Wendy B. Puryear

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.

Environmental contaminant mixtures modulate in vitro influenza infection

Environmental chemicals, particularly organochlorinated contaminants (OCs), are associated with a ranged of adverse health effects, including impairment of the immune system and antiviral immunity. Influenza A virus (IAV) is an infectious disease of major global public health concern and exposure to OCs can increase the susceptibility, morbidity, and mortality to disease. It is however unclear how pollutants are interacting and affecting the outcome of viral infections at the cellular level. In this study, we investigated the effects of a mixture of environmentally relevant OCs on IAV infectivity upon in vitro exposure in Madin Darby Canine Kidney (MDCK) cells and human lung epithelial cells (A549). Exposure to OCs reduced IAV infectivity in MDCK and A549 cells during both short (18-24h) and long-term (72h) infections at 0.05 and 0.5ppm, and effects were more pronounced in cells co-treated with OCs and IAV than pre-treated with OCs prior to IAV (p<0.001). Pre-treatment of host cells with OCs did not affect IAV cell surface attachment or entry. Visualization of IAV by transmission electron microscopy revealed increased envelope deformations and fewer intact virions during OC exposure. Taken together, our results suggest that disruption of IAV infection upon in vitro exposure to OCs was not due to host-cell effects influencing viral attachment and entry, but perhaps mediated by direct effects on viral particles or cellular processes involved in host-virus interactions. In vitro infectivity studies such as ours can shed light on the complex processes underlying host-pathogen-pollutant interactions.

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.

Evaluating the use of stable isotope analysis to infer the feeding ecology of a growing US gray seal (Halichoerus grypus) population

Gray seals (Halichoerus grypus) have been rapidly recolonizing the Northeast US coast, eliciting concern from the fishing industry. However, the ecological effect of this recovery is still unknown and as such, research is needed to better understand how the diet composition of gray seals in US waters will contribute to the ecological impact. While previous research on seal diets has focused on the analysis of hard prey remains, stable isotope analysis presents an alternative method that can be used to describe marine mammal diets when direct observation is impossible. To address this issue, we used stable isotope analysis of gray seal pup vibrissae and lanugo from Monomoy Island, Cape Cod, MA during the 2015/2016 winter breeding season to estimate adult female diet composition during pregnancy. Stable isotope mixing models (SIMM) suggested adult female gray seals were consuming greater amounts of cephalopod prey and less sand lance than previously indicated from analysis of hard prey remains. However, using SIMMs to estimate the diet composition of gray seals remains difficult due to the large number of isotopically similar prey species and uncertainty in tissue-specific, stable isotope trophic enrichment factors. Even so, by combining prey sources into ecologically informative groups and integrating prior information into SIMMs it is possible to obtain additional insights into the diet of this generalist predator.

Genomic signatures of population bottleneck and recovery in Northwest Atlantic pinnipeds

Abstract Population increases over the past several decades provide natural settings in which to study the evolutionary processes that occur during bottleneck, growth, and spatial expansion. We used parallel natural experiments of historical decline and subsequent recovery in two sympatric pinniped species in the Northwest Atlantic, the gray seal (Halichoerus grypus atlantica) and harbor seal (Phoca vitulina vitulina), to study the impact of recent demographic change in genomic diversity. Using restriction site‐associated DNA sequencing, we assessed genomic diversity at over 8,700 polymorphic gray seal loci and 3,700 polymorphic harbor seal loci in samples from multiple cohorts collected throughout recovery over the past half‐century. Despite significant differences in the degree of genetic diversity assessed in the two species, we found signatures of historical bottlenecks in the contemporary genomes of both gray and harbor seals. We evaluated temporal trends in diversity across cohorts, as well as compared samples from sites at both the center and edge of a recent gray seal range expansion, but found no significant change in genomewide diversity following recovery. We did, however, find that the variance and degree of allele frequency change measured over the past several decades were significantly different from neutral expectations of drift under population growth. These two cases of well‐described demographic history provide opportunities for critical evaluation of current approaches to simulating and understanding the genetic effects of historical demographic change in natural populations.

Genetic diversity from pre-bottleneck to recovery in two sympatric pinniped species in the Northwest Atlantic

Conservation successes of the past several decades provide natural settings to study post-bottleneck evolutionary processes in species undergoing recovery. Here, we study the impact of demographic change on genetic diversity in parallel natural experiments of historical decline and subsequent recovery in two sympatric pinniped species in the Northwest Atlantic, the gray seal (Halichoerus grypus atlantica) and harbor seal (Phoca vitulina concolor). We compare genetic diversity at the mitochondrial control region today to diversity in archaeological specimens, which represent the populations prior to the regional bounties of the late 1800s to mid-1900s that drastically reduced population sizes and led to local extirpations. We further assess genetic diversity throughout recovery, using biological collections from ongoing long-term studies of both species. Overall, the genetic data are consistent with the historical presence of large, genetically diverse populations of pinnipeds prior to human exploitation, and suggest that gray seals were more dramatically impacted by historical bottlenecks than harbor seals in the Northwest Atlantic. Current mitochondrial diversity in both species is relatively high, and we observe little change over the past several decades during a period of roughly parallel rapid population increases. However, there remain large differences in haplotype composition between pinniped populations of pre-exploitation and today, a lasting genetic signature of historical exploitation that is likely to persist into the future.

Maintenance of influenza A viruses and antibody response in mallards (Anas platyrhynchos) sampled during the non-breeding season in Alaska

Prevalence of influenza A virus (IAV) infections in northern-breeding waterfowl has previously been reported to reach an annual peak during late summer or autumn; however, little is known about IAV infection dynamics in waterfowl populations persisting at high-latitude regions such as Alaska, during winter. We captured mallards (Anas platyrhynchos) throughout the non-breeding season (August–April) of 2012–2015 in Fairbanks and Anchorage, the two largest cities in Alaska, to assess patterns of IAV infection and antibody production using molecular methods and a standard serologic assay. In addition, we used virus isolation, genetic sequencing, and a virus microneutralization assay to characterize viral subtypes and to evaluate the immune response of mallards captured on multiple occasions through time. We captured 923 mallards during three successive sampling years: Fairbanks in 2012/13 and 2013/14, and Anchorage in 2014/15. Prevalence varied by age, season, and year/site with high and relatively stable estimates throughout the non-breeding season. Infected birds were detected in all locations/seasons except early-winter in Fairbanks during 2013/14. IAVs with 17 combinations of hemagglutinin (H1–5, H7–9, H11, H12) and neuraminidase (N1–6, N8, N9) subtypes were isolated. Antibodies to IAVs were detected throughout autumn and winter for all sampling locations and years, however, seroprevalence was higher among adults and varied among years. Mallards exhibited individual heterogeneity with regard to immune response, providing instances of both seroconversion and seroreversion to detected viral subtypes. The probability that an individual transitioned from one serostatus to another varied by age, with juvenile mallards having higher rates of seroconversion and seroreversion than adults. Our study provides evidence that a diversity of IAVs circulate in populations of mallards wintering at urban locations in Alaska, and we suggest waterfowl wintering at high-latitudes may play an important role in maintenance of viruses across breeding seasons.