Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard-a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used beta-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. beta-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.

Background Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied. Aim and Methods In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naive Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype. Results and Conclusion Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.

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 mu g/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.

Studies of parasitism in chinstrap penguins (Pygoscelis antarctica) are infrequent and mainly refer to the identification and description of its parasites, with little ecological data. In an attempt to address that lack of knowledge, we collected endo- and ecto-parasites from 326 live and four dead of chinstrap penguins, in three different localities of Antarctica not studied before. Three species of endoparasites and two of ectoparasites were found parasitizing birds: two tapeworms, Tetrabothrius pauliani (Cestoda: Tetrabothriidae) and Parorchites zederi (Cestoda: Dilepididae); one roundworm, Stegophorus macronectes (Nematoda: Acuariidae), and one feather louse: Austrogoniodes gressitti (Insecta: Phthiraptera: Philopteridae). Ticks (Ixodes uriae-Acari: Ixodidae) were collected from the ground near the penguin nesting colonies at two localities, Shirreff Cape and Narebsky Point. New ecological data are given for the two species of ectoparasites. No parasites were found in the blood collected from 300 live penguins.

The vertebrate innate immune system provides hosts with a rapid, non-specific response to a wide range of invading pathogens. However, the speed and duration of innate responses will be influenced by the co-evolutionary dynamics of specific host-pathogen combinations. Here, we show that low pathogenic avian influenza virus (LPAI) subtype H1N1 elicits a strong but extremely transient innate immune response in its main wildlife reservoir, the mallard (Anas platyrhynchos). Using a series of experimental and methodological improvements over previous studies, we followed the expression of retinoic acid inducible gene 1 (RIG-I) and myxovirus resistance gene (Mx) in mallards semi-naturally infected with low pathogenic H1N1. One day post infection, both RIG-I and Mx were significantly upregulated in all investigated tissues. By two days post infection, the expression of both genes had generally returned to basal levels, and remained so for the remainder of the experiment. This is despite the fact that birds continued to actively shed viral particles throughout the study period. We additionally show that the spleen plays a particularly active role in the innate immune response to LPAI. Waterfowl and avian influenza viruses have a long co-evolutionary history, suggesting that the mallard innate immune response has evolved to provide a minimum effective response to LPAIs such that the viral infection is brought under control while minimising the damaging effects of a sustained immune response.

This article provides data on primer sequences used to amplify the innate immune genes RIG-I and Mx and a set of normalizing reference genes in mallards (Anal platyrhynchos), and shows which reference genes are stable, per tissue, for our experimental settings. Data on the expressional changes of these two genes over a time-course of infection with low pathogenic avian influenza virus (LPAI) are provided. Individual-level data are also presented, including LPAI infection load, and per tissue gene expression of RIG-I and Mx. Gene expression in two outlier individuals is explored in more depth. (C) 2018 The Authors. Published by Elsevier Inc.

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 island of Newfoundland, Canada, is at the eastern edge of North America and has migratory bird connections with the continental mainland as well as across the North Atlantic Ocean. Here, we report a 4-year avian influenza virus (AIV) epidemiological study in ducks in the St. John's region of Newfoundland. The overall prevalence of AIV detection in ducks during this study was 7.2%, with American Black Ducks contributing the vast majority of the collected samples and the AIV positives. The juvenile ducks showed a significantly higher AIV detection rate (10.6%) compared with adults (3.4%). Seasonally, AIV prevalence rates were higher in the autumn (8.4%), but positives were still detected in the winter (4.6%). Preliminary serology tests showed a high incidence of previous AIV infection (20/38, 52.6%). A total of 43 viruses were characterized for their HA-NA or HA subtypes, which revealed a large diversity of AIV subtypes and little recurrence of subtypes from year to year. Investigation of the movement patterns of ducks in this region showed that it is a largely non-migratory duck population, which may contribute to the observed pattern of high AIV subtype turnover. Phylogenetic analysis of 4 H1N1 and one H5N4 AIVs showed these viruses were highly similar to other low pathogenic AIV sequences from waterfowl in North America and assigned all gene segments into American-avian clades. Notably, the H1N1 viruses, which were identified in consecutive years, possessed homologous genomes. Such detection of homologous AIV genomes across years is rare, but indicates the role of the environmental reservoir in viral perpetuation.

Wild birds, including waterfowl such as ducks, are reservoir hosts of influenza A viruses. Despite the increased number of avian influenza virus (AIV) genome sequences available, our understanding of AIV genetic structure and transmission through space and time in waterfowl in North America is still limited. In particular, AIVs in ducks of the Atlantic flyway of North America have not been thoroughly investigated. To begin to address this gap, we analyzed 109 AIV genome sequences from ducks in the Atlantic flyway to determine their genetic structure and to document the extent of gene flow in the context of sequences from other locations and other avian and mammalian host groups. The analyses included 25 AIVs from ducks from Newfoundland, Canada, from 2008-2011 and 84 available reference duck AIVs from the Atlantic flyway from 2006-2011. A vast diversity of viral genes and genomes was identified in the 109 viruses. The genetic structure differed amongst the 8 viral segments with predominant single lineages found for the PB2, PB1 and M segments, increased diversity found for the PA, NP and NS segments (2, 3 and 3 lineages, respectively), and the highest diversity found for the HA and NA segments (12 and 9 lineages, respectively). Identification of inter-hemispheric transmissions was rare with only 2% of the genes of Eurasian origin. Virus transmission between ducks and other bird groups was investigated, with 57.3% of the genes having highly similar (≥99% nucleotide identity) genes detected in birds other than ducks. Transmission between North American flyways has been frequent and 75.8% of the genes were highly similar to genes found in other North American flyways. However, the duck AIV genes did display spatial distribution bias, which was demonstrated by the different population sizes of specific viral genes in one or two neighbouring flyways compared to more distant flyways.

Within the growing order of Chlamydiales, there are a number of pathogens. One is Chlamydia psittaci, a zoonotic pathogen, with birds as natural hosts that may be transmitted to humans and cause severe respiratory disease, psittacosis. The prevalence of this pathogen in Antarctic birds is almost unknown as well as the ramifications of its potential spread in na < ve bird populations. To investigate the prevalence of chlamydia organisms, cloacal and fecal samples were collected from 264 penguins and 263 seabirds on the Antarctic Peninsula and in Southern Chile. No C. psittaci could be detected by 23S rRNA real-time PCR. However, DNA sequencing of the 16S rRNA 298-bp signature sequence revealed a Chlamydiaceae-like bacterium previously found in seabirds from the subarctic zone, demonstrating that this not yet fully characterized bacterium is widespread. In conclusion, the prevalence of C. psittaci among wild birds on the Antarctic Peninsula seems to be low, but other types of chlamydial organisms are common. Further studies are required to taxonomically define and finally understand the role of these non-classified Chlamydiae.

Wild waterbirds, specifically waterfowl, gulls, and shorebirds, are recognized as the primordial reservoir of influenza A viruses (IAVs). However, the role of seabirds, an abundant, diverse, and globally distributed group of birds, in the perpetuation and transmission of IAVs is less clear. Here we summarize published and publicly available data for influenza viruses in seabirds, which for the purposes of this study are defined as birds that exhibit a largely or exclusively pelagic lifestyle and exclude waterfowl, gulls, and shorebirds, and we review this collective dataset to assess the role of seabirds in the influenza A ecology. Since 1961, more than 40,000 samples have been collected worldwide from the seabirds considered here and screened, using a variety of techniques, for evidence of active or past IAV infection. From these data, the overall prevalence of active infection has been estimated to be very low; however, serological data provide evidence that some seabird species are more frequently exposed to IAVs. Sequence data for viruses from seabirds are limited, except for murres (common murre, Uria aalge, and thick-billed murre, Uria lomvia; family Alcidae) for which there are full or partial genome sequences available for more than 80 viruses. Characterization of these viruses suggests that murres are infected with Group 1 hemagglutinin subtype viruses more frequently as compared to Group 2 and also indicates that these northern, circumpolar birds are frequently infected by intercontinental reassortant viruses. Greater temporal and spatial sampling and characterization of additional viruses are required to better understand the role of seabirds in global IAV dynamics.

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.

Avian influenza viruses (AIVs) continue to impose a negative impact on animal and human health worldwide. In particular, the emergence of highly pathogenic AIV H5 and, more recently, the emergence of low pathogenic AIV H7N9 have led to enormous socioeconomical losses in the poultry industry and resulted in fatal human infections. While H5N1 remains infamous, the number of zoonotic infections with H7N9 has far surpassed those attributed to H5. Despite the clear public health concerns posed by AIV H7, it is unclear why specifically this virus subtype became endemic in poultry and emerged in humans. In this review, we bring together data on global patterns of H7 circulation, evolution and emergence in humans. Specifically, we discuss data from the wild bird reservoir, expansion and epidemiology in poultry, significant increase in their zoonotic potential since 2013 and genesis of highly pathogenic H7. In addition, we analysed available sequence data from an evolutionary perspective, demonstrating patterns of introductions into distinct geographic regions and reassortment dynamics. The integration of all aspects is crucial in the optimisation of surveillance efforts in wild birds, poultry and humans, and we emphasise the need for a One Health approach in controlling emerging viruses such as AIV H7.

During recent years, extensive amounts of data have become available regarding influenza A virus (IAV) in wild birds in northern Europe, while information from southern Europe is more limited. Here, we present an IAV surveillance study conducted in western Portugal 2008-2009, analyzing 1653 samples from six different species of waterfowl, with the majority of samples taken from Mallards (Anas platyrhynchos). Overall 4.4% of sampled birds were infected. The sampling results revealed a significant temporal variation in the IAV prevalence, including a pronounced peak among predominantly young birds in June, indicating that IAV circulate within breeding populations in the wetlands of western Portugal. The H10N7 and H9N2 subtypes were predominant among isolated viruses. Phylogenetic analyses of the hemagglutinin and neuraminidase sequences of H10N7, H9N2 and H11N3 virus showed that sequences from Portugal were closely related to viral sequences from Central Europe as well as to IAVs isolated in the southern parts of Africa, reflecting Portugal's position on the European-African bird migratory flyway. This study highlights the importance of Portugal as a migratory crossroad for IAV, connecting breeding stationary waterfowl with birds migrating between continents which enable transmission and spread of IAV.

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.

Background: Newcastle disease virus (NDV) is the causative agent of the Newcastle disease, a severe disease in birds associated with substantial economic losses to the poultry industry worldwide. Sweden is situated along the Western European waterfowl flyway and applies a non-vaccination policy combined with directives of immediate euthanisation of NDV infected flocks. During the last decades there have been several outbreaks with NDV in poultry in Sweden. However, less is known about the virus prevalence in the wild bird population including waterfowl, a well-established reservoir of avian paramyxovirus type 1 (APMV-1), the paramyxovirus serotype that include pathogenic NDV. Methods: The survey constituted of 2332 samples from Mallards (Anas platyrhynchos), trapped in the southern part of Sweden during autumn migration in 2010. These samples were screened for APMV-1 by real-time reverse transcription PCR, and viral strains from positive samples were isolated and characterized by sequence analysis of the fusion gene and by phylogenetic analysis. Conclusions: Twenty of these samples were positive for APMV-1, hence a virus prevalence of 0.9% (95% Confidence Interval [95% CI]=0.54%, 1.35%). The highest APMV-1 prevalence was detected in juvenile Mallards sampled in November (n=887, prevalence 1.24% ([95% CI])=0.67%, 2.24%). Sequence analysis and evaluation of phylogenetic relatedness indicated that isolated APMV-1 strains were lentogenic, and phylogenetically most closely related to genotype Ib strains within the clade of class II viruses. The sampling system employed enabled us to follow APMV-1 infections and the shedding of one particular viral strain in one individual bird over several days. Furthermore, combining previous screening results with the APMV-1 detections in this study showed that more than 50% of Mallards that tested positive for APMV-1 RNA were co-infected with influenza A virus.

Low pathogenic influenza A virus (LPIAV) prevalence and subtype distribution differs between and across bird taxa. A crucial factor in the epidemiology of these viruses and virus subtypes is the ability to transmit between and within different host taxa and individuals. Successful viral transmission depends on availability of susceptible hosts and exposure of host to virus. Exposure to viruses and susceptibility to virus infection and/or disease are shaped by both host and virus traits. In this review we have identified key host and virus traits that can affect LPIAV transmission, both in terms of exposure and susceptibility. Furthermore we highlight current challenges in assessment of these traits and identify methodological considerations for future studies.

This thesis explores the evolution of avian influenza A viruses (IAV), as well as host-pathogen interactions between these viruses and their main reservoir host, the Mallard (Anas platyrhynchos). IAV is a genetically diverse, multi-host virus and wild birds, particularly dabbling ducks, are the natural reservoir. At our study site, up to 30% of migratory Mallards are infected with IAV during an autumn season, and host a large number of virus subtypes. IAV diversity is driven by two main mechanisms: mutation, driving genetic drift; and reassortment following co-infection, resulting in genetic shift.

Reassortment is pervasive within an autumn season, both across multiple subtypes and within a single subtype. It is a key genetic feature in long-term maintenance of common subtypes, as it allows for independent lineage turn-over, generating novel genetic constellations. I hypothesize that the decoupling of successful constellations and generation of novel annual constellations enables viruses to escape herd immunity; these genetic changes must confer antigenic change for the process to be favourable. Indeed, in an experiment utilizing vaccines, circulating viruses escaped homosubtypic immunity, resulting in the proliferation of infections with the same subtype as the vaccine. While the host plays an important role in shaping IAV evolutionary genetics, one must consider that Mallards are infected with a multitude of other microorganisms. Here, Mallards were infected with IAV, gamma coronaviruses, and avian paramyxovirus type 1 simultaneously, and we found a putative synergistic interaction between IAV and gamma coronaviruses.

Mallards occupy the interface between humans, poultry, and wild birds, and are the reservoir of IAV diversity. New incursions of highly pathogenic H5 viruses to both Europe and North America reaffirms the role of wild birds, particularly waterfowl, in diffusion of viruses spatially. Using European low pathogenic viruses and Mallard model, this thesis contributes to aspects of epidemiology, ecology, and evolutionary dynamics of waterfowl viruses, particularly IAV

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 Coy 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. (C) 2014 Elsevier B.V. All rights reserved.

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; Leach's 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.

Influenza A virus (IAV) is ubiquitous in waterfowl. In the northern hemisphere IAV prevalence is highest during the autumn and coincides with a peak in viral subtype diversity. Although haemagglutinin subtypes H1-H12 are associated with waterfowl hosts, subtypes H8-H12 are detected very infrequently. To better understand the role of waterfowl in the maintenance of these rare subtypes, we sequenced H8-H12 viruses isolated from Mallards (Anas platyrhynchos) from 2002 to 2009. These rare viruses exhibited varying ecological and phylodynamic features. The Eurasian clades of H8 and H12 phylogenies were dominated by waterfowl sequences; mostly viruses sequenced in this study. H11, once believed to be a subtype that infected charadriiformes (shorebirds), exhibited patterns more typical of common virus subtypes. Finally, subtypes H9 and H10, which have maintained lineages in poultry, showed markedly different patterns: H10 was associated with all possible NA subtypes and this drove HA lineage diversity within years. Rare viruses belonging to subtypes H8-H12 were highly reassorted, indicating that these rare subtypes are part of the broader IAV pool. Our results suggest that waterfowl play a role in the maintenance of these rare subtypes, but we recommend additional sampling of non-traditional hosts to better understand the reservoirs of these rare viruses.

The Mallard (Anas platyrhynchos) is an important reservoir species for influenza A viruses (IAV), and in this host, prevalence and virus diversity are high. Studies have demonstrated the presence of homosubtypic immunity, where individuals are unlikely to be reinfected with the same subtype within an autumn season. Further, evidence for heterosubtypic immunity exists, whereby immune responses specific for one subtype offer partial or complete protection against related HA subtypes. We utilized a natural experimental system to determine whether homo- or heterospecific immunity could be induced following experimental vaccination. Thirty Mallards were vaccinated with an inactivated H3, H6 or a sham vaccine and after seroconversion were exposed to naturally infected wild conspecifics. All ducks were infected within 2days and had both primary and secondary infections. Overall, there was no observable difference between groups; all individuals were infected with H3 and H10 IAV. At the cessation of the experiment, most individuals had anti-NP antibodies and neutralizing antibodies against H10. Not all individuals had H3 neutralizing antibodies. The isolated H3 IAVs revealed genetic dissimilarity to the H3 vaccine strain, specifically substitutions in the vicinity of the receptor-binding site. There was no evidence of vaccine-induced homosubtypic immunity to H3, a likely result of both a poor H3 immune response in the ducks and H3 immune escape. Likewise, there was no observed heterosubtypic protection related to H6 vaccination. This study highlights the need for experimental approaches to assess how exposure to pathogens and resulting immune processes translates to individual and population disease dynamics.

A hallmark of disease is that most pathogens are able to infect more than one host species. However, for most pathogens, we still have a limited understanding of how this affects epidemiology, persistence and virulence of infections—including several zoonotic pathogens that reside in wild animal reservoirs and spillover into humans. In this chapter, we review the current knowledge of mallard (Anas platyrhynchos) as host for pathogens. This species is widely distributed, often occupying habitats close to humans and livestock, and is an important game bird species and the ancestor to domestic ducks—thereby being an excellent model species to highlight aspects of the wildlife, domestic animal interface and the relevance for human health. We discuss mallard as host for a range of pathogens but focus more in depth of it as a reservoir host for influenza A virus (IAV). Over the last decades, IAV research has surged, prompted in part to the genesis and spread of highly pathogenic virus variants that have been devastating to domestic poultry and caused a number of human spillover infections. The aim of this chapter is to synthesise and review the intricate interactions of virus, host and environmental factors governing IAV epidemiology and evolution.

Coronaviruses (CoVs) are found in a wide variety of wild and domestic animals, and constitute a risk for zoonotic and emerging infectious disease. In poultry, the genetic diversity, evolution, distribution and taxonomy of some coronaviruses have been well described, but little is known about the features of CoVs in wild birds. In this study we screened 764 samples from 22 avian species of the orders Anseriformes and Charadriiformes in Sweden collected in 2006/2007 for CoV, with an overall CoV prevalence of 18.7%, which is higher than many other wild bird surveys. The highest prevalence was found in the diving duck-smainly Greater Scaup (Aythya marila; 51.5%)-and the dabbling duck Mallard (Anas platyrhynchos; 19.2%). Sequences from two of the Greater Scaup CoV fell into an infrequently detected lineage, shared only with a Tufted Duck (Aythya fuligula) CoV. Coronavirus sequences from Mallards in this study were highly similar to CoV sequences from the sample species and location in 2011, suggesting long-term maintenance in this population. A single Black-headed Gull represented the only positive sample from the order Charadriiformes. Globally, Anas species represent the largest fraction of avian CoV sequences, and there seems to be no host species, geographical or temporal structure. To better understand the eitiology, epidemiology and ecology of these viruses more systematic surveillance of wild birds and subsequent sequencing of detected CoV is imperative.

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 ofnew 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.

Avian influenza A virus (AIV) is ubiquitous in waterfowl and is detected annually at high prevalence in waterfowl during the Northern Hemisphere autumn. Some AIV subtypes are globally common in waterfowl, such as H3N8, H4N6, and H6N2, and are detected in the same populations at a high frequency, annually. In order to investigate genetic features associated to the long-term maintenance of common subtypes in migratory ducks, we sequenced 248 H4 viruses isolated across 8 years (2002-9) from mallards (Anas platyrhynchos) sampled in southeast Sweden. Phylogenetic analyses showed that both H4 and N6 sequences fell into three distinct lineages, structured by year of isolation. Specifically, across the 8 years of the study, we observed lineage replacement, whereby a different HA lineage circulated in the population each year. Analysis of deduced amino acid sequences of the HA lineages illustrated key differences in regions of the globular head of hemagglutinin that overlap with established antigenic sites in homologous hemagglutinin H3, suggesting the possibility of antigenic differences among these HA lineages. Beyond HA, lineage replacement was common to all segments, such that novel genome constellations were detected across years. A dominant genome constellation would rapidly amplify in the duck population, followed by unlinking of gene segments as a result of reassortment within 2-3 weeks following introduction. These data help reveal the evolutionary dynamics exhibited by AIV on both annual and decadal scales in an important reservoir host.

Detection of influenza virus in oropharyngeal swabs collected during wild bird surveillance is assumed to representrespiratory infection, although intestine is the main site of infection. We tested this assumption by histologicalexamination of the respiratory tract of wild Mallards with virus-positive oropharyngeal swabs. Thirty-two of 125Mallards tested had viral-RNA positive oropharyngeal swabs. The respiratory tracts of four Mallards with the mostvirus were examined in detail by immunohistochemistry. None had detectable virus antigen in the respiratory tract,suggesting it was not infected. An alternative explanation is that the oropharynx was contaminated with virusthrough feeding in surface water or through preening.