Rebecca L. Poulson

Avian influenza virus in water: infectivity is dependent on pH, salinity and temperature.

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoir for avian influenza (AI) viruses. Transmission within these aquatic bird populations occurs through an indirect fecal-oral route involving contaminated water on shared aquatic habitats. In order to better understand the influence that aquatic environments exert on AI transmission and maintenance in the wild-bird reservoir system, we determined the duration of persistence for 12 wild-bird origin AI viruses under natural ranges of pH, salinity, and temperature. Viral persistence was measured using a laboratory-based distilled water model system. The AI viruses varied in their response to each of the examined variables, but, generally, the viruses were most stable at a slightly basic pH (7.4-8.2), low temperatures (<17 degrees C), and fresh to brackish salinities (0-20,000 parts per million (ppm)). Alternatively, the AI viruses had a much shorter duration of persistence in acidic conditions (pH<6.6), warmer temperatures (>32 degrees C), and high salinity (>25,000 ppm). The results of this research suggest that the pH, temperature, and salinity in natural aquatic habitats can influence the ability of AI viruses to remain infective within these environments. Furthermore, these results provide insight into chemical and physical properties of water that could enhance or restrict AI virus transmission on an aquatic bird habitat.

Influenza-A Viruses in Ducks in Northwestern Minnesota: Fine Scale Spatial and Temporal Variation in Prevalence and Subtype Diversity

Waterfowl from northwestern Minnesota were sampled by cloacal swabbing for Avian Influenza Virus (AIV) from July – October in 2007 and 2008. AIV was detected in 222 (9.1%) of 2,441 ducks in 2007 and in 438 (17.9%) of 2,452 ducks in 2008. Prevalence of AIV peaked in late summer. We detected 27 AIV subtypes during 2007 and 31 during 2008. Ten hemagglutinin (HA) subtypes were detected each year (i.e., H1, 3–8, and 10–12 during 2007; H1-8, 10 and 11 during 2008). All neuraminidase (NA) subtypes were detected during each year of the study. Subtype diversity varied between years and increased with prevalence into September. Predominant subtypes during 2007 (comprising ≥5% of subtype diversity) included H1N1, H3N6, H3N8, H4N6, H7N3, H10N7, and H11N9. Predominant subtypes during 2008 included H3N6, H3N8, H4N6, H4N8, H6N1, and H10N7. Additionally, within each HA subtype, the same predominant HA/NA subtype combinations were detected each year and included H1N1, H3N8, H4N6, H5N2, H6N1, H7N3, H8N4, H10N7, and H11N9. The H2N3 and H12N5 viruses also predominated within the H2 and H12 subtypes, respectively, but only were detected during a single year (H2 and H12 viruses were not detected during 2007 and 2008, respectively). Mallards were the predominant species sampled (63.7% of the total), and 531 AIV were isolated from this species (80.5% of the total isolates). Mallard data collected during both years adequately described the observed temporal and spatial prevalence from the total sample and also adequately represented subtype diversity. Juvenile mallards also were adequate in describing the temporal and spatial prevalence of AIV as well as subtype diversity.

Avian Influenza Virus in Aquatic Habitats: What Do We Need to Learn?

Abstract Although aquatic habitats utilized by wild and domestic birds potentially can provide a bridge for avian influenza virus (AIV) transmission among many diverse hosts, the factors controlling environmental persistence and transmission via these habitats are poorly understood. AIV has been detected in water samples collected in the field, and under experimental laboratory conditions, these viruses can remain infective in water for periods of time that would be consistent with an environmental reservoir. However, the application of laboratory results to field realities is complicated by the complexity and scale of these systems. In this brief review, we present a summary of existing research on the environmental tenacity of AIV, provide an example of the challenges associated with the application of laboratory results to the field realities associated with detection of AIV from environmental sources, and identify gaps in our current understanding of the factors potentially affecting AIV infectivity in the environment, specifically from aquatic habitats utilized by wild birds.

DETECTION OF AVIAN INFLUENZA VIRUSES FROM SHOREBIRDS: EVALUATION OF SURVEILLANCE AND TESTING APPROACHES

Although influenza A viruses have been isolated from numerous shorebird species (Family: Scolopacidae) worldwide, our understanding of natural history of these viruses in this diverse group is incomplete. Gaining this information can be complicated by sampling difficulties related to live capture, the need for large sample sizes related to a potentially low prevalence of infection, and the need to maintain flexibility in diagnostic approaches related to varied capabilities and resources. To provide information relevant to improving sampling and testing of shorebirds for influenza A viruses, we retrospectively evaluated a combined data set from Delaware Bay, USA, collected from 2000 to 2009. Our results indicate that prevalence trends and subtype diversity can be effectively determined by either direct sampling of birds or indirect sampling of feces; however, the extent of detected subtype diversity is a function of the number of viruses recovered during that year. Even in cases where a large number of viruses are identified, an underestimate of true subtype diversity is likely. Influenza A virus isolation from Ruddy Turnstones can be enhanced by testing both cloacal and tracheal samples, and matrix real-time PCR can be used as an effective screening tool. Serologic testing to target species of interest also has application to shorebird surveillance. Overall, all of the sampling and diagnostic approaches have utility as applied to shorebird surveillance, but all are associated with inherent biases that need to be considered when comparing results from independent studies.

Intestinal excretion of a wild bird-origin H3N8 low pathogenic avian influenza virus in mallards (Anas Platyrhynchos).

Mallards (Anas platyrhynchos) and other dabbling ducks in the genus Anas are an important component of the wild bird reservoir for avian influenza (AI) virus; these viruses are maintained in migratory duck populations through a fecal-oral transmission route. We provide a detailed characterization of intestinal viral shedding in Mallards infected with a wild bird-origin low pathogenic (LP) AI virus. Five of eight, 1-mo-old Mallards inoculated with a high dose of an H3N8 LP AI virus became infected as determined by reisolation and seroconversion. Infected birds excreted high concentrations of virus for up to 14 days postinoculation (DPI) without exhibiting overt clinical signs of disease. The pattern of viral shedding was relatively consistent between individual birds, with peak shedding on 2–3 DPI and a progressive decline over the remainder of infection. Detection of viral shedding varied depending on sample type (excrement sample or cloacal swab) and diagnostic test (virus isolation or real-time quantitative reverse transcription polymerase chain reaction). Our data provide detailed insights into the intestinal excretion of an H3N8 LP AI virus in Mallards and the performance of diagnostic assays commonly used in wild bird surveillance. Such information is valuable for estimating potential risks for spillover of LP AI viruses from Mallards to domestic animals, developing accurate transmission models for Mallard populations and facilitating the interpretation and comparison of surveillance results from different studies.

The enigma of the apparent disappearance of Eurasian highly pathogenic H5 clade 2.3.4.4 influenza A viruses in North American waterfowl

Significance The role of wild aquatic birds in perpetuating highly pathogenic avian influenza viruses (HPAIVs) is unresolved. We examined whether the subtype H5 clade 2.3.4.4 HPAIV that devastated the US poultry industry in 2015 is perpetuated in wild aquatic birds. Virologic surveillance in 2014/15 and over the previous 43 y failed to detect HPAIVs in wild aquatic birds before or after the poultry outbreak, supporting the premise that there are unresolved mechanisms preventing wild aquatic birds from perpetuating HPAIVs. The significance of these findings is that timely and efficient strategies used to successfully prevent and eradicate HPAIVs infecting poultry, without the use of vaccines, appear to complement natural biological mechanisms in disrupting the perpetuation and possible spread of HPAIVs by wild aquatic birds. One of the major unresolved questions in influenza A virus (IAV) ecology is exemplified by the apparent disappearance of highly pathogenic (HP) H5N1, H5N2, and H5N8 (H5Nx) viruses containing the Eurasian hemagglutinin 2.3.4.4 clade from wild bird populations in North America. The introduction of Eurasian lineage HP H5 clade 2.3.4.4 H5N8 IAV and subsequent reassortment with low-pathogenic H?N2 and H?N1 North American wild bird-origin IAVs in late 2014 resulted in widespread HP H5Nx IAV infections and outbreaks in poultry and wild birds across two-thirds of North America starting in November 2014 and continuing through June 2015. Although the stamping out strategies adopted by the poultry industry and animal health authorities in Canada and the United States—which included culling, quarantining, increased biosecurity, and abstention from vaccine use—were successful in eradicating the HP H5Nx viruses from poultry, these activities do not explain the apparent disappearance of these viruses from migratory waterfowl. Here we examine current and historical aquatic bird IAV surveillance and outbreaks of HP H5Nx in poultry in the United States and Canada, providing additional evidence of unresolved mechanisms that restrict the emergence and perpetuation of HP avian influenza viruses in these natural reservoirs.

Evidence for seasonal patterns in the relative abundance of avian influenza virus subtypes in blue-winged teal ( Anas discors )

Abstract Seasonal dynamics of influenza A viruses (IAVs) are driven by host density and population immunity. Through an analysis of subtypic data for IAVs isolated from Blue-winged Teal (Anas discors), we present evidence for seasonal patterns in the relative abundance of viral subtypes in spring and summer/autumn.

Susceptibility of Avian Species to North American H13 Low Pathogenic Avian Influenza Viruses

SUMMARY. Gulls are widely recognized reservoirs for low pathogenic avian influenza (LPAI) viruses; however, the subtypes maintained in these populations and/or the transmission mechanisms involved are poorly understood. Although, a wide diversity of influenza viruses have been isolated from gulls, two hemagglutinin subtypes (H13 and H16) are rarely detected in other avian groups, and existing surveillance data suggests they are maintained almost exclusively within gull populations. In order to evaluate the host range of these gull-adapted influenza subtypes and to characterize viral infection in the gull host, we conducted a series of challenge experiments, with multiple North American strains of H13 LPAI virus in ring-billed gulls (Larus delawarensis), mallards (Anas platyrhynchos), chickens (Gallus domesticus), and turkeys (Meleagris gallopavo). The susceptibility to H13 LPAI viruses varied between species and viral strain. Gulls were highly susceptible to H13 LPAI virus infection and excreted virus via the oropharynx and cloaca for several days. The quantity and duration of shedding was similar between the two routes. Turkeys and ducks were resistant to infection with most strains of H13 LPAI virus, but low numbers of inoculated birds were infected after challenge with specific viral strains. Chickens were refractory to infection with all strains of H13 LPAI virus they were challenged with. The experimental results presented herein are consistent with existing surveillance data on H13 LPAI viruses in birds, and indicate that influenza viruses of the H13 subtype are strongly host-adapted to gulls, but rare spill-over into aberrant hosts (i.e., turkeys and ducks) can occur.

Genomic Characterization of H14 Subtype Influenza A Viruses in New World Waterfowl and Experimental Infectivity in Mallards (Anas platyrhynchos)

Recent repeated isolation of H14 hemagglutinin subtype influenza A viruses (IAVs) in the New World waterfowl provides evidence to suggest that host and/or geographic ranges for viruses of this subtype may be expanding. In this study, we used genomic analyses to gain inference on the origin and evolution of H14 viruses in New World waterfowl and conducted an experimental challenge study in mallards (Anas platyrhynchos) to evaluate pathogenicity, viral replication, and transmissibility of a representative viral strain in a natural host species. Genomic characterization of H14 subtype IAVs isolated from New World waterfowl, including three isolates sequenced specifically for this study, revealed high nucleotide identity among individual gene segments (e.g. ≥95% shared identity among H14 HA gene segments). In contrast, lower shared identity was observed among internal gene segments. Furthermore, multiple neuraminidase subtypes were observed for H14 IAVs isolated in the New World. Gene segments of H14 viruses isolated after 2010 shared ancestral genetic lineages with IAVs isolated from wild birds throughout North America. Thus, genomic characterization provided evidence for viral evolution in New World waterfowl through genetic drift and genetic shift since purported introduction from Eurasia. In the challenge study, no clinical disease or lesions were observed among mallards experimentally inoculated with A/blue-winged teal/Texas/AI13-1028/2013(H14N5) or exposed via contact with infected birds. Titers of viral shedding for mallards challenged with the H14N5 IAV were highest at two days post-inoculation (DPI); however shedding was detected up to nine DPI using cloacal swabs. The distribution of viral antigen among mallards infected with H14N5 IAV was largely restricted to enterocytes lining the villi in the lower intestinal tract and in the epithelium of the bursa of Fabricius. Characterization of the infectivity of A/blue-winged teal/Texas/AI13-1028/2013(H14N5) in mallards provides support for similarities in viral replication and shedding as compared to previously described waterfowl-adapted, low pathogenic IAV strains in ducks.

ARE PASSERINE BIRDS RESERVOIRS FOR INFLUENZA A VIRUSES

Abstract Although peridomestic passerine species have been involved in influenza A virus (IAV) outbreaks in poultry, there is little evidence to indicate they serve as reservoirs for these viruses under natural conditions. Recent molecular-based detections of IAV in terrestrial wild birds have challenged this paradigm, and it has been suggested that additional research is warranted to better define the role of these birds as IAV hosts. To address this need, we reviewed the published literature reporting results from IAV surveillance of passerines. We also conducted prospective virologic and serologic surveillance of North American passerines for IAVs. The literature review included 60 publications from 1975–2013 that reported results from 829 species of passerines and other terrestrial birds. In our prospective study during 2010 and 2011, 3,868 serum samples and 900 swab samples were collected and tested from 102 terrestrial wild bird species from Georgia, New Jersey, Delaware, and Minnesota, USA. Antibodies to the nucleoprotein of IAV were detected with a commercial blocking enzyme-linked immunosorbent assay in 4/3,868 serum samples (0.1%); all positive samples were from Minnesota. No virus was detected in 900 swab samples by virus isolation in embryonated chicken eggs or matrix real-time reverse transcriptase PCR. Our results are consistent with historic literature; although passerines and terrestrial wild birds may have a limited role in the epidemiology of IAV when associated with infected domestic poultry or other aberrant hosts, there is no evidence supporting their involvement as natural reservoirs for IAV.