Michelle Wille

Long-term variation in influenza A virus prevalence and subtype diversity in migratory mallards in northern Europe.

Data on long-term circulation of pathogens in wildlife populations are seldom collected, and hence understanding of spatial–temporal variation in prevalence and genotypes is limited. Here, we analysed a long-term surveillance series on influenza A virus (IAV) in mallards collected at an important migratory stopover site from 2002 to 2010, and characterized seasonal dynamics in virus prevalence and subtype diversity. Prevalence dynamics were influenced by year, but retained a common pattern for all years whereby prevalence was low in spring and summer, but increased in early autumn with a first peak in August, and a second more pronounced peak during October–November. A total of 74 haemagglutinin (HA)/neuraminidase (NA) combinations were isolated, including all NA and most HA (H1–H12) subtypes. The most common subtype combinations were H4N6, H1N1, H2N3, H5N2, H6N2 and H11N9, and showed a clear linkage between specific HA and NA subtypes. Furthermore, there was a temporal structuring of subtypes within seasons based on HA phylogenetic relatedness. Dissimilar HA subtypes tended to have different temporal occurrence within seasons, where the subtypes that dominated in early autumn were rare in late autumn, and vice versa. This suggests that build-up of herd immunity affected IAV dynamics in this system.

Individual Variation in Influenza A Virus Infection Histories and Long-Term Immune Responses in Mallards

Wild dabbling ducks (genus Anas) are the main reservoir for influenza A virus (IAV) in the Northern Hemisphere. Current understanding of disease dynamics and epidemiology in this virus-host system has primarily been based on population-level surveillance studies and infection experiments conducted in laboratory settings. Using a combined experimental-natural approach with wild-strain captive mallards (Anas platyrhynchos), we monitored individual IAV infection histories and immunological responses of 10 birds over the course of 15 months. This is the first detailed study to track natural IAV infection histories over several seasons amongst the same individuals growing from juvenile to adults. The general trends in the infection histories of the monitored birds reflected seasonal variation in prevalence at the population level. However, within the study group there were significant differences between individuals in infection frequency as well as in short and long term anti-IAV antibody response. Further observations included individual variation in the number of infecting virus subtypes, and a strong tendency for long-lasting hemagglutinin-related homosubtypic immunity. Specifically, all infections in the second autumn, except one, were of different subtypes compared to the first autumn. The variation among birds concerning these epidemiologically important traits illustrates the necessity for IAV studies to move from the level of populations to examine individuals in order to further our understanding of IAV disease and epidemiology.

Extensive Geographic Mosaicism in Avian Influenza Viruses from Gulls in the Northern Hemisphere

Due to limited interaction of migratory birds between Eurasia and America, two independent avian influenza virus (AIV) gene pools have evolved. There is evidence of low frequency reassortment between these regions, which has major implications in global AIV dynamics. Indeed, all currently circulating lineages of the PB1 and PA segments in North America are of Eurasian origin. Large-scale analyses of intercontinental reassortment have shown that viruses isolated from Charadriiformes (gulls, terns, and shorebirds) are the major contributor of these outsider events. To clarify the role of gulls in AIV dynamics, specifically in movement of genes between geographic regions, we have sequenced six gull AIV isolated in Alaska and analyzed these along with 142 other available gull virus sequences. Basic investigations of host species and the locations and times of isolation reveal biases in the available sequence information. Despite these biases, our analyses reveal a high frequency of geographic reassortment in gull viruses isolated in America. This intercontinental gene mixing is not found in the viruses isolated from gulls in Eurasia. This study demonstrates that gulls are important as vectors for geographically reassorted viruses, particularly in America, and that more surveillance effort should be placed on this group of birds.

Reassortment of American and Eurasian genes in an influenza A virus isolated from a great black-backed gull (Larus marinus), a species demonstrated to move between these regions

The primary hosts for influenza A viruses are waterfowl, although gulls and shorebirds are also important in global avian influenza dynamics. Avian influenza virus genes are separated phylogenetically into two geographic clades, American and Eurasian, which is caused by the geographic separation of the host species between these two regions. We surveyed a gregarious and cosmopolitan species, the Great Black-backed Gull (Larus marinus), in Newfoundland, Canada, for the presence of avian influenza viruses. We have isolated and determined the complete genome sequence of an H13N2 virus, A/Great Black-backed Gull/Newfoundland/296/2008(H13N2), from one of these birds. Phylogenetic analysis revealed that this virus contained two genes in the American gull clade (PB1, HA), two genes in the American avian clade (PA, NA), and four genes in the Eurasian gull clade (PB2, NP, M, NS). We analyzed bird band recovery information and found the first evidence of trans-Atlantic migration from Newfoundland to Europe (UK, Spain and Portugal) for this species. Thus, great black-backed gulls could be important for movement of avian influenza viruses across the Atlantic Ocean and within North America.

Frequency and patterns of reassortment in natural influenza A virus infection in a reservoir host

Influenza A viruses (IAV) can dramatically alter both genotype and phenotype at a rapid rate as a product of co-infection and reassortment. Avian IAV exhibit high levels of phylogenetic incongruence, suggesting high levels of reassortment in the virus reservoir. Using a natural-experimental system, we reconstructed relationships amongst 92 viruses across 15 subtypes from 10 Mallards in an autumn season. Phylogenetic analyses estimated that 56% of the isolated viruses were reassorted. Network analysis demonstrated different patterns of reassortment and limited exchange of segments between primary and secondary infections. No clear patterns of linkage between segments were found, and patterns within a season were likely the consequence of continued introduction of new constellations, high viral load and diversity in the wild bird reservoir, and co-infections. This is the first IAV study to implement multiple tools available for elucidating factors governing reassortment patterns in naturally infected Mallards.

Perpetuation and reassortment of gull influenza A viruses in Atlantic North America.

Gulls are important hosts of avian influenza A viruses (AIVs) and gull AIVs often contain gene segments of mixed geographic and host lineage origins. In this study, the prevalence of AIV in gulls of Newfoundland, Canada from 2008 to 2011 was analyzed. Overall prevalence was low (30/1645, 1.8%) but there was a distinct peak of infection in the fall. AIV seroprevalence was high in Newfoundland gulls, with 50% of sampled gulls showing evidence of previous infection. Sequences of 16 gull AIVs were determined and analyzed to shed light on the transmission, reassortment and persistence dynamics of gull AIVs in Atlantic North America. Intercontinental and waterfowl lineage reassortment was prevalent. Of particular note were a wholly Eurasian AIV and another with an intercontinental reassortant waterfowl lineage virus. These patterns of geographic and inter-host group transmission highlight the importance of characterization of gull AIVs as part of attempts to understand global AIV dynamics.

The genome sequence of an H11N2 avian influenza virus from a Thick-billed Murre (Uria lomvia) shows marine-specific and regional patterns of relationships to other viruses.

Influenza A viruses infect a range of host species, including various mammals and more than 100 species of birds. For avian influenza viruses (AIV), prevalence varies between different groups of birds, with waterfowl showing the highest prevalence. We have sequenced the complete genome of A/Thick-billed Murre/Newfoundland/031/2007(H11N2), an AIV identified in the pelagic seabird, Thick-billed Murre (Uria lomvia). This represents the first complete genome sequence of an AIV from this host species, and only the second complete genome sequence from a seabird in the alcid group. All of the virus segments fall within the American avian lineage. Several of the segments show a close relationship to AIV identified in other marine host species, and also a strong geographic association with other AIV sequences from the northeastern coast of North America from recent years. The identification of this virus, and the growing number of AIV identified in seabird species, indicates these marine birds could be underappreciated host species. This has potential consequences for global influenza dynamics because of the seasonal distributions and migratory patterns of this group of birds.

Temporal dynamics, diversity, and interplay in three components of the virodiversity of a Mallard population: influenza A virus, avian paramyxovirus and avian coronavirus.

Abstract Multiple infections, or simultaneous infection of a host with multiple parasites, are the rule rather than the exception. Interactions between co-occurring pathogens in a population may be mutualistic, competitive or facilitative. For some pathogen combinations, these interrelated effects will have epidemiological consequences; however this is as yet poorly incorporated into practical disease ecology. For example, screening of Mallards for influenza A viruses (IAV) have repeatedly revealed high prevalence and large subtype diversity in the Northern Hemisphere. Other studies have identified avian paramyxovirus type 1 (APMV-1) and coronaviruses (CoVs) in Mallards, but without making inferences on the larger viral assemblage. In this study we followed 144 wild Mallards across an autumn season in a natural stopover site and constructed infection histories of IAV, APMV-1 and CoV. There was a high prevalence of IAV, comprising of 27 subtype combinations, while APMV-1 had a comparatively low prevalence (with a peak of 2%) and limited strain variation, similar to previous findings. Avian CoVs were common, with prevalence up to 12%, and sequence analysis identified different putative genetic lineages. An investigation of the dynamics of co-infections revealed a synergistic effect between CoV and IAV, whereby CoV prevalence was higher given that the birds were co-infected with IAV. There were no interactive effects between IAV and APMV-1. Disease dynamics are the result of an interplay between parasites, host immune responses, and resources; and is imperative that we begin to include all factors to better understand infectious disease risk.

Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water

ABSTRACT Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

Evaluation of seabirds in Newfoundland and Labrador, Canada, as hosts of influenza A viruses.

Abstract Influenza A viruses infect a wide range of hosts, including many species of birds. Avian influenza A virus (AIV) infection appears to be most common in Anseriformes (ducks, geese, and swans) and some Charadriiformes (shorebirds and gulls), but many other birds also serve as hosts of AIV. Here, we evaluated the role of seabirds as hosts for AIV. We tested 3,160 swab samples from 13 seabird species between May 2008 and December 2011 in Newfoundland and Labrador, Canada. We also tested 156 serum samples for evidence of previous infection of AIV in Common Murres (Uria aalge) and Atlantic Puffins (Fratercula arctica). Avian influenza A virus was detected in breeding Common Murres and nonbreeding Thick-billed Murres (Uria lomvia), and Common Murres also had high antibody prevalence (44%). From these findings, combined with other studies showing AIV infection in murres, we conclude that murres are important for the ecology of AIV. For other species (Razorbill, Alca torda; Leachs Storm-Petrel, Oceanodroma leucorhoa; Black-legged Kittiwake, Rissa tridactyla; Atlantic Puffin) with good coverage (>100 samples) we did not detect AIV. However, serology indicates infection does occur in Atlantic Puffins, with 22% antibody prevalence found. The possibility of virus spread through dense breeding colonies and the long distance movements of these hosts make a more thorough evaluation of the role for seabirds as hosts of AIV important.