Two reassortant H16 influenza A viruses were isolated from gulls in California. Seven of the eight segments were most closely related to H16 and H13 isolates from eastern North America and Iceland. Of note is a C-terminal truncation of the nonstructural 1 (NS1) protein in one of the isolates that is usually found in swine H1N1 virus.

Influenza A viruses (IAVs) utilize sialylated host glycans as ligands for binding and infection. The glycan-binding preference of IAV hemagglutinin (HA) is an important determinant of host specificity. Propagation of IAV in embryonated chicken eggs and cultured mammalian cells yields viruses with amino acid substitutions in the HA that can alter the binding specificity. Therefore, it is important to determine the binding specificity of IAV directly in primary samples since it reflects the actual tropism of virus in nature. We developed a novel platform for analysis of IAV binding specificity in samples that contain very low virus titers. This platform consists of a high-density flexible glycan display on magnetic beads, which promotes multivalent interactions with the viral HA. Glycan-bound virus is detected by quantifying the viral neuraminidase activity via a fluorogenic reporter, 2'-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid. This method eliminates the need for labeling the virus and significantly enhances the sensitivity of detection.

We report the complete genome sequence of a reassortant H14N2 avian influenza virus isolated in 2011 from a northern shoveler in California. This introduced Eurasian subtype acquired seven segments from North American viruses and circulated in the Pacific Flyway 1 year after its detection in the Mississippi Flyway.

A high prevalence and diversity of avian influenza (AI) viruses were detected in a population of wild mallards sampled during summer 2011 in California, providing an opportunity to compare results obtained before and after virus culture. We tested cloacal swab samples prior to culture by matrix real-time PCR, and by amplifying and sequencing a 640bp portion of the hemagglutinin (HA) gene. Each sample was also inoculated into embryonated chicken eggs, and full genome sequences were determined for cultured viruses. While low matrix Ct values were a good predictor of virus isolation from eggs, samples with high or undetectable Ct values also yielded isolates. Furthermore, a single passage in eggs altered the occurrence and detection of viral strains, and mixed infections (different HA subtypes) were detected less frequently after culture. There is no gold standard or perfect reference comparison for surveillance of unknown viruses, and true negatives are difficult to distinguish from false negatives. This study showed that sequencing samples prior to culture increases the detection of mixed infections and enhances the identification of viral strains and sequences that may have changed or even disappeared during culture.

From 2011-2018, we conducted surveillance in marine mammals along the California coast for influenza A virus (IAV), frequently detecting anti-influenza antibodies and intermittently detecting IAV. In spring 2019, this pattern changed. Despite no change in surveillance intensity, we detected IAV RNA in 10 samples in March and April, mostly in nasal and rectal swabs from northern elephant seals (Mirounga angustirostris). Although virus isolation was unsuccessful, IAV sequenced from one northern elephant seal nasal swab showed close genetic identity with pandemic H1N1 IAV subclade 6B.1A.1 that was concurrently circulating in humans in the 2018/19 influenza season. This represents the first report of human A(H1N1)pdm09 IAV in northern elephant seals since 2010, suggesting IAV continues to spill over from humans to pinnipeds.

Environmental contamination is widespread and can negatively impact wildlife health. Some contaminants, including heavy metals, have immunosuppressive effects, but prior studies have rarely measured contamination and disease simultaneously, which limits our understanding of how contaminants and pathogens interact to influence wildlife health. Here, we measured mercury concentrations, influenza infection, influenza antibodies and body condition in 749 individuals from 11 species of wild ducks overwintering in California. We found that the odds of prior influenza infection increased more than fivefold across the observed range of blood mercury concentrations, while accounting for species, age, sex and date. Influenza infection prevalence was also higher in species with higher average mercury concentrations. We detected no relationship between influenza infection and body fat content. This positive relationship between influenza prevalence and mercury concentrations in migratory waterfowl suggests that immunotoxic effects of mercury contamination could promote the spread of avian influenza along migratory flyways, especially if influenza has minimal effects on bird health and mobility. More generally, these results show that the effects of environmental contamination could extend beyond the geographical area of contamination itself by altering the prevalence of infectious diseases in highly mobile hosts.

We used surveillance data collected in California before, concurrent with, and subsequent to an outbreak of highly pathogenic (HP) clade 2.3.4.4 influenza A viruses (IAVs) in 2014-2015 to (i) evaluate IAV prevalence in waterfowl, (ii) assess the evidence for spill-over infections in marine mammals and (iii) genetically characterize low-pathogenic (LP) and HP IAVs to refine inference on the spatiotemporal extent of HP genome constellations and to evaluate possible evolutionary pathways. We screened samples from 1496 waterfowl and 1142 marine mammals collected from April 2014 to August 2015 and detected IAV RNA in 159 samples collected from birds (n=157) and pinnipeds (n=2). HP IAV RNA was identified in three samples originating from American wigeon (Anas americana). Genetic sequence data were generated for a clade 2.3.4.4 HP IAV-positive diagnostic sample and 57 LP IAV isolates. Phylogenetic analyses revealed that the HP IAV was a reassortant H5N8 virus with gene segments closely related to LP IAVs detected in mallards (Anas platyrhynchos) sampled in California and other IAVs detected in wild birds sampled within the Pacific Americas Flyway. In addition, our analysis provided support for common ancestry between LP IAVs recovered from waterfowl sampled in California and gene segments of reassortant HP H5N1 IAVs detected in British Columbia, Canada and Washington, USA. Our investigation provides evidence that waterfowl are likely to have played a role in the evolution of reassortant HP IAVs in the Pacific Americas Flyway during 2014-2015, whereas we did not find support for spill-over infections in potential pinniped hosts.

To understand susceptibility of wild California sea lions and Northern elephant seals to influenza A virus (IAV), we developed an ex vivo respiratory explant model and used it to compare infection kinetics for multiple IAV subtypes. We first established the approach using explants from colonized rhesus macaques, a model for human IAV. Trachea, bronchi, and lungs from 11 California sea lions, 2 Northern elephant seals, and 10 rhesus macaques were inoculated within 24 h postmortem with 6 strains representing 4 IAV subtypes. Explants from the 3 species showed similar IAV infection kinetics, with peak viral titers 48 to 72 h post-inoculation that increased by 2 to 4 log₁₀ PFU/explant relative to the inoculum. Immunohistochemistry localized IAV infection to apical epithelial cells. These results demonstrate that respiratory tissue explants from wild marine mammals support IAV infection. In the absence of the ability to perform experimental infections of marine mammals, this ex vivo culture of respiratory tissues mirrors the in vivo environment and serves as a tool to study IAV susceptibility, host range, and tissue tropism. IMPORTANCE Although influenza A virus can infect marine mammals, a dearth of marine mammal cell lines and ethical and logistical challenges prohibiting experimental infections of living marine mammals mean that little is known about IAV infection kinetics in these species. We circumvented these limitations by adapting a respiratory tract explant model first to establish the approach with rhesus macaques and then for use with explants from wild marine mammals euthanized for nonrespiratory medical conditions. We observed that multiple strains representing 4 IAV subtypes infected trachea, bronchi, and lungs of macaques and marine mammals with variable peak titers and kinetics. This ex vivo model can define infection dynamics for IAV in marine mammals. Further, use of explants from animals euthanized for other reasons reduces use of animals in research.

Avian influenza viruses can pose serious risks to agricultural production, human health, and wildlife. An understanding of viruses in wild reservoir species across time and space is important to informing surveillance programs, risk models, and potential population impacts for vulnerable species. Although it is recognized that influenza A virus prevalence peaks in reservoir waterfowl in late summer through autumn, temporal and spatial variation across species has not been fully characterized. We combined two large influenza databases for North America and applied spatiotemporal models to explore patterns in prevalence throughout the annual cycle and across the continental United States for 30 waterfowl species. Peaks in prevalence in late summer through autumn were pronounced for dabbling ducks in the genera Anas and Spatula, but not Mareca. Spatially, areas of high prevalence appeared to be related to regional duck density, with highest predicted prevalence found across the upper Midwest during early fall, though further study is needed. We documented elevated prevalence in late winter and early spring, particularly in the Mississippi Alluvial Valley. Our results suggest that spatiotemporal variation in prevalence outside autumn staging areas may also represent a dynamic parameter to be considered in IAV ecology and associated risks.