Neus Latorre-Margalef

Surveillance of influenza A virus in migratory waterfowl in northern Europe

Ducks may maintain influenza virus from 1 year to the next.

Effects of influenza A virus infection on migrating mallard ducks.

The natural reservoir of influenza A virus is waterfowl, particularly dabbling ducks (genus Anas). Although it has long been assumed that waterfowl are asymptomatic carriers of the virus, a recent study found that low-pathogenic avian influenza (LPAI) infection in Bewicks swans (Cygnus columbianus bewickii) negatively affected stopover time, body mass and feeding behaviour. In the present study, we investigated whether LPAI infection incurred ecological or physiological costs to migratory mallards (Anas platyrhynchos) in terms of body mass loss and staging time, and whether such costs could influence the likelihood for long-distance dispersal of the avian influenza virus by individual ducks. During the autumn migrations of 2002–2007, we collected faecal samples (n=10 918) and biometric data from mallards captured and banded at Ottenby, a major staging site in a flyway connecting breeding and wintering areas of European waterfowl. Body mass was significantly lower in infected ducks than in uninfected ducks (mean difference almost 20 g over all groups), and the amount of virus shed by infected juveniles was negatively correlated with body mass. There was no general effect of infection on staging time, except for juveniles in September, in which birds that shed fewer viruses stayed shorter than birds that shed more viruses. LPAI infection did not affect speed or distance of subsequent migration. The data from recaptured individuals showed that the maximum duration of infection was on average 8.3 days (s.e. 0.5), with a mean minimum duration of virus shedding of only 3.1 days (s.e. 0.1). Shedding time decreased during the season, suggesting that mallards acquire transient immunity for LPAI infection. In conclusion, deteriorated body mass following infection was detected, but it remains to be seen whether this has more long-term fitness effects. The short virus shedding time suggests that individual mallards are less likely to spread the virus at continental or intercontinental scales.

Influenza virus in a natural host, the mallard: experimental infection data.

Wild waterfowl, particularly dabbling ducks such as mallards (Anas platyrhynchos), are considered the main reservoir of low-pathogenic avian influenza viruses (LPAIVs). They carry viruses that may evolve and become highly pathogenic for poultry or zoonotic. Understanding the ecology of LPAIVs in these natural hosts is therefore essential. We assessed the clinical response, viral shedding and antibody production of juvenile mallards after intra-esophageal inoculation of two LPAIV subtypes previously isolated from wild congeners. Six ducks, equipped with data loggers that continually monitored body temperature, heart rate and activity, were successively inoculated with an H7N7 LPAI isolate (day 0), the same H7N7 isolate again (day 21) and an H5N2 LPAI isolate (day 35). After the first H7N7 inoculation, the ducks remained alert with no modification of heart rate or activity. However, body temperature transiently increased in four individuals, suggesting that LPAIV strains may have minor clinical effects on their natural hosts. The excretion patterns observed after both re-inoculations differed strongly from those observed after the primary H7N7 inoculation, suggesting that not only homosubtypic but also heterosubtypic immunity exist. Our study suggests that LPAI infection has minor clinically measurable effects on mallards and that mallard ducks are able to mount immunological responses protective against heterologous infections. Because the transmission dynamics of LPAIVs in wild populations is greatly influenced by individual susceptibility and herd immunity, these findings are of high importance. Our study also shows the relevance of using telemetry to monitor disease in animals.

Sampling for low-pathogenic avian influenza A virus in wild Mallard ducks: oropharyngeal versus cloacal swabbing.

Sampling for low-pathogenic avian influenza A virus in wild Mallard ducks : oropharyngeal versus cloacal swabbing

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.

Heterosubtypic Immunity to Influenza A Virus Infections in Mallards May Explain Existence of Multiple Virus Subtypes

Wild birds, particularly duck species, are the main reservoir of influenza A virus (IAV) in nature. However, knowledge of IAV infection dynamics in the wild bird reservoir, and the development of immune responses, are essentially absent. Importantly, a detailed understanding of how subtype diversity is generated and maintained is lacking. To address this, 18,679 samples from 7728 Mallard ducks captured between 2002 and 2009 at a single stopover site in Sweden were screened for IAV infections, and the resulting 1081 virus isolates were analyzed for patterns of immunity. We found support for development of homosubtypic hemagglutinin (HA) immunity during the peak of IAV infections in the fall. Moreover, re-infections with the same HA subtype and related prevalent HA subtypes were uncommon, suggesting the development of natural homosubtypic and heterosubtypic immunity (p-value = 0.02). Heterosubtypic immunity followed phylogenetic relatedness of HA subtypes, both at the level of HA clades (p-value = 0.04) and the level of HA groups (p-value = 0.05). In contrast, infection patterns did not support specific immunity for neuraminidase (NA) subtypes. For the H1 and H3 Clades, heterosubtypic immunity showed a clear temporal pattern and we estimated within-clade immunity to last at least 30 days. The strength and duration of heterosubtypic immunity has important implications for transmission dynamics of IAV in the natural reservoir, where immune escape and disruptive selection may increase HA antigenic variation and explain IAV subtype diversity.

Environmental levels of the antiviral oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards.

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC50 for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.

Disease Dynamics and Bird Migration—Linking Mallards Anas platyrhynchos and Subtype Diversity of the Influenza A Virus in Time and Space

The mallard Anas platyrhynchos is a reservoir species for influenza A virus in the northern hemisphere, with particularly high prevalence rates prior to as well as during its prolonged autumn migration. It has been proposed that the virus is brought from the breeding grounds and transmitted to conspecifics during subsequent staging during migration, and so a better understanding of the natal origin of staging ducks is vital to deciphering the dynamics of viral movement pathways. Ottenby is an important stopover site in southeast Sweden almost halfway downstream in the major Northwest European flyway, and is used by millions of waterfowl each year. Here, mallards were captured and sampled for influenza A virus infection, and positive samples were subtyped in order to study possible links to the natal area, which were determined by a novel approach combining banding recovery data and isotopic measurements (δ2H) of feathers grown on breeding grounds. Geographic assignments showed that the core natal areas of studied mallards were in Estonia, southern and central Finland, and northwestern Russia. This study demonstrates a clear temporal succession of latitudes of natal origin during the course of autumn migration. We also demonstrate a corresponding and concomitant shift in virus subtypes. Acknowledging that these two different patterns were based in part upon different data, a likely interpretation worth further testing is that the early arriving birds with more proximate origins have different influenza A subtypes than the more distantly originating late autumn birds. If true, this knowledge would allow novel insight into the origins and transmission of the influenza A virus among migratory hosts previously unavailable through conventional approaches.

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.

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.