wild birds in Japan suggests multiple reassortment

Low-pathogenic avian influenza viruses (LPAIVs) of the H5 subtype can mutate to highly pathogenic forms, potentially destabilizing the poultry industry. Wild migratory birds are considered a natural reservoir of LPAIVs capable of dispersing both high- and low-pathogenic forms of the virus. Therefore, surveillance and characterization of AIV in wild birds are essential. Here, we report on the isolation and genetic characterization of 10 AIVs of the H5N2 subtype obtained through surveillance in Hokkaido, Japan, during 2009 and 2011. Full-genome sequencing revealed that the H5 and N2 genes of these isolates are all closely related to each other, belonging to the Eurasian avian-like lineage, but they are unrelated to H5 highly pathogenic strains of clade 2.3.4.4. The internal genes of the isolates were found to be diverse, consistent with our hypothesis that these H5N2 strains have undergone multiple reassortment events. Even though all of the H5N2 isolates were characterized as LPAIV based on the amino acid sequences at the HA cleavage site, this analysis demonstrates a diverse pool of precursors that may seed future outbreaks in poultry and possible human transmissions, suggesting the need for high-quality surveillance.

Host-directed combinatorial RNAi improves inhibition of diverse strains of influenza A virus in human respiratory epithelial cells

Influenza A virus infections are important causes of morbidity and mortality worldwide, and currently available prevention and treatment methods are suboptimal. In recent years, genome-wide investigations have revealed numerous host factors that are required for influenza to successfully complete its life cycle. However, only a select, small number of influenza strains were evaluated using this platform, and there was considerable variation in the genes identified across different investigations. In an effort to develop a universally efficacious therapeutic strategy with limited potential for the emergence of resistance, this study was performed to investigate the effect of combinatorial RNA interference (RNAi) on inhibiting the replication of diverse influenza A virus subtypes and strains. Candidate genes were selected for targeting based on the results of multiple previous independent genome-wide studies. The effect of single and combinatorial RNAi on the replication of 12 diverse influenza A viruses, including three strains isolated from birds and one strain isolated from seals, was then evaluated in primary normal human bronchial epithelial cells. After excluding overly toxic siRNA, two siRNA combinations were identified that reduced mean viral replication by greater than 79 percent in all mammalian strains, and greater than 68 percent in all avian strains. Host-directed combinatorial RNAi effectively prevents growth of a broad range of influenza virus strains in vitro, and is a potential therapeutic candidate for further development and future in vivo studies.

Bayesian Analysis of High Throughput Data

Duplicate or triplicate experimental replicates are commonplace in the high throughput literature. However, it has not been tested whether this is statistically defensible or not. To address this issue, we use probabilistic programming to develop a simple hierarchical model for analyzing high throughput measurement data. With the model and simulated data, we show that a small increase in replicate experiments can quantitatively improve accuracy in measurement. We also provide posterior densities for statistical parameters used in the evaluation of HT data. Finally, we provide an extensible open source implementation that ingests data structured in a simple format and produces posterior densities of estimated measurement and assay evaluation parameters.

Genetic characterization of a rare H12N3 avian influenza virus isolated from a green-winged teal in Japan

This study reports on the genetic characterization of an avian influenza virus, subtype H12N3, isolated from an Eurasian green-winged teal (Anas crecca) in Japan in 2009. The entire genome sequence of the isolate was analyzed, and phylogenetic analyses were conducted to characterize the evolutionary history of the isolate. Phylogenetic analysis of the hemagglutinin and neuraminidase genes indicated that the virus belonged to the Eurasian-like avian lineage. Molecular dating indicated that this H12 virus is likely a multiple reassortant influenza A virus. This is the first reported characterization of influenza A virus subtype H12N3 isolated in Japan and these data contribute to the accumulation of knowledge on the genetic diversity and generation of novel influenza A viruses.