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Wild birds are considered to be the natural reservoir hosts of avian influenza viruses (AIVs). Wild bird-origin AIVs may spill over into new hosts and overcome species barriers after evolutionary adaptation. H13N8 AIVs used to be considered primarily circulated in multispecies gulls but have recently been shown to possess cross-species infectivity. In this study, we analyzed the genetic changes that occurred in the process of the evolution of H13 AIVs. Phylogenetic analysis revealed that H13 AIVs underwent complex reassortment events. Based on the full genomic diversity, we divided H13 AIVs into 81 genotypes. Reassortment experiments indicated that basic polymerase 2 (PB2) and nucleoprotein (NP) genes of the H9N2 AIV significantly enhanced the polymerase activity of the H13N8 AIV. Using the replication-incompetent virus screening system, we identified two mutations, PB2-I76T and PB2-I559T, which could enhance the polymerase activity of the H13N8 AIV in mammalian cells. Notably, these mutations had been acquired by circulating H13N8 AIVs in 2015. These findings suggest that H13N8 AIVs are about to cross the host barrier. Occasional genetic reassortments with other AIVs and natural mutation events could promote this process. It is imperative to intensify monitoring efforts for H13N8 AIVs.

The use of wild animals in research is complicated due to the capture and housing conditions, as well as to legal aspects, making it difficult to develop in vivo and in vitro models for the study of pathologies that affect these species. Here we validate an in vitro model of tendon-derived mesenchymal cells (TDSC) from Eurasian blackbird (Turdus merula) cadaveric samples. Through the expression of surface markers and the ability to differentiate into multiple lineages, the nature of the cells was confirmed. We then evaluated Mesenchymal Stem Cells (MSCs) as an infection model for the Usutu Flavivirus. To this aim, blackbird TDSCs were compared to Vero E6 cells, commonly used in Flavivirus studies. Both cells showed permissiveness to USUV infection as confirmed by immunocytochemistry. Moreover, TDSCs exhibited replication kinetics similar to, although slightly lower than, Vero E6, confirming these cells as a pertinent study model for the study of the pathogenesis of USUV. In this work, we isolated and characterized tendon-derived mesenchymal stem cells, which represent an interesting and convenient in vitro model for the study of wildlife species in laboratories.

In October 2021, a wild bird-origin H3N8 influenza virus-A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8)-was isolated from Chinese pond heron in China. Phylogenetic and molecular analyses were performed to characterize the genetic origin of the H3N8 strain. Phylogenetic analysis revealed that eight gene segments of this avian influenza virus H3N8 belong to Eurasian lineages. HA gene clustered with avian influenza viruses is circulating in poultry in southern China. The NA gene possibly originated from wild ducks in South Korea and has the highest homology (99.3%) with A/Wild duck/South Korea/KNU2020-104/2020 (H3N8), while other internal genes have a complex and wide range of origins. The HA cleavage site is PEKQTR↓GLF with one basic amino acid, Q226 and T228 at HA preferentially bind to the alpha-2,3-linked sialic acid receptor, non-deletion of the stalk region in the NA gene and no mutations at E627K and D701N of the PB2 protein, indicating that isolate A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) was a typical avian influenza with low pathogenicity. However, there are some mutations that may increase pathogenicity and transmission in mammals, such as N30D, T215A of M1 protein, and P42S of NS1 protein. In animal studies, A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) replicates inefficiently in the mouse lung and does not adapt well to the mammalian host. Overall, A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) is a novel wild bird-origin H3N8 influenza virus reassortant from influenza viruses of poultry and wild birds. This wild bird-origin avian influenza virus is associated with wild birds along the East Asian-Australasian flyway. Therefore, surveillance of avian influenza viruses in wild birds should be strengthened to assess their mutation and pandemic risk in advance.

The seasonal migration of wild aquatic birds plays a critical role in the maintenance, transmission, and incursion of the avian influenza virus (AIV). AIV surveillance was performed during 2020–2021 in two national nature reserves with abundant wild bird resources in Yunnan, China. Four H5N8 AIVs isolates from the common crane were identified by next-generation sequencing. Phylogenetic analysis demonstrated that all eight gene segments of these H5N8 AIVs belonged to clade 2.3.4.4b high-pathogenic AIV (HPAIV) and shared high nucleotide sequence similarity with the strains isolated in Hubei, China, and Siberia, Russia, in 2020–2021. The H5N8 HPAIVs from common cranes were characterized by both human and avian dual-receptor specificity in the hemagglutinin (HA) protein. Moreover, possessing the substitutions contributes to overcoming transmission barriers of mammalian hosts in polymerase basic 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acid (PA), and exhibiting the long stalk in the neck region of the neuraminidase (NA) protein contributes to adaptation in wild birds. Monitoring AIVs in migratory birds, at stopover sites and in their primary habitats, i.e., breeding or wintering grounds, could provide insight into potential zoonosis caused by AIVs.

Arctic regions are ecologically significant for the environmental persistence and geographic dissemination of influenza A viruses (IAVs) by avian hosts and other wildlife species. Data describing the epidemiology and ecology of IAVs among wildlife in the arctic are less frequently published compared to southern temperate regions, where prevalence and subtype diversity are more routinely documented. Following PRISMA guidelines, this systematic review addresses this gap by describing the prevalence, spatiotemporal distribution, and ecological characteristics of IAVs detected among wildlife and the environment in this understudied region of the globe. The literature search was performed in PubMed and Google Scholar using a set of pre-defined search terms to identify publications reporting on IAVs in Arctic regions between 1978 and February 2022. A total of 2125 articles were initially screened, 267 were assessed for eligibility, and 71 articles met inclusion criteria. IAVs have been detected in multiple wildlife species in all Arctic regions, including seabirds, shorebirds, waterfowl, seals, sea lions, whales, and terrestrial mammals, and in the environment. Isolates from wild birds comprise the majority of documented viruses derived from wildlife; however, among all animals and environmental matrices, 26 unique low and highly pathogenic subtypes have been characterized in the scientific literature from Arctic regions. Pooled prevalence across studies indicates 4.23% for wild birds, 3.42% among tested environmental matrices, and seroprevalences of 9.29% and 1.69% among marine and terrestrial mammals, respectively. Surveillance data are geographically biased, with most data from the Alaskan Arctic and many fewer reports from the Russian, Canadian, North Atlantic, and Western European Arctic. We highlight multiple important aspects of wildlife host, pathogen, and environmental ecology of IAVs in Arctic regions, including the role of avian migration and breeding cycles for the global spread of IAVs, evidence of inter-species and inter-continental reassortment at high latitudes, and how climate change-driven ecosystem shifts, including changes in the seasonal availability and distribution of dietary resources, have the potential to alter host–pathogen–environment dynamics in Arctic regions. We conclude by identifying gaps in knowledge and propose priorities for future research.

Wild birds are considered to be the natural reservoir hosts of avian influenza viruses (AIVs). Wild bird-origin AIVs may spill over into new hosts and overcome species barriers after evolutionary adaptation. H13N8 AIVs used to be considered primarily circulated in multispecies gulls but have recently been shown to possess cross-species infectivity. In this study, we analyzed the genetic changes that occurred in the process of the evolution of H13 AIVs. Phylogenetic analysis revealed that H13 AIVs underwent complex reassortment events. Based on the full genomic diversity, we divided H13 AIVs into 81 genotypes. Reassortment experiments indicated that basic polymerase 2 (PB2) and nucleoprotein (NP) genes of the H9N2 AIV significantly enhanced the polymerase activity of the H13N8 AIV. Using the replication-incompetent virus screening system, we identified two mutations, PB2-I76T and PB2-I559T, which could enhance the polymerase activity of the H13N8 AIV in mammalian cells. Notably, these mutations had been acquired by circulating H13N8 AIVs in 2015. These findings suggest that H13N8 AIVs are about to cross the host barrier. Occasional genetic reassortments with other AIVs and natural mutation events could promote this process. It is imperative to intensify monitoring efforts for H13N8 AIVs.

Zoonotic infection with avian influenza viruses (AIVs) of subtype H7, such as H7N9 and H7N4, has raised concerns worldwide. During the winter of 2020–2021, five novel H7 low pathogenic AIVs (LPAIVs) containing different neuraminidase (NA) subtypes, including two H7N3, an H7N8, and two H7N9, were detected in wild bird feces in South Korea. Complete genome sequencing and phylogenetic analysis showed that the novel H7Nx AIVs were reassortants containing two gene segments (hemagglutinin (HA) and matrix) that were related to the zoonotic Jiangsu–Cambodian H7 viruses causing zoonotic infection and six gene segments originating from LPAIVs circulating in migratory birds in Eurasia. A genomic constellation analysis demonstrated that all H7 isolates contained a mix of gene segments from different viruses, indicating that multiple reassortment occurred. The well-known mammalian adaptive substitution (E627K and D701N) in PB2 was not detected in any of these isolates. The detection of multiple reassortant H7Nx AIVs in wild birds highlights the need for intensive surveillance in both wild birds and poultry in Eurasia.

H7 low pathogenic avian influenza viruses (LPAIVs) can mutate into highly pathogenic avian influenza viruses (HPAIVs). In addition to avian species, H7 avian influenza viruses (AIVs) also infect humans. In this study, two AIVs, H7N9 (20X-20) and H7N7 (34X-2), isolated from the feces of wild birds in South Korea in 2021, were genetically analyzed. The HA cleavage site of the two H7 Korean viruses was confirmed to be ELPKGR/GLF, indicating they are LPAIVs. There were no amino acid substitutions at the receptor-binding site of the HA gene of two H7 Korean viruses compared to that of A/Anhui/1/2013 (H7N9), which prefer human receptors. In the phylogenetic tree analysis, the HA gene of the two H7 Korean viruses shared the highest nucleotide similarity with the Korean H7 subtype AIVs. In addition, the HA gene of the two H7 Korean viruses showed high nucleotide similarity to that of the A/Jiangsu/1/2018(H7N4) virus, which is a human influenza virus originating from avian influenza virus. Most internal genes (PB2, PB1, PA, NP, NA, M, and NS) of the two H7 Korean viruses belonged to the Eurasian lineage, except for the M gene of 34X-2. This result suggests that active reassortment occurred among AIVs. In pathogenicity studies of mice, the two H7 Korean viruses replicated in the lungs of mice. In addition, the body weight of mice infected with 34X-2 decreased 7 days post-infection (dpi) and inflammation was observed in the peribronchiolar and perivascular regions of the lungs of mice. These results suggest that mammals can be infected with the two H7 Korean AIVs. Our data showed that even low pathogenic H7 AIVs may infect mammals, including humans, as confirmed by the A/Jiangsu/1/2018(H7N4) virus. Therefore, continuous monitoring and pathogenicity assessment of AIVs, even of LPAIVs, are required.

Since 2014, H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIV) have caused outbreaks in wild birds and poultry in multiple continents, including Asia, Europe, Africa, and North America. Wild birds were suspected to be the sources of the local and global spreads of HPAIV. This study evaluated the infectivity, pathogenicity, and transmissibility of clade 2.3.4.4 H5N6 HPAIV in mandarin ducks (Aixgalericulata) and domestic pigeons (Columbia livia domestica). None of the birds used in this study, 20 mandarin ducks or 8 pigeons, showed clinical signs or mortality due to H5N6 HPAI infection. Two genotypes of H5N6 HPAIV showed replication and transmission by direct and indirect contact between mandarin ducks. H5N6 HPAIV replicated and transmitted by direct contact between pigeons, although the viral shedding titer and duration were relatively lower and shorter than those in mandarin ducks. Influenza virus antigen was detected in various internal organs of infected mandarin ducks and pigeons, indicating systemic infection. Therefore, our results indicate mandarin ducks and pigeons can be subclinically infected with clade 2.3.4.4 H5N6 HPAIV and transfer the virus to adjacent birds. The role of mandarin ducks and pigeons in the spread and prevalence of clade 2.3.4.4 H5N6 viruses should be carefully monitored.

During October 2020–January 2021, we isolated a total of 67 highly pathogenic avian influenza (HPAI) H5N8 viruses from wild birds and outbreaks in poultry in South Korea. We sequenced the isolates and performed phylogenetic analysis of complete genome sequences to determine the origin, evolution, and spread patterns of these viruses. Phylogenetic analysis of the hemagglutinin (HA) gene showed that all the isolates belong to H5 clade 2.3.4.4 subgroup B (2.3.4.4b) and form two distinct genetic clusters, G1 and G2. The cluster G1 was closely related to the 2.3.4.4b H5N8 HPAI viruses detected in Europe in early 2020, while the cluster G2 had a close genetic relationship with the 2.3.4.4b H5N8 viruses that circulated in Europe in late 2020. A total of seven distinct genotypes were identified, including five novel reassortants carrying internal genes of low pathogenic avian influenza viruses. Our Bayesian discrete trait phylodynamic analysis between host types suggests that the viruses initially disseminated from migratory waterfowl to domestic duck farms in South Korea. Subsequently, domestic duck farms most likely contributed to the transmission of HPAI viruses to chicken and minor poultry farms, highlighting the need for enhanced, high levels of biosecurity measures at domestic duck farms to effectively prevent the introduction and spread of HPAI.

Wild aquatic birds, a natural reservoir of avian influenza viruses (AIVs), transmit AIVs to poultry farms, causing huge economic losses. Therefore, the prevalence and genetic characteristics of AIVs isolated from wild birds in South Korea from October 2019 to March 2020 were investigated and analyzed. Fresh avian fecal samples (3256) were collected by active monitoring of 11 wild bird habitats. Twenty-eight AIVs were isolated. Seven HA and eight NA subtypes were identified. All AIV hosts were Anseriformes species. The HA cleavage site of 20 representative AIVs was encoded by non-multi-basic amino acid sequences. Phylogenetic analysis of the eight segment genes of the AIVs showed that most genes clustered within the Eurasian lineage. However, the HA gene of H10 viruses and NS gene of four viruses clustered within the American lineage, indicating intercontinental reassortment of AIVs. Representative viruses likely to infect mammals were selected and evaluated for pathogenicity in mice. JB21-58 (H5N3), JB42-93 (H9N2), and JB32-81 (H11N2) were isolated from the lungs, but JB31-69 (H11N9) was not isolated from the lungs until the end of the experiment at 14 dpi. None of infected mice showed clinical sign and histopathological change in the lung. In addition, viral antigens were not detected in lungs of all mice at 14 dpi. These data suggest that LPAIVs derived from wild birds are unlikely to be transmitted to mammals. However, because LPAIVs can reportedly infect mammals, including humans, continuous surveillance and monitoring of AIVs are necessary, despite their low pathogenicity.

In October 2020, a highly pathogenic avian influenza (HPAI) subtype H5N8 virus was identified from a fecal sample of a wild mandarin duck (Aix galericulata) in South Korea. We sequenced all eight genome segments of the virus, designated as A/Mandarin duck/Korea/K20-551-4/2020(H5N8), and conducted genetic characterization and comparative phylogenetic analysis to track its origin. Genome sequencing and phylogenetic analysis show that the hemagglutinin gene belongs to H5 clade 2.3.4.4 subgroup B. All genes share high levels of nucleotide identity with H5N8 HPAI viruses identified from Europe during early 2020. Enhanced active surveillance in wild and domestic birds is needed to monitor the introduction and spread of HPAI via wild birds and to inform the design of improved prevention and control strategies.

West Nile (WNV) and Usutu (USUV) viruses are two mosquito-borne viruses belonging to the family Flaviviridae and genus Flavivirus. The natural transmission cycle of WNV and USUV involves mosquitoes and birds, while mammals are thought to be accidental hosts. The goal of this study was to report-in the context of "off-season monitoring" and passive surveillance-the detection of WNV and USUV RNA in wild birds. To this end, we analyzed biological samples of wild birds in Northern Italy, from October to May, hence outside of the regional monitoring period (June-September). The virological investigations for the detection of USUV and WNV RNA were performed using real-time PCR on frozen samples of the brain, myocardium, kidney, and spleen. In a total sample of 164 wild birds belonging to 27 different species, sequences of both viruses were detected: four birds (2.44%) were positive for WNV and five (3.05%) for USUV. Off-season infections of WNV and especially USUV are still widely discussed and only a few studies have been published to date. To the best of our knowledge, this study is the first report on the detection of USUV RNA until December 22nd. Although further studies are required, our results confirm the viral circulation out-of-season of Flavivirus in wild birds, suggesting reconsidering the epidemiological monitoring period based on each individual climate zone and taking into consideration global warming which will play an important role in the epidemiology of vector-borne diseases.

Wild birds play an important role as reservoir hosts and vectors for zoonotic arboviruses and foster their spread. Usutu virus (USUV) has been circulating endemically in Germany since 2011, while West Nile virus (WNV) was first diagnosed in several bird species and horses in 2018. In 2017 and 2018, we screened 1709 live wild and zoo birds with real-time polymerase chain reaction and serological assays. Moreover, organ samples from bird carcasses submitted in 2017 were investigated. Overall, 57 blood samples of the live birds (2017 and 2018), and 100 organ samples of dead birds (2017) were positive for USUV-RNA, while no WNV-RNA-positive sample was found. Phylogenetic analysis revealed the first detection of USUV lineage Europe 2 in Germany and the spread of USUV lineages Europe 3 and Africa 3 towards Northern Germany. USUV antibody prevalence rates were high in Eastern Germany in both years. On the contrary, in Northern Germany, high seroprevalence rates were first detected in 2018, with the first emergence of USUV in this region. Interestingly, high WNV-specific neutralizing antibody titers were observed in resident and short-distance migratory birds in Eastern Germany in 2018, indicating the first signs of a local WNV circulation.

Avian coronaviruses (ACoVs) are continuously evolving and causing serious economic consequences in the poultry industry and around the globe. Owing to their extensive genetic diversity and high mutation rates, controlling ACoVs has become a challenge. In this context, the potential contribution of wild birds in the disease dynamics, especially in domesticated birds, remains largely unknown. In the present study, five hundred fifty-seven (n = 557) cloacal/fecal swabs were collected from four different wild bird species from eight Egyptian governorates during 2016 and a total of fourteen positive isolates were used for phylodynamics and evolutionary analysis. Genetic relatedness based on spike (S1) gene demonstrated the clustering of majority of these isolates where nine isolates grouped within Egy/variant 2 (IS/885 genotype) and five isolates clustered within Egy/variant 1 (IS/1494/06 genotype). Interestingly, these isolates showed noticeable genetic diversity and were clustered distal to the previously characterized Egy/variant 1 and Egy/variant 2 in Egyptian commercial poultry. The S1 gene based comparison of nucleotide identity percentages revealed that all fourteen isolates reported in this study were genetically related to the variant GI-23 lineage with 92–100% identity. Taken together, our results demonstrate that ACoVs are circulating in Egyptian wild birds and highlight their possible contributions in the disease dynamics. The study also proposes that regular monitoring of the ACoVs in wild birds is required to effectively assess the role of wild birds in disease spread, and the emergence of ACoVs strains in the country.

The use of wild animals in research is complicated due to the capture and housing conditions, as well as to legal aspects, making it difficult to develop in vivo and in vitro models for the study of pathologies that affect these species. Here we validate an in vitro model of tendon-derived mesenchymal cells (TDSC) from Eurasian blackbird ( Turdus merula ) cadaveric samples. Through the expression of surface markers and the ability to differentiate into multiple lineages, the nature of the cells was confirmed. We then evaluated Mesenchymal Stem Cells (MSCs) as an infection model for the Usutu Flavivirus. To this aim, blackbird TDSCs were compared to Vero E6 cells, commonly used in Flavivirus studies. Both cells showed permissiveness to USUV infection as confirmed by immunocytochemistry. Moreover, TDSCs exhibited replication kinetics similar to, although slightly lower than, Vero E6, confirming these cells as a pertinent study model for the study of the pathogenesis of USUV. In this work, we isolated and characterized tendon-derived mesenchymal stem cells, which represent an interesting and convenient in vitro model for the study of wildlife species in laboratories., Competing Interests: The authors declare no conflict of interest.

In October 2021, a wild bird-origin H3N8 influenza virus-A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8)-was isolated from Chinese pond heron in China. Phylogenetic and molecular analyses were performed to characterize the genetic origin of the H3N8 strain. Phylogenetic analysis revealed that eight gene segments of this avian influenza virus H3N8 belong to Eurasian lineages. HA gene clustered with avian influenza viruses is circulating in poultry in southern China. The NA gene possibly originated from wild ducks in South Korea and has the highest homology (99.3%) with A/Wild duck/South Korea/KNU2020-104/2020 (H3N8), while other internal genes have a complex and wide range of origins. The HA cleavage site is PEKQTR↓GLF with one basic amino acid, Q226 and T228 at HA preferentially bind to the alpha-2,3-linked sialic acid receptor, non-deletion of the stalk region in the NA gene and no mutations at E627K and D701N of the PB2 protein, indicating that isolate A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) was a typical avian influenza with low pathogenicity. However, there are some mutations that may increase pathogenicity and transmission in mammals, such as N30D, T215A of M1 protein, and P42S of NS1 protein. In animal studies, A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) replicates inefficiently in the mouse lung and does not adapt well to the mammalian host. Overall, A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) is a novel wild bird-origin H3N8 influenza virus reassortant from influenza viruses of poultry and wild birds. This wild bird-origin avian influenza virus is associated with wild birds along the East Asian-Australasian flyway. Therefore, surveillance of avian influenza viruses in wild birds should be strengthened to assess their mutation and pandemic risk in advance.

The seasonal migration of wild aquatic birds plays a critical role in the maintenance, transmission, and incursion of the avian influenza virus (AIV). AIV surveillance was performed during 2020-2021 in two national nature reserves with abundant wild bird resources in Yunnan, China. Four H5N8 AIVs isolates from the common crane were identified by next-generation sequencing. Phylogenetic analysis demonstrated that all eight gene segments of these H5N8 AIVs belonged to clade 2.3.4.4b high-pathogenic AIV (HPAIV) and shared high nucleotide sequence similarity with the strains isolated in Hubei, China, and Siberia, Russia, in 2020-2021. The H5N8 HPAIVs from common cranes were characterized by both human and avian dual-receptor specificity in the hemagglutinin (HA) protein. Moreover, possessing the substitutions contributes to overcoming transmission barriers of mammalian hosts in polymerase basic 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acid (PA), and exhibiting the long stalk in the neck region of the neuraminidase (NA) protein contributes to adaptation in wild birds. Monitoring AIVs in migratory birds, at stopover sites and in their primary habitats, i.e., breeding or wintering grounds, could provide insight into potential zoonosis caused by AIVs.

Arctic regions are ecologically significant for the environmental persistence and geographic dissemination of influenza A viruses (IAVs) by avian hosts and other wildlife species. Data describing the epidemiology and ecology of IAVs among wildlife in the arctic are less frequently published compared to southern temperate regions, where prevalence and subtype diversity are more routinely documented. Following PRISMA guidelines, this systematic review addresses this gap by describing the prevalence, spatiotemporal distribution, and ecological characteristics of IAVs detected among wildlife and the environment in this understudied region of the globe. The literature search was performed in PubMed and Google Scholar using a set of pre-defined search terms to identify publications reporting on IAVs in Arctic regions between 1978 and February 2022. A total of 2125 articles were initially screened, 267 were assessed for eligibility, and 71 articles met inclusion criteria. IAVs have been detected in multiple wildlife species in all Arctic regions, including seabirds, shorebirds, waterfowl, seals, sea lions, whales, and terrestrial mammals, and in the environment. Isolates from wild birds comprise the majority of documented viruses derived from wildlife; however, among all animals and environmental matrices, 26 unique low and highly pathogenic subtypes have been characterized in the scientific literature from Arctic regions. Pooled prevalence across studies indicates 4.23% for wild birds, 3.42% among tested environmental matrices, and seroprevalences of 9.29% and 1.69% among marine and terrestrial mammals, respectively. Surveillance data are geographically biased, with most data from the Alaskan Arctic and many fewer reports from the Russian, Canadian, North Atlantic, and Western European Arctic. We highlight multiple important aspects of wildlife host, pathogen, and environmental ecology of IAVs in Arctic regions, including the role of avian migration and breeding cycles for the global spread of IAVs, evidence of inter-species and inter-continental reassortment at high latitudes, and how climate change-driven ecosystem shifts, including changes in the seasonal availability and distribution of dietary resources, have the potential to alter host-pathogen-environment dynamics in Arctic regions. We conclude by identifying gaps in knowledge and propose priorities for future research.

Zoonotic infection with avian influenza viruses (AIVs) of subtype H7, such as H7N9 and H7N4, has raised concerns worldwide. During the winter of 2020-2021, five novel H7 low pathogenic AIVs (LPAIVs) containing different neuraminidase (NA) subtypes, including two H7N3, an H7N8, and two H7N9, were detected in wild bird feces in South Korea. Complete genome sequencing and phylogenetic analysis showed that the novel H7Nx AIVs were reassortants containing two gene segments (hemagglutinin (HA) and matrix) that were related to the zoonotic Jiangsu-Cambodian H7 viruses causing zoonotic infection and six gene segments originating from LPAIVs circulating in migratory birds in Eurasia. A genomic constellation analysis demonstrated that all H7 isolates contained a mix of gene segments from different viruses, indicating that multiple reassortment occurred. The well-known mammalian adaptive substitution (E627K and D701N) in PB2 was not detected in any of these isolates. The detection of multiple reassortant H7Nx AIVs in wild birds highlights the need for intensive surveillance in both wild birds and poultry in Eurasia.

H7 low pathogenic avian influenza viruses (LPAIVs) can mutate into highly pathogenic avian influenza viruses (HPAIVs). In addition to avian species, H7 avian influenza viruses (AIVs) also infect humans. In this study, two AIVs, H7N9 (20X-20) and H7N7 (34X-2), isolated from the feces of wild birds in South Korea in 2021, were genetically analyzed. The HA cleavage site of the two H7 Korean viruses was confirmed to be ELPKGR/GLF, indicating they are LPAIVs. There were no amino acid substitutions at the receptor-binding site of the HA gene of two H7 Korean viruses compared to that of A/Anhui/1/2013 (H7N9), which prefer human receptors. In the phylogenetic tree analysis, the HA gene of the two H7 Korean viruses shared the highest nucleotide similarity with the Korean H7 subtype AIVs. In addition, the HA gene of the two H7 Korean viruses showed high nucleotide similarity to that of the A/Jiangsu/1/2018(H7N4) virus, which is a human influenza virus originating from avian influenza virus. Most internal genes (PB2, PB1, PA, NP, NA, M, and NS) of the two H7 Korean viruses belonged to the Eurasian lineage, except for the M gene of 34X-2. This result suggests that active reassortment occurred among AIVs. In pathogenicity studies of mice, the two H7 Korean viruses replicated in the lungs of mice. In addition, the body weight of mice infected with 34X-2 decreased 7 days post-infection (dpi) and inflammation was observed in the peribronchiolar and perivascular regions of the lungs of mice. These results suggest that mammals can be infected with the two H7 Korean AIVs. Our data showed that even low pathogenic H7 AIVs may infect mammals, including humans, as confirmed by the A/Jiangsu/1/2018(H7N4) virus. Therefore, continuous monitoring and pathogenicity assessment of AIVs, even of LPAIVs, are required.

Since 2014, H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIV) have caused outbreaks in wild birds and poultry in multiple continents, including Asia, Europe, Africa, and North America. Wild birds were suspected to be the sources of the local and global spreads of HPAIV. This study evaluated the infectivity, pathogenicity, and transmissibility of clade 2.3.4.4 H5N6 HPAIV in mandarin ducks (Aix galericulata ). None of the birds used in this study, 20 mandarin ducks or 8 pigeons, showed clinical signs or mortality due to H5N6 HPAI infection. Two genotypes of H5N6 HPAIV showed replication and transmission by direct and indirect contact between mandarin ducks. H5N6 HPAIV replicated and transmitted by direct contact between pigeons, although the viral shedding titer and duration were relatively lower and shorter than those in mandarin ducks. Influenza virus antigen was detected in various internal organs of infected mandarin ducks and pigeons, indicating systemic infection. Therefore, our results indicate mandarin ducks and pigeons can be subclinically infected with clade 2.3.4.4 H5N6 HPAIV and transfer the virus to adjacent birds. The role of mandarin ducks and pigeons in the spread and prevalence of clade 2.3.4.4 H5N6 viruses should be carefully monitored.Columbia livia domestica ). None of the birds used in this study, 20 mandarin ducks or 8 pigeons, showed clinical signs or mortality due to H5N6 HPAI infection. Two genotypes of H5N6 HPAIV showed replication and transmission by direct and indirect contact between mandarin ducks. H5N6 HPAIV replicated and transmitted by direct contact between pigeons, although the viral shedding titer and duration were relatively lower and shorter than those in mandarin ducks. Influenza virus antigen was detected in various internal organs of infected mandarin ducks and pigeons, indicating systemic infection. Therefore, our results indicate mandarin ducks and pigeons can be subclinically infected with clade 2.3.4.4 H5N6 HPAIV and transfer the virus to adjacent birds. The role of mandarin ducks and pigeons in the spread and prevalence of clade 2.3.4.4 H5N6 viruses should be carefully monitored.

During October 2020-January 2021, we isolated a total of 67 highly pathogenic avian influenza (HPAI) H5N8 viruses from wild birds and outbreaks in poultry in South Korea. We sequenced the isolates and performed phylogenetic analysis of complete genome sequences to determine the origin, evolution, and spread patterns of these viruses. Phylogenetic analysis of the hemagglutinin (HA) gene showed that all the isolates belong to H5 clade 2.3.4.4 subgroup B (2.3.4.4b) and form two distinct genetic clusters, G1 and G2. The cluster G1 was closely related to the 2.3.4.4b H5N8 HPAI viruses detected in Europe in early 2020, while the cluster G2 had a close genetic relationship with the 2.3.4.4b H5N8 viruses that circulated in Europe in late 2020. A total of seven distinct genotypes were identified, including five novel reassortants carrying internal genes of low pathogenic avian influenza viruses. Our Bayesian discrete trait phylodynamic analysis between host types suggests that the viruses initially disseminated from migratory waterfowl to domestic duck farms in South Korea. Subsequently, domestic duck farms most likely contributed to the transmission of HPAI viruses to chicken and minor poultry farms, highlighting the need for enhanced, high levels of biosecurity measures at domestic duck farms to effectively prevent the introduction and spread of HPAI.

Wild aquatic birds, a natural reservoir of avian influenza viruses (AIVs), transmit AIVs to poultry farms, causing huge economic losses. Therefore, the prevalence and genetic characteristics of AIVs isolated from wild birds in South Korea from October 2019 to March 2020 were investigated and analyzed. Fresh avian fecal samples (3256) were collected by active monitoring of 11 wild bird habitats. Twenty-eight AIVs were isolated. Seven HA and eight NA subtypes were identified. All AIV hosts were Anseriformes species. The HA cleavage site of 20 representative AIVs was encoded by non-multi-basic amino acid sequences. Phylogenetic analysis of the eight segment genes of the AIVs showed that most genes clustered within the Eurasian lineage. However, the HA gene of H10 viruses and NS gene of four viruses clustered within the American lineage, indicating intercontinental reassortment of AIVs. Representative viruses likely to infect mammals were selected and evaluated for pathogenicity in mice. JB21-58 (H5N3), JB42-93 (H9N2), and JB32-81 (H11N2) were isolated from the lungs, but JB31-69 (H11N9) was not isolated from the lungs until the end of the experiment at 14 dpi. None of infected mice showed clinical sign and histopathological change in the lung. In addition, viral antigens were not detected in lungs of all mice at 14 dpi. These data suggest that LPAIVs derived from wild birds are unlikely to be transmitted to mammals. However, because LPAIVs can reportedly infect mammals, including humans, continuous surveillance and monitoring of AIVs are necessary, despite their low pathogenicity.

In October 2020, a highly pathogenic avian influenza (HPAI) subtype H5N8 virus was identified from a fecal sample of a wild mandarin duck ( Aix galericulata ) in South Korea. We sequenced all eight genome segments of the virus, designated as A/Mandarin duck/Korea/K20-551-4/2020(H5N8), and conducted genetic characterization and comparative phylogenetic analysis to track its origin. Genome sequencing and phylogenetic analysis show that the hemagglutinin gene belongs to H5 clade 2.3.4.4 subgroup B. All genes share high levels of nucleotide identity with H5N8 HPAI viruses identified from Europe during early 2020. Enhanced active surveillance in wild and domestic birds is needed to monitor the introduction and spread of HPAI via wild birds and to inform the design of improved prevention and control strategies.

Wild birds play an important role as reservoir hosts and vectors for zoonotic arboviruses and foster their spread. Usutu virus (USUV) has been circulating endemically in Germany since 2011, while West Nile virus (WNV) was first diagnosed in several bird species and horses in 2018. In 2017 and 2018, we screened 1709 live wild and zoo birds with real-time polymerase chain reaction and serological assays. Moreover, organ samples from bird carcasses submitted in 2017 were investigated. Overall, 57 blood samples of the live birds (2017 and 2018), and 100 organ samples of dead birds (2017) were positive for USUV-RNA, while no WNV-RNA-positive sample was found. Phylogenetic analysis revealed the first detection of USUV lineage Europe 2 in Germany and the spread of USUV lineages Europe 3 and Africa 3 towards Northern Germany. USUV antibody prevalence rates were high in Eastern Germany in both years. On the contrary, in Northern Germany, high seroprevalence rates were first detected in 2018, with the first emergence of USUV in this region. Interestingly, high WNV-specific neutralizing antibody titers were observed in resident and short-distance migratory birds in Eastern Germany in 2018, indicating the first signs of a local WNV circulation.

Avian coronaviruses (ACoVs) are continuously evolving and causing serious economic consequences in the poultry industry and around the globe. Owing to their extensive genetic diversity and high mutation rates, controlling ACoVs has become a challenge. In this context, the potential contribution of wild birds in the disease dynamics, especially in domesticated birds, remains largely unknown. In the present study, five hundred fifty-seven ( n = 557) cloacal/fecal swabs were collected from four different wild bird species from eight Egyptian governorates during 2016 and a total of fourteen positive isolates were used for phylodynamics and evolutionary analysis. Genetic relatedness based on spike (S1) gene demonstrated the clustering of majority of these isolates where nine isolates grouped within Egy/variant 2 (IS/885 genotype) and five isolates clustered within Egy/variant 1 (IS/1494/06 genotype). Interestingly, these isolates showed noticeable genetic diversity and were clustered distal to the previously characterized Egy/variant 1 and Egy/variant 2 in Egyptian commercial poultry. The S1 gene based comparison of nucleotide identity percentages revealed that all fourteen isolates reported in this study were genetically related to the variant GI-23 lineage with 92⁻100% identity. Taken together, our results demonstrate that ACoVs are circulating in Egyptian wild birds and highlight their possible contributions in the disease dynamics. The study also proposes that regular monitoring of the ACoVs in wild birds is required to effectively assess the role of wild birds in disease spread, and the emergence of ACoVs strains in the country.