Characterization of low-pathogenic H5 subtype influenza viruses from Eurasia: implications for the origin of highly pathogenic H5N1 viruses

Highly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries, resulting in repeated outbreaks in poultry and increased cases of human infection. The immediate precursor of these HPAI viruses is believed to be A/goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses first detected in Guangdong, China, in 1996. From 2000 onwards, many novel reassortant H5N1 influenza viruses or genotypes have emerged in southern China. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low-pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also show reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza virus gene pool among poultry in Eurasia.     

Genetic evidence of intercontinental movement of avian influenza in a migratory bird: the northern pintail (Anas acuta)

The role of migratory birds in the movement of the highly pathogenic (HP) avian influenza H5N1 remains a subject of debate. Testing hypotheses regarding intercontinental movement of low pathogenic avian influenza (LPAI) viruses will help evaluate the potential that wild birds could carry Asian-origin strains of HP avian influenza to North America during migration. Previous North American assessments of LPAI genetic variation have found few Asian reassortment events. Here, we present results from whole-genome analyses of LPAI isolates collected in Alaska from the northern pintail (Anas acuta), a species that migrates between North America and Asia. Phylogenetic analyses confirmed the genetic divergence between Asian and North American strains of LPAI, but also suggested inter-continental virus exchange and at a higher frequency than previously documented. In 38 isolates from Alaska, nearly half (44.7%) had at least one gene segment more closely related to Asian than to North American strains of LPAI. Additionally, sequences of several Asian LPAI isolates from GenBank clustered more closely with North American northern pintail isolates than with other Asian origin viruses. Our data support the role of wild birds in the intercontinental transfer of influenza viruses, and reveal a higher degree of transfer in Alaska than elsewhere in North America.     

Homo- and heterosubtypic low pathogenic avian influenza exposure on H5N1 highly pathogenic avian influenza virus infection in wood ducks (Aix sponsa)

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin) and heterosubtypic (heterologous hemagglutinin) low pathogenic avian influenza (LPAI) viruses on the outcome of a H5N1 HPAI virus infection in wood ducks (Aix sponsa) was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations.     

Interspecific exchange of avian influenza virus genes in Alaska: the influence of trans-hemispheric migratory tendency and breeding ground sympatry

The movement and transmission of avian influenza viral strains via wild migratory birds may vary by host species as a result of migratory tendency and sympatry with other infected individuals. To examine the roles of host migratory tendency and species sympatry on the movement of Eurasian low-pathogenic avian influenza (LPAI) genes into North America, we characterized migratory patterns and LPAI viral genomic variation in mallards (Anas platyrhynchos) of Alaska in comparison with LPAI diversity of northern pintails (Anas acuta). A 50-year band-recovery data set suggests that unlike northern pintails, mallards rarely make trans-hemispheric migrations between Alaska and Eurasia. Concordantly, fewer (14.5%) of 62 LPAI isolates from mallards contained Eurasian gene segments compared to those from 97 northern pintails (35%), a species with greater inter-continental migratory tendency. Aerial survey and banding data suggest that mallards and northern pintails are largely sympatric throughout Alaska during the breeding season, promoting opportunities for interspecific transmission. Comparisons of full-genome isolates confirmed near-complete genetic homology (>99.5%) of seven viruses between mallards and northern pintails. This study found viral segments of Eurasian lineage at a higher frequency in mallards than previous studies, suggesting transmission from other avian species migrating inter-hemispherically or the common occurrence of endemic Alaskan viruses containing segments of Eurasian origin. We conclude that mallards are unlikely to transfer Asian-origin viruses directly to North America via Alaska but that they are likely infected with Asian-origin viruses via interspecific transfer from species with regular migrations to the Eastern Hemisphere.     

Novel H7N7 avian influenza viruses detected in migratory wild birds in eastern China between 2018 and 2020

Wild birds are the natural reservoirs of avian influenza viruses, and surveillance and assessment of these viruses in wild birds provide valuable information for early warning and control of animal diseases. In this study, we isolated 19 H7N7 avian influenza viruses from wild bird between 2018 and 2020. Full genomic analysis revealed that these viruses bear a single basic amino acid in the cleavage site of their hemagglutinin gene, and formed four different genotypes by actively reassorting other avian influenza viruses circulating in wild birds and ducks. The H7N7 viruses bound to both avian-type and human-type receptors, although their affinity for human-type receptors was markedly lower than that for avian-type receptors. Moreover, we found that the H7N7 viruses could replicate efficiently in the upper respiratory tract and caecum of domestic ducks, and that the H5/H7 inactivated vaccine used in poultry in China provided complete protection against H7N7 wild bird virus challenge in ducks. Our findings demonstrate that wild bird H7N7 viruses pose a substantial threat to the poultry industry across the East Asian-Australian migratory flyway, emphasize the importance of influenza virus surveillance in both wild and domestic birds, and support the development of active control strategies against H7N7 virus.     

Highly pathogenic avian influenza outbreak mitigated by seasonal low pathogenic strains: Insights from dynamic modeling

The spread of highly pathogenic avian influenza (HPAI) H5N1 remains a threat for both wild and domestic bird populations, while low pathogenic avian influenza (LPAI) strains have been reported to induce partial immunity to HPAI in poultry and some wild birds inoculated with both HPAI and LPAI strains. Here, based on the reported data and experiments, we develop a two-strain avian influenza model to examine the extent to which this partial immunity observed at the individual level can affect the outcome of the outbreaks among migratory birds in the wild at the population level during different seasons. We find a distinct mitigating effect of LPAI on the death toll induced by HPAI strain, and this effect is particularly important for populations previously exposed to and recovered from LPAI. We further investigate the effect of the dominant mode of transmission of an HPAI strain on the outcome of the epidemic. Four combinations of contact based direct transmission and indirect fecal-to-oral (or environmental) routes are examined. For a given infection peak of HPAI, indirect fecal-to-oral transmission of HPAI can lead to a higher death toll than that associated with direct transmission. The mitigating effect of LPAI can, in turn, be dependent on the route of infection of HPAI.     

Highly (H5N1) and low (H7N2) pathogenic avian influenza virus infection in falcons via nasochoanal route and ingestion of experimentally infected prey

An experimental infection with highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) viruses was carried out on falcons in order to examine the effects of these viruses in terms of pathogenesis, viral distribution in tissues and viral shedding. The distribution pattern of influenza virus receptors was also assessed. Captive-reared gyr-saker (Falco rusticolus x Falco cherrug) hybrid falcons were challenged with a HPAI H5N1 virus (A/Great crested grebe/Basque Country/06.03249/2006) or a LPAI H7N2 virus (A/Anas plathyrhynchos/Spain/1877/2009), both via the nasochoanal route and by ingestion of previously infected specific pathogen free chicks. Infected falcons exhibited similar infection dynamics despite the different routes of exposure, demonstrating the effectiveness of in vivo feeding route. H5N1 infected falcons died, or were euthanized, between 5-7 days post-infection (dpi) after showing acute severe neurological signs. Presence of viral antigen in several tissues was confirmed by immunohistochemistry and real time RT-PCR (RRT-PCR), which were generally associated with significant microscopical lesions, mostly in the brain. Neither clinical signs, nor histopathological findings were observed in any of the H7N2 LPAI infected falcons, although all of them had seroconverted by 11 dpi. Avian receptors were strongly present in the upper respiratory tract of the falcons, in accordance with the consistent oral viral shedding detected by RRT-PCR in both H5N1 HPAI and H7N2 LPAI infected falcons. The present study demonstrates that gyr-saker hybrid falcons are highly susceptible to H5N1 HPAI virus infection, as previously observed, and that they may play a major role in the spreading of both HPAI and LPAI viruses. For the first time in raptors, natural infection by feeding on infected prey was successfully reproduced. The use of avian prey species in falconry husbandry and wildlife rehabilitation facilities could put valuable birds of prey and humans at risk and, therefore, this practice should be closely monitored.     

Migration of Whooper Swans and Outbreaks of Highly Pathogenic Avian Influenza H5N1 Virus in Eastern Asia

Evaluating the potential involvement of wild avifauna in the emergence of highly pathogenic avian influenza H5N1 (hereafter H5N1) requires detailed analyses of temporal and spatial relationships between wild bird movements and disease emergence. The death of wild swans (Cygnus spp.) has been the first indicator of the presence of H5N1 in various Asian and European countries; however their role in the geographic spread of the disease remains poorly understood. We marked 10 whooper swans (Cygnus cygnus) with GPS transmitters in northeastern Mongolia during autumn 2006 and tracked their migratory movements in relation to H5N1 outbreaks. The prevalence of H5N1 outbreaks among poultry in eastern Asia during 2003–2007 peaked during winter, concurrent with whooper swan movements into regions of high poultry density. However outbreaks involving poultry were detected year round, indicating disease perpetuation independent of migratory waterbird presence. In contrast, H5N1 outbreaks involving whooper swans, as well as other migratory waterbirds that succumbed to the disease in eastern Asia, tended to occur during seasons (late spring and summer) and in habitats (areas of natural vegetation) where their potential for contact with poultry is very low to nonexistent. Given what is known about the susceptibility of swans to H5N1, and on the basis of the chronology and rates of whooper swan migration movements, we conclude that although there is broad spatial overlap between whooper swan distributions and H5N1 outbreak locations in eastern Asia, the likelihood of direct transmission between these groups is extremely low. Thus, our data support the hypothesis that swans are best viewed as sentinel species, and moreover, that in eastern Asia, it is most likely that their infections occurred through contact with asymptomatic migratory hosts (e.g., wild ducks) at or near their breeding grounds.     

Evidence of infection with H4 and H11 avian influenza viruses among Lebanese chicken growers

Human infections with H5, H7, and H9 avian influenza viruses are well documented. Exposure to poultry is the most important risk factor for humans becoming infected with these viruses. Data on human infection with other low pathogenicity avian influenza viruses is sparse but suggests that such infections may occur. Lebanon is a Mediterranean country lying under two major migratory birds flyways and is home to many wild and domestic bird species. Previous reports from this country demonstrated that low pathogenicity avian influenza viruses are in circulation but highly pathogenic H5N1 viruses were not reported. In order to study the extent of human infection with avian influenza viruses in Lebanon, we carried out a seroprevalence cross-sectional study into which 200 poultry-exposed individuals and 50 non-exposed controls were enrolled. We obtained their sera and tested it for the presence of antibodies against avian influenza viruses types H4 through H16 and used a questionnaire to collect exposure data. Our microneutralization assay results suggested that backyard poultry growers may have been previously infected with H4 and H11 avian influenza viruses. We confirmed these results by using a horse red blood cells hemagglutination inhibition assay. Our data also showed that farmers with antibodies against each virus type clustered in a small geographic area suggesting that unrecognized outbreaks among birds may have led to these human infections. In conclusion, this study suggests that occupational exposure to chicken is a risk factor for infection with avian influenza especially among backyard growers and that H4 and H11 influenza viruses may possess the ability to cross the species barrier to infect humans.     

Evidence of infection by H5N2 highly pathogenic avian influenza viruses in healthy wild waterfowl

The potential existence of a wild bird reservoir for highly pathogenic avian influenza (HPAI) has been recently questioned by the spread and the persisting circulation of H5N1 HPAI viruses, responsible for concurrent outbreaks in migratory and domestic birds over Asia, Europe, and Africa. During a large-scale surveillance programme over Eastern Europe, the Middle East, and Africa, we detected avian influenza viruses of H5N2 subtype with a highly pathogenic (HP) viral genotype in healthy birds of two wild waterfowl species sampled in Nigeria. We monitored the survival and regional movements of one of the infected birds through satellite telemetry, providing a rare evidence of a non-lethal natural infection by an HP viral genotype in wild birds. Phylogenetic analysis of the H5N2 viruses revealed close genetic relationships with H5 viruses of low pathogenicity circulating in Eurasian wild and domestic ducks. In addition, genetic analysis did not reveal known gallinaceous poultry adaptive mutations, suggesting that the emergence of HP strains could have taken place in either wild or domestic ducks or in non-gallinaceous species. The presence of coexisting but genetically distinguishable avian influenza viruses with an HP viral genotype in two cohabiting species of wild waterfowl, with evidence of non-lethal infection at least in one species and without evidence of prior extensive circulation of the virus in domestic poultry, suggest that some strains with a potential high pathogenicity for poultry could be maintained in a community of wild waterfowl.     

Genetically Diverse Low Pathogenicity Avian Influenza A Virus Subtypes Co-Circulate among Poultry in Bangladesh

Influenza virus surveillance, poultry outbreak investigations and genomic sequencing were assessed to understand the ecology and evolution of low pathogenicity avian influenza (LPAI) A viruses in Bangladesh from 2007 to 2013. We analyzed 506 avian specimens collected from poultry in live bird markets and backyard flocks to identify influenza A viruses. Virus isolation-positive specimens (n = 50) were subtyped and their coding-complete genomes were sequenced. The most frequently identified subtypes among LPAI isolates were H9N2, H11N3, H4N6, and H1N1. Less frequently detected subtypes included H1N3, H2N4, H3N2, H3N6, H3N8, H4N2, H5N2, H6N1, H6N7, and H7N9. Gene sequences were compared to publicly available sequences using phylogenetic inference approaches. Among the 14 subtypes identified, the majority of viral gene segments were most closely related to poultry or wild bird viruses commonly found in Southeast Asia, Europe, and/or northern Africa. LPAI subtypes were distributed over several geographic locations in Bangladesh, and surface and internal protein gene segments clustered phylogenetically with a diverse number of viral subtypes suggesting extensive reassortment among these LPAI viruses. H9N2 subtype viruses differed from other LPAI subtypes because genes from these viruses consistently clustered together, indicating this subtype is enzootic in Bangladesh. The H9N2 strains identified in Bangladesh were phylogenetically and antigenically related to previous human-derived H9N2 viruses detected in Bangladesh representing a potential source for human infection. In contrast, the circulating LPAI H5N2 and H7N9 viruses were both phylogenetically and antigenically unrelated to H5 viruses identified previously in humans in Bangladesh and H7N9 strains isolated from humans in China. In Bangladesh, domestic poultry sold in live bird markets carried a wide range of LPAI virus subtypes and a high diversity of genotypes. These findings, combined with the seven year timeframe of sampling, indicate a continuous circulation of these viruses in the country.     

Influenza in migratory birds and evidence of limited intercontinental virus exchange

Migratory waterfowl of the world are the natural reservoirs of influenza viruses of all known subtypes. However, it is unknown whether these waterfowl perpetuate highly pathogenic (HP) H5 and H7 avian influenza viruses. Here we report influenza virus surveillance from 2001 to 2006 in wild ducks in Alberta, Canada, and in shorebirds and gulls at Delaware Bay (New Jersey), United States, and examine the frequency of exchange of influenza viruses between the Eurasian and American virus clades, or superfamilies. Influenza viruses belonging to each of the subtypes H1 through H13 and N1 through N9 were detected in these waterfowl, but H14 and H15 were not found. Viruses of the HP Asian H5N1 subtypes were not detected, and serologic studies in adult mallard ducks provided no evidence of their circulation. The recently described H16 subtype of influenza viruses was detected in American shorebirds and gulls but not in ducks. We also found an unusual cluster of H7N3 influenza viruses in shorebirds and gulls that was able to replicate well in chickens and kill chicken embryos. Genetic analysis of 6,767 avian influenza gene segments and 248 complete avian influenza viruses supported the notion that the exchange of entire influenza viruses between the Eurasian and American clades does not occur frequently. Overall, the available evidence does not support the perpetuation of HP H5N1 influenza in migratory birds and suggests that the introduction of HP Asian H5N1 to the Americas by migratory birds is likely to be a rare event.     

Highly pathogenic avian influenza is an emerging disease threat to wild birds in North America

Prior to the emergence of the A/goose/Guangdong/1/1996 (Gs/GD) H5N1 influenza A virus, the long-held and well-supported paradigm was that highly pathogenic avian influenza (HPAI) outbreaks were restricted to poultry, the result of cross-species transmission of precursor viruses from wild aquatic birds that subsequently gained pathogenicity in domestic birds. Therefore, management agencies typically adopted a prevention, control, and eradication strategy that included strict biosecurity for domestic bird production, isolation of infected and exposed flocks, and prompt depopulation. In most cases, this strategy has proved sufficient for eradicating HPAI. Since 2002, this paradigm has been challenged with many detections of viral descendants of the Gs/GD lineage among wild birds, most of which have been associated with sporadic mortality events. Since the emergence and evolution of the genetically distinct clade 2.3.4.4 Gs/GD lineage HPAI viruses in approximately 2010, there have been further increases in the occurrence of HPAI in wild birds and geographic spread through migratory bird movement. A prominent example is the introduction of clade 2.3.4.4 Gs/GD HPAI viruses from East Asia to North America via migratory birds in autumn 2014 that ultimately led to the largest outbreak of HPAI in the history of the United States. Given the apparent maintenance of Gs/GD lineage HPAI viruses in a global avian reservoir; bidirectional virus exchange between wild and domestic birds facilitating the continued adaptation of Gs/GD HPAI viruses in wild bird hosts; the current frequency of HPAI outbreaks in wild birds globally, and particularly in Eurasia where Gs/GD HPAI viruses may now be enzootic; and ongoing dispersal of AI viruses from East Asia to North America via migratory birds, HPAI now represents an emerging disease threat to North American wildlife. This recent paradigm shift implies that management of HPAI in domestic birds alone may no longer be sufficient to eradicate HPAI viruses from a given country or region. Rather, agencies managing wild birds and their habitats may consider the development or adoption of mitigation strategies to minimize introductions to poultry, to reduce negative impacts on wild bird populations, and to diminish adverse effects to stakeholders using wildlife resources. The main objective of this review is, therefore, to provide information that will assist wildlife managers in developing mitigation strategies or approaches for dealing with outbreaks of Gs/GD HPAI in wild birds in the form of preparedness, surveillance, research, communications, and targeted management actions. Resultant outbreak response plans and actions may represent meaningful steps of wildlife managers toward the use of collaborative and multi-jurisdictional One Health approaches when it comes to the detection, investigation, and mitigation of emerging viruses at the human-domestic animal-wildlife interface.     

Complete Genome Sequence of a Novel H4N1 Influenza Virus Isolated from a Pig in Central China

Pigs are proposed to be “mixing vessel” hosts that can produce genetically novel reassortant viruses with pandemic potential. The appearance of any novel influenza viruses among pigs should pose concerns for human health. Here, we report the complete genome sequence of a novel H4N1 influenza virus [A/Swine/HuBei/06/2009(H4N1)] isolated from a pig in Central China in 2009. The genomic sequence analysis indicates that this virus is a wholly avian-original influenza virus. Each gene may come from different avian influenza viruses outside mainland China, suggesting the role of migratory birds in the dispersal of influenza virus.     

Characterization of the 2012 Highly Pathogenic Avian Influenza H7N3 Virus Isolated from Poultry in an Outbreak in Mexico: Pathobiology and Vaccine Protection

In June of 2012, an H7N3 highly pathogenic avian influenza (HPAI) virus was identified as the cause of a severe disease outbreak in commercial laying chicken farms in Mexico. The purpose of this study was to characterize the Mexican 2012 H7N3 HPAI virus (A/chicken/Jalisco/CPA1/2012) and determine the protection against the virus conferred by different H7 inactivated vaccines in chickens. Both adult and young chickens intranasally inoculated with the virus became infected and died at between 2 and 4 days postinoculation (p.i.). High virus titers and viral replication in many tissues were demonstrated at 2 days p.i. in infected birds. The virus from Jalisco, Mexico, had high sequence similarity of greater than 97% to the sequences of wild bird viruses from North America in all eight gene segments. The hemagglutinin gene of the virus contained a 24-nucleotide insert at the hemagglutinin cleavage site which had 100% sequence identity to chicken 28S rRNA, suggesting that the insert was the result of nonhomologous recombination with the host genome. For vaccine protection studies, both U.S. H7 low-pathogenic avian influenza (LPAI) viruses and a 2006 Mexican H7 LPAI virus were tested as antigens in experimental oil emulsion vaccines and injected into chickens 3 weeks prior to challenge. All H7 vaccines tested provided ≥90% protection against clinical disease after challenge and decreased the number of birds shedding virus and the titers of virus shed. This study demonstrates the pathological consequences of the infection of chickens with the 2012 Mexican lineage H7N3 HPAI virus and provides support for effective programs of vaccination against this virus in poultry.     

Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses

The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture.     

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.