Transmission of avian influenza A viruses among species in an artificial barnyard

Waterfowl and shorebirds harbor and shed all hemagglutinin and neuraminidase subtypes of influenza A viruses and interact in nature with a broad range of other avian and mammalian species to which they might transmit such viruses. Estimating the efficiency and importance of such cross-species transmission using epidemiological approaches is difficult. We therefore addressed this question by studying transmission of low pathogenic H5 and H7 viruses from infected ducks to other common animals in a quasi-natural laboratory environment designed to mimic a common barnyard. Mallards (Anas platyrhynchos) recently infected with H5N2 or H7N3 viruses were introduced into a room housing other mallards plus chickens, blackbirds, rats and pigeons, and transmission was assessed by monitoring virus shedding (ducks) or seroconversion (other species) over the following 4 weeks. Additional animals of each species were directly inoculated with virus to characterize the effect of a known exposure. In both barnyard experiments, virus accumulated to high titers in the shared water pool. The H5N2 virus was transmitted from infected ducks to other ducks and chickens in the room either directly or through environmental contamination, but not to rats or blackbirds. Ducks infected with the H7N2 virus transmitted directly or indirectly to all other species present. Chickens and blackbirds directly inoculated with these viruses shed significant amounts of virus and seroconverted; rats and pigeons developed antiviral antibodies, but, except for one pigeon, failed to shed virus.     

Risks of Avian Influenza Transmission in Areas of Intensive Free-Ranging Duck Production with Wild Waterfowl

For decades, southern China has been considered to be an important source for emerging influenza viruses since key hosts live together in high densities in areas with intensive agriculture. However, the underlying conditions of emergence and spread of avian influenza viruses (AIV) have not been studied in detail, particularly the complex spatiotemporal interplay of viral transmission between wild and domestic ducks, two major actors of AIV epidemiology. In this synthesis, we examine the risks of avian influenza spread in Poyang Lake, an area of intensive free-ranging duck production and large numbers of wild waterfowl. Our synthesis shows that farming of free-grazing domestic ducks is intensive in this area and synchronized with wild duck migration. The presence of juvenile domestic ducks in harvested paddy fields prior to the arrival and departure of migrant ducks in the same fields may amplify the risk of AIV circulation and facilitate the transmission between wild and domestic populations. We provide evidence associating wild ducks migration with the spread of H5N1 in the spring of 2008 from southern China to South Korea, Russia, and Japan, supported by documented wild duck movements and phylogenetic analyses of highly pathogenic avian influenza H5N1 sequences. We suggest that prevention measures based on a modification of agricultural practices may be implemented in these areas to reduce the intensity of AIV transmission between wild and domestic ducks. This would require involving all local stakeholders to discuss feasible and acceptable solutions.     

Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.     

Assessing changes in genomic divergence following a century of human‐mediated secondary contact among wild and captive‐bred ducks

Along with manipulating habitat, the direct release of domesticated individuals into the wild is a practice used worldwide to augment wildlife populations. We test between possible outcomes of human‐mediated secondary contact using genomic techniques at both historical and contemporary timescales for two iconic duck species. First, we sequence several thousand ddRAD‐seq loci for contemporary mallards (Anas platyrhynchos) throughout North America and two domestic mallard types (i.e., known game‐farm mallards and feral Khaki Campbell's). We show that North American mallards may well be becoming a hybrid swarm due to interbreeding with domesticated game‐farm mallards released for hunting. Next, to attain a historical perspective, we applied a bait‐capture array targeting thousands of loci in century‐old (1842–1915) and contemporary (2009–2010) mallard and American black duck (Anas rubripes) specimens. We conclude that American black ducks and mallards have always been closely related, with a divergence time of ~600,000 years before present, and likely evolved through prolonged isolation followed by limited bouts of gene flow (i.e., secondary contact). They continue to maintain genetic separation, a finding that overturns decades of prior research and speculation suggesting the genetic extinction of the American black duck due to contemporary interbreeding with mallards. Thus, despite having high rates of hybridization, actual gene flow is limited between mallards and American black ducks. Conversely, our historical and contemporary data confirm that the intensive stocking of game‐farm mallards during the last ~100 years has fundamentally changed the genetic integrity of North America's wild mallard population, especially in the east. It thus becomes of great interest to ask whether the iconic North American mallard is declining in the wild due to introgression of maladaptive traits from domesticated forms. Moreover, we hypothesize that differential gene flow from domestic game‐farm mallards into the wild mallard population may explain the overall temporal increase in differentiation between wild black ducks and mallards, as well as the uncoupling of genetic diversity and effective population size estimates across time in our results. Finally, our findings highlight how genomic methods can recover complex population histories by capturing DNA preserved in traditional museum specimens.     

Possible waterborne transmission and maintenance of influenza viruses in domestic ducks.

Two duck farms in Hong Kong were examined monthly for 1 year for the occurrence and persistence of influenza viruses within the duck communities. The predominant virus in one community was H3N2, a virus antigenically related to the pandemic Hong Kong strain. This virus was isolated monthly throughout the year from feces or pond water or both, indicating a cycle of waterborne transmission. Viruses of the same antigenic combination were isolated 1 and 2 years after the last sampling occasion, implying persistence in the community. Infection was asymptomatic. Maintenance of virus appeared to be dependent upon the continual introduction of ducklings susceptible to infection onto virus-contaminated water; the feces of ducks 70 to 80 days old were generally free of detectable virus despite the exposure of the ducks to virus in pond water. In the second community, in which ducklings were not introduced after the initial sampling, the prevailing viruses, H7N1 and H7N2, also present asymptomatically, ceased to be detected once the ducks were 70 to 80 days old. The normal practice of raising ducks of different ages on the same farm, wherein the water supplies are shared, as typified by the first community, appears to be instrumental in maintaining a large reservoir of influenza viruses in the duck population of southern China.     

Birds and Viruses at a Crossroad - Surveillance of Influenza A Virus in Portuguese Waterfowl

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.     

Influenza-A viruses in ducks in northwestern Minnesota: fine scale spatial and temporal variation in prevalence and subtype diversity

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

Flying Over an Infected Landscape: Distribution of Highly Pathogenic Avian Influenza H5N1 Risk in South Asia and Satellite Tracking of Wild Waterfowl

Highly pathogenic avian influenza (HPAI) H5N1 virus persists in Asia, posing a threat to poultry, wild birds, and humans. Previous work in Southeast Asia demonstrated that HPAI H5N1 risk is related to domestic ducks and people. Other studies discussed the role of migratory birds in the long distance spread of HPAI H5N1. However, the interplay between local persistence and long-distance dispersal has never been studied. We expand previous geospatial risk analysis to include South and Southeast Asia, and integrate the analysis with migration data of satellite-tracked wild waterfowl along the Central Asia flyway. We find that the population of domestic duck is the main factor delineating areas at risk of HPAI H5N1 spread in domestic poultry in South Asia, and that other risk factors, such as human population and chicken density, are associated with HPAI H5N1 risk within those areas. We also find that satellite tracked birds (Ruddy Shelduck and two Bar-headed Geese) reveal a direct spatio-temporal link between the HPAI H5N1 hot-spots identified in India and Bangladesh through our risk model, and the wild bird outbreaks in May-June-July 2009 in China (Qinghai Lake), Mongolia, and Russia. This suggests that the continental-scale dynamics of HPAI H5N1 are structured as a number of persistence areas delineated by domestic ducks, connected by rare transmission through migratory waterfowl.     

Viral replication, persistence in water and genetic characterization of two influenza A viruses isolated from surface lake water

Water-borne transmission has been suggested as an important transmission mechanism for Influenza A (IA) viruses in wild duck populations; however, relatively few studies have attempted to detect IA viruses from aquatic habitats. Water-isolated viruses have rarely been genetically characterized and evaluation for persistence in water and infectivity in natural hosts has never been documented. In this study, we focused on two IA viruses (H3N8 and H4N6 subtypes) isolated from surface lake water in Minnesota, USA. We investigated the relative prevalence of the two virus subtypes in wild duck populations at the sampling site and their genetic relatedness to IA viruses isolated in wild waterbirds in North America. Viral persistence under different laboratory conditions (temperature and pH) and replication in experimentally infected Mallards (Anas platyrhynchos) were also characterized. Both viruses were the most prevalent subtype one year following their isolation in lake water. The viruses persisted in water for an extended time period at constant temperature (several weeks) but infectivity rapidly reduced under multiple freeze-thaw cycles. Furthermore, the two isolates efficiently replicated in Mallards. The complete genome characterization supported that these isolates originated from genetic reassortments with other IA viruses circulating in wild duck populations during the year of sampling. Based on phylogenetic analyses, we couldn't identify genetically similar viruses in duck populations in the years following their isolation from lake water. Our study supports the role for water-borne transmission for IA viruses but also highlights that additional field and experimental studies are required to support inter-annual persistence in aquatic habitats.     

Susceptibility of wood ducks to H5N1 highly pathogenic avian influenza virus

Since 2002, H5N1 highly pathogenic avian influenza (HPAI) viruses have caused mortality in numerous species of wild birds; this is atypical for avian influenza virus (AIV) infections in these avian species, especially for species within the order Anseriformes. Although these infections document the susceptibility of wild birds to H5N1 HPAI viruses and the spillover of these viruses from infected domestic birds to wild birds, it is unknown whether H5N1 HPAI viruses can persist in free-living avian populations. In a previous study, we established that wood ducks (Aix sponsa) are highly susceptible to infection with H5N1 HPAI viruses. To quantify this susceptibility and further evaluate the likelihood of H5N1 HPAI viral maintenance in a wild bird population, we determined the concentration of virus required to produce infection in wood ducks. To accomplish this, 25 wood ducks were inoculated intranasally at 12-16 wk of age with decreasing concentrations of a H5N1 HPAI virus (A/Whooper Swan/Mongolia/244/05 [H5N1]). The median infectious dose and the lethal dose of H5N1 HPAI virus in wood ducks were very low (10(0.95) and 10(1.71) median embryo infectious dose [EID(50)]/ml, respectively) and less than that of chickens (10(2.80) and 10(2.80) EID(50)/ml). These results confirm that wood ducks are highly susceptible to infection with H5N1 HPAI virus. The data from this study, combined with what is known experimentally about H5N1 HPAI virus infection in wood ducks and viral persistence in aquatic environments, suggest that the wood duck would represent a sensitive indicator species for H5N1 HPAI. Results also suggest that the potential for decreased transmission efficiency associated with reduced viral shedding (especially from the cloaca) and a loss of environmental fitness (in water), may be offset by the ability of this virus to be transmitted through a very low infectious dose.     

Genetic structure of Eurasian and North American mallard ducks based on mtDNA data

To elucidate the origin and genetic structure of the domesticated duck in Eurasia and North America, we sequenced 114 duck D-loop sequences and retrieved 489 D-loop sequences from GenBank. In total, 603 ducks including 50 duck breeds/populations from eight countries (China, France, Russia, India, Kazakhstan, Mongolia, Thailand and USA) were used in this study. One hundred and thirty-four haplotypes and 81 variable sites were detected. H49 was the predominant haplotype, which was considered to be the same dominant haplotype found in the previous studies, and was found in 309 birds. The smallest values for both genetic differentiation index (F(ST), 0.04156) and the number of the net nucleotide substitutions between two populations (D(A), 0.00018) were observed between Eurasian domestic ducks and Eurasian mallards. No geography, breed or population clusters were observed in the Eurasian domestic ducks and mallards. Five haplotypes were shared by USA mallards and Eurasian domestic duck/Eurasian mallards. Only one haplotype (H49) was shared by Eurasian domestic ducks and China spot-billed ducks. By combining phylogenetic analyses, haplotype network profile, genetic distances and shared haplotypes, we can draw two major conclusions: (i) Eurasian and North American mallards show a clear geographic distribution pattern; (ii) Eurasian domestic ducks are derived from the Eurasian mallards, not from the spot-billed ducks.     

Origin and domestication history of Peking ducks deltermined through microsatellite and mitochondrial marker analysis

In order to elucidate the domestication history of Peking ducks, 190 blood samples from six Chinese indigenous duck breeds were collected with186 individualsgenotyped by 15 microsatellite markers. Both the F_ST and Nei’s standard genetic distances (D_s) from the microsatellite data indicated high genetic differentiation between Peking duck and other Chinese indigenous breeds. The haplotype network with mtDNA data showed that most of the Peking duck haplotypes were distinctly different from those of other domestic breeds. Although the H01 haplotype was shared by all domesticated duck breeds, Peking ducks displayed 12 specific domestic duck haplotypes, including four similar haplotypes H02, H04, H08 and H22, that formed a single haplogroup (A). Both H02 and H22 haplotypes were also shared by mallard and Peking ducks, indicating that Peking ducks originated from wild mallard ducks.     

A duck enteritis virus-vectored bivalent live vaccine provides fast and complete protection against H5N1 avian influenza virus infection in ducks

Ducks play an important role in the maintenance of highly pathogenic H5N1 avian influenza viruses (AIVs) in nature, and the successful control of AIVs in ducks has important implications for the eradication of the disease in poultry and its prevention in humans. The inactivated influenza vaccine is expensive, labor-intensive, and usually needs 2 to 3 weeks to induce protective immunity in ducks. Live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine is used routinely to control lethal DEV infections in many duck-producing areas. Here, we first established a system to generate the DEV vaccine strain by using the transfection of overlapping fosmid DNAs. Using this system, we constructed two recombinant viruses, rDEV-ul41HA and rDEV-us78HA, in which the hemagglutinin (HA) gene of the H5N1 virus A/duck/Anhui/1/06 was inserted and stably maintained within the ul41 gene or between the us7 and us8 genes of the DEV genome. Duck studies indicated that rDEV-us78HA had protective efficacy similar to that of the live DEV vaccine against lethal DEV challenge; importantly, a single dose of 10(6) PFU of rDEV-us78HA induced complete protection against a lethal H5N1 virus challenge in as little as 3 days postvaccination. The protective efficacy against both lethal DEV and H5N1 challenge provided by rDEV-ul41HA inoculation in ducks was slightly weaker than that provided by rDEV-us78HA. These results demonstrate, for the first time, that recombinant DEV is suitable for use as a bivalent live attenuated vaccine, providing rapid protection against both DEV and H5N1 virus infection in ducks.     

Establishment and lineage replacement of H6 influenza viruses in domestic ducks in southern China

Domestic ducks in southern China act as an important reservoir for influenza viruses and have also facilitated the establishment of multiple H6 influenza virus lineages. To understand the continuing evolution of these established lineages, 297 H6 viruses isolated from domestic ducks during 2006 and 2007 were genetically and antigenically analyzed. Phylogenetic analyses showed that group II duck H6 viruses had replaced the previously predominant group I lineage and extended their geographic distribution from coastal to inland regions. Group II H6 virus showed that the genesis and development of multiple types of deletions in the neuraminidase (NA) stalk region could occur in the influenza viruses from domestic ducks. A gradual replacement of the N2 NA subtype with N6 was observed. Significant antigenic changes occurred within group II H6 viruses so that they became antigenically distinguishable from group I and gene pool viruses. Gene exchange between group II H6 viruses and the established H5N1, H9N2, or H6N1 virus lineages in poultry in the region was very limited. These findings suggest that domestic ducks can facilitate significant genetic and antigenic changes in viruses established in this host and highlight gaps in our knowledge of influenza virus ecology and even the evolutionary behavior of this virus family in its aquatic avian reservoirs.     

Range-wide genetic population structure of common pochard (Aythya ferina): a potentially important vector of highly pathogenic avian influenza viruses

An understanding of the distribution and spatial structure of the natural vectors of zoonothic pathogens is of interest for effective disease control and prevention. Here, we investigate the range-wide population genetic structure of common pochard (Aythya ferina), a long-distance migratory duck and potential vector of highly pathogenic avian influenza. We collected several hundred samples from breeding and wintering grounds across Eurasia including some H5N1-positive individuals and generated partial sequences of the mitochondrial control region and multilocus microsatellite genotypes. Genetic differentiation among breeding populations was significant for both marker types but higher for maternally inherited mtDNA than for biparentally inherited nuclear markers. There was only weak genetic divergence between ducks sampled in Europe and East Asia, and genetic differentiation between populations was not generally associated with geographical distance. No evidence of genetic substructure was detected for ducks sampled on the European wintering grounds. Our results suggest limited breeding-site fidelity, especially in females, but extensive population admixture on the wintering grounds. The specific role of pochards as natural vectors of zoonotic pathogens and in particular H5N1 remains to be clarified but our results point to wintering grounds as potential hotspots for disease transmission.     

Genetics,Receptor Binding,and Virulence in Mice of H10N8 Influenza Viruses Isolated from Ducks and Chickens in Live Poultry Markets in China

We analyzed eight H10N8 viruses isolated from ducks and chickens in live poultry markets from 2009 to 2013 in China. These viruses showed distinct genetic diversity and formed five genotypes: the four duck isolates formed four different genotypes, whereas the four chicken viruses belong to a single genotype. The viruses bound to both human- and avian-type receptors, and four of the viruses caused 12.7% to 22.5% body weight loss in mice.     

Role of quail in the interspecies transmission of H9 influenza A viruses: molecular changes on HA that correspond to adaptation from ducks to chickens

H9 influenza viruses have become endemic in land-based domestic poultry in Asia and have sporadically crossed to pigs and humans. To understand the molecular determinants of their adaptation to land-based birds, we tested the replication and transmission of several 1970s duck H9 viruses in chickens and quail. Quail were more susceptible than chickens to these viruses, and generation of recombinant H9 viruses by reverse genetics showed that changes in the HA gene are sufficient to initiate efficient replication and transmission in quail. Seven amino acid positions on the HA molecule corresponded to adaptation to land-based birds. In quail H9 viruses, the pattern of amino acids at these seven positions is intermediate between those of duck and chicken viruses; this fact may explain the susceptibility of quail to duck H9 viruses. Our findings suggest that quail provide an environment in which the adaptation of influenza viruses from ducks generates novel variants that can cross the species barrier.     

Mallard–Black Duck Hybridization and Population Genetic Structure in North Carolina

North Carolina, USA, represents the southern extent of the American black duck's (Anas rubripes) breeding range. Mallards (A. platyrhynchos) are present on the breeding grounds of the American black duck and hybridization is observed between these species; therefore, we assessed the genetic integrity, hybridization rates, and population structure of this local breeding population. We extracted genomic and mitochondrial DNA from chorioallantoic membranes and contour feathers from monitored black duck nests. We then prepared the extracted DNA for analysis using high-throughput DNA sequencing methods (ddRAD-seq). First, we assessed nuclear and mitochondrial population structure, genetic diversity, and differentiation across samples from North Carolina, and compared them against 199 genetically vetted mallards, black ducks, and mallard × black duck hybrids that served as genetic references. Next, we tested for parentage and sibling relationship and overall relatedness of black ducks in North Carolina. We recovered strong population structure and high co-ancestry across genetic markers due to interrelatedness among sampled nests in North Carolina and concluded that black ducks have been locally breeding in this area for a prolonged period of time. Despite a high level of interrelatedness among our samples, nucleotide diversity was similar to the reference continental black duck population, suggesting little effect of genetic drift, including inbreeding. Additionally, we conclude that molecular diversity of black ducks in North Carolina is maintained at reference population levels through the influx of genetic material from unrelated, migrating male black ducks. Finally, we report a hybridization level of 47.5%, covering 3 filial generations. Of identified hybrids, 54.7% and 53% were the direct result of interbreeding between black ducks and captive-reared or wild mallards, respectively. We conclude that because of high rates of interspecific hybridization and successive backcrossing events, introgression from wild and feral mallards is occurring into this population of breeding black ducks and requires careful consideration in future management efforts. © 2021 The Wildlife Society.