Human influenza A virus H1N1 in marine mammals in California, 2019.

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.

Avian influenza antibody prevalence increases with mercury contamination in wild waterfowl.

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.

Spatiotemporal changes in influenza A virus prevalence among wild waterfowl inhabiting the continental United States throughout the annual cycle.

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.

Respiratory Tract Explant Infection Dynamics of Influenza A Virus in California Sea Lions, Northern Elephant Seals, and Rhesus Macaques.

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.

Surveillance for highly pathogenic influenza A viruses in California during 2014-2015 provides insights into viral evolutionary pathways and the spatiotemporal extent of viruses in the Pacific Americas Flyway.

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.

Serologic Detection of Subtype-specific Antibodies to Influenza A Viruses in Southern Sea Otters (Enhydra lutris nereis).

There are approximately 3,000 southern sea otters (Enhydra lutris nereis) in the nearshore environment along the California coast, US, and the species is classified as Threatened under the Endangered Species Act. We tested sera from 661 necropsied southern sea otters sampled from 1997 to 2015 to determine overall exposure to influenza A viruses (IAVs) and to identify subtype-specific antibody responses. Using an enzyme-linked immunosorbent assay (ELISA), antibodies to IAV nucleoproteins were detected in 160 (24.2%) otters, with seropositive animals found in every year except 2008. When the ELISA-positive samples were tested by virus microneutralization, antibody responses were detected to avian-origin hemagglutinin subtypes H1, H3, H4, H5, H6, H7, H9, and H11. Strong antibody responses to pandemic H1N1 (pdmH1N1) were also detected, indicating that epizootic transmission of pdmH1N1 occurred among the southern sea otter population after the emergence of this human-origin virus in 2009. We conclude that southern sea otters are susceptible to infection with avian and human-origin IAV and that exposure to a wide array of subtypes likely occurs during a given otter's 10- to 15-yr life span. Important unanswered questions include what effect, if any, IAV infection has on sea otter health, and how these animals become infected in their nearshore environment.

Capture and characterization of influenza A virus from primary samples using glycan bead arrays.

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.

Complete genome sequences of the first reported california h16 influenza a viruses.

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.

Avian influenza: mixed infections and missing viruses.

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.

Complete Genome Sequence of a Reassortant H14N2 Avian Influenza Virus from California.

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.

Evaluation of phenotypic markers in full genome sequences of avian influenza isolates from California.

We evaluated phenotypic markers in full-genome sequences of avian influenza isolates to identify avian strains with increased potential for transmission and pathogenicity in mammals. Of 149 markers examined, 67 were positive in the consensus sequences from 206 avian isolates. Analysis of deep sequencing data in a subset of 24 isolates revealed that 344 subpopulations occurred at marker positions. Markers in subpopulations were significantly more likely to be negative (258/344) than positive (86/344), but nearly all of the marker-positive subpopulations (78/86) were associated with marker-negative consensus sequences. Our analysis revealed significant variation in important markers among avian isolates, and showed that consensus sequences do not fully convey an isolate's potential for increased transmissibility and pathogenicity in mammals.

Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production.

The quality-by-design (QbD) regulatory initiative promotes the development of process design spaces describing the multidimensional effects and interactions of process variables on critical quality attributes of therapeutic products. However, because of the complex nature of production processes, strategies must be devised to provide for design space development with reasonable allocation of resources while maintaining highly dependable results. Here, we discuss strategies for the determination of design spaces for viral clearance by anion exchange chromatography (AEX) during purification of monoclonal antibodies. We developed a risk assessment for AEX using a formalized method and applying previous knowledge of the effects of certain variables and the mechanism of action for virus removal by this process. We then use design-of-experiments (DOE) concepts to perform a highly fractionated factorial experiment and show that varying many process parameters simultaneously over wide ranges does not affect the ability of the AEX process to remove endogenous retrovirus-like particles from CHO-cell derived feedstocks. Finally, we performed a full factorial design and observed that a high degree of viral clearance was obtained for three different model viruses when the most significant process parameters were varied over ranges relevant to typical manufacturing processes. These experiments indicate the robust nature of viral clearance by the AEX process as well as the design space where removal of viral impurities and contaminants can be assured. In addition, the concepts and methodology presented here provides a general approach for the development of design spaces to assure that quality of biotherapeutic products is maintained.

Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products.

The mammalian cell-lines used to produce biopharmaceutical products are known to produce endogenous retrovirus-like particles and have the potential to foster adventitious viruses as well. To ensure product safety and regulatory compliance, recovery processes must be capable of removing or inactivating any viral impurities or contaminants which may be present. Anion exchange chromatography (AEX) is a common process in the recovery of monoclonal antibody products and has been shown to be effective for viral removal. To further characterize the robustness of viral clearance by AEX with respect to process variations, we have investigated the ability of an AEX process to remove three model viruses using various combinations of mAb products, feedstock conductivities and compositions, equilibration buffers, and pooling criteria. Our data indicate that AEX provides complete or near-complete removal of all three model viruses over a wide range of process conditions, including those typically used in manufacturing processes. Furthermore, this process provides effective viral clearance for different mAb products, using a variety of feedstocks, equilibration buffers, and different pooling criteria. Viral clearance is observed to decrease when feedstocks with sufficiently high conductivities are used, and the limit at which the decrease occurs is dependent on the salt composition of the feedstock. These data illustrate the robust nature of the AEX recovery process for removal of viruses, and they indicate that proper design of AEX processes can ensure viral safety of mAb products.