Avian Influenza Virus and Avian Paramyxoviruses in Wild Waterfowl of the Western Coast of the Caspian Sea (2017-2020).

The flyways of many different wild waterfowl pass through the Caspian Sea region. The western coast of the middle Caspian Sea is an area with many wetlands, where wintering grounds with large concentrations of birds are located. It is known that wild waterfowl are a natural reservoir of the influenza A virus. In the mid-2000s, in the north of this region, the mass deaths of swans, gulls, and pelicans from high pathogenicity avian influenza virus (HPAIV) were noted. At present, there is still little known about the presence of avian influenza virus (AIVs) and different avian paramyxoviruses (APMVs) in the region's waterfowl bird populations. Here, we report the results of monitoring these viruses in the wild waterfowl of the western coast of the middle Caspian Sea from 2017 to 2020. Samples from 1438 individuals of 26 bird species of 7 orders were collected, from which 21 strains of AIV were isolated, amounting to a 1.46% isolation rate of the total number of samples analyzed (none of these birds exhibited external signs of disease). The following subtypes were determined and whole-genome nucleotide sequences of the isolated strains were obtained: H1N1 (n = 2), H3N8 (n = 8), H4N6 (n = 2), H7N3 (n = 2), H8N4 (n = 1), H10N5 (n = 1), and H12N5 (n = 1). No high pathogenicity influenza virus H5 subtype was detected. Phylogenetic analysis of AIV genomes did not reveal any specific pattern for viruses in the Caspian Sea region, showing that all segments belong to the Eurasian clades of classic avian-like influenza viruses. We also did not find the amino acid substitutions in the polymerase complex (PA, PB1, and PB2) that are critical for the increase in virulence or adaptation to mammals. In total, 23 hemagglutinating viruses not related to influenza A virus were also isolated, of which 15 belonged to avian paramyxoviruses. We were able to sequence 12 avian paramyxoviruses of three species, as follows: Newcastle disease virus (n = 4); Avian paramyxovirus 4 (n = 5); and Avian paramyxovirus 6 (n = 3). In the Russian Federation, the Newcastle disease virus of the VII.1.1 sub-genotype was first isolated from a wild bird (common pheasant) in the Caspian Sea region. The five avian paramyxovirus 4 isolates obtained belonged to the common clade in Genotype I, whereas phylogenetic analysis of three isolates of Avian paramyxovirus 6 showed that two isolates, isolated in 2017, belonged to Genotype I and that an isolate identified in 2020 belonged to Genotype II. The continued regular monitoring of AIVs and APMVs, the obtaining of data on the biological properties of isolated strains, and the accumulation of information on virus host species will allow for the adequate planning of epidemiological measures, suggest the most likely routes of spread of the virus, and assist in the prediction of the introduction of the viruses in the western coastal region of the middle Caspian Sea.

Genetics of H5N1 and H5N8 High-Pathogenicity Avian Influenza Viruses Isolated in Japan in Winter 2021-2022.

In winter 2021-2022, H5N1 and H5N8 high-pathogenicity avian influenza (HPAI) viruses (HPAIVs) caused serious outbreaks in Japan: 25 outbreaks of HPAI at poultry farms and 107 cases in wild birds or in the environment. Phylogenetic analyses divided H5 HPAIVs isolated in Japan in the winter of 2021-2022 into three groups-G2a, G2b, and G2d-which were disseminated at different locations and times. Full-genome sequencing analyses of these HPAIVs revealed a strong relationship of multiple genes between Japan and Siberia, suggesting that they arose from reassortment events with avian influenza viruses (AIVs) in Siberia. The results emphasize the complex of dissemination and reassortment events with the movement of migratory birds, and the importance of continual monitoring of AIVs in Japan and Siberia for early alerts to the intrusion of HPAIVs.

Experimental Infection of Chickens with H5N8 High Pathogenicity Avian Influenza Viruses Isolated in Japan in the Winter of 2020-2021.

During the winter of 2020-2021, numerous outbreaks of high pathogenicity avian influenza (HPAI) were caused by viruses of the subtype H5N8 in poultry over a wide region in Japan. The virus can be divided into five genotypes-E1, E2, E3, E5, and E7. The major genotype responsible for the outbreaks was E3, followed by E2. To investigate the cause of these outbreaks, we experimentally infected chickens with five representative strains of each genotype. We found that the 50% chicken infectious dose differed by up to 75 times among the five strains, and the titer of the E3 strains (102.75 50% egg infectious dose (EID50)) was the lowest, followed by that of the E2 strains (103.50 EID50). In viral transmission experiments, in addition to the E3 and E2 strains, the E5 strain was transmitted to naïve chickens with high efficiency (>80%), whereas the other strains had low efficiencies (<20%). We observed a clear difference in the virological characteristics among the five strains isolated in the same season. The higher infectivity of the E3 and E2 viruses in chickens may have caused the large number of HPAI outbreaks in Japan during this season.

Detection of H5N1 High Pathogenicity Avian Influenza Viruses in Four Raptors and Two Geese in Japan in the Fall of 2022.

In the fall of 2022, high pathogenicity avian influenza viruses (HPAIVs) were detected from raptors and geese in Japan, a month earlier than in past years, indicating a shift in detection patterns. In this study, we conducted a phylogenetic analysis on H5N1 HPAIVs detected from six wild birds during the 2022/2023 season to determine their genetic origins. Our findings revealed that these HPAIVs belong to the G2 group within clade 2.3.4.4b, with all isolates classified into three subgroups: G2b, G2d, and G2c. The genetic background of the G2b virus (a peregrine falcon-derived strain) and G2d viruses (two raptors and two geese-derived strains) were the same as those detected in Japan in the 2021/2022 season. Since no HPAI cases were reported in Japan during the summer of 2022, it is probable that migratory birds reintroduced the G2b and G2d viruses. Conversely, the G2c virus (a raptor-derived strain) was first recognized in Japan in the fall of 2022. This strain might share a common ancestor with HPAIVs from Asia and West Siberia observed in the 2021/2022 season. The early migration of waterfowl to Japan in the fall of 2022 could have facilitated the early invasion of HPAIVs.

Virological and Genetic Characterization of the Unusual Avian Influenza H14Nx Viruses in the Northern Asia.

Wild aquatic birds are generally identified as a natural reservoir of avian influenza viruses (AIVs), where a high diversity of subtypes has been detected. Some AIV subtypes are considered to have relatively low prevalence in wild bird populations. Six-year AIV surveillance in Siberia revealed sporadic cases of the rarely identified H14-subtype AIV circulation. Complete genome sequencing of three H14 isolates were performed, and the analysis indicated interconnections between low pathogenic avian influenza (LPAI) viruses. We conducted hemagglutination inhibition and virus neutralization assays, estimated the susceptibility of isolates to neuraminidase inhibitors, and characterized receptor specificity. Our study revealed circulation of a new H14N9 subtype described for the first time. However, the low prevalence of the H14-subtype AIV population may be the reason for the underestimation of the diversity of H14-subtype AIVs. According to the available data, a region in which H14-subtype viruses were detected several times in 2007-2022 in the Eastern Hemisphere is Western Siberia, while the virus was also detected once in South Asia (Pakistan). Phylogenetic analysis of HA segment sequences revealed the circulation of two clades of H14-subtype viruses originated from initial 1980s Eurasian clade; the first was detected in Northern America and the second in Eurasia.

Different Infectivity and Transmissibility of H5N8 and H5N1 High Pathogenicity Avian Influenza Viruses Isolated from Chickens in Japan in the 2021/2022 Season.

H5N8 and H5N1 high pathogenicity avian influenza viruses (HPAIVs) caused outbreaks in poultry farms in Japan from November 2021 to May 2022. Hemagglutinin genes of these viruses belong to clade 2.3.4.4B and can be divided phylogenetically into the following groups: 20A, 20E, and 21E. In this study, we compared the infectivity and transmissibility of HPAIVs from three groups of chickens. Representative strains from 20A, 20E, and 21E groups are A/chicken/Akita/7C/2021(H5N8)(Akita7C), A/chicken/Kagoshima/21A6T/2021(H5N1)(Kagoshima6T), and A/chicken/Iwate/21A7T/2022(H5N1)(Iwate7T), respectively. Fifty percent lethal dose of Akita7C in chickens (103.83 fifty percent egg infectious dose (EID50)) was up to seven times lower than those of Kagoshima6T and Iwate7T (104.50 and 104.68 EID50, respectively). Mean death times for Akita7C- and Kagoshima6T-infected chickens (3.45 and 3.30 days, respectively) were at least a day longer than that of Iwate7T (2.20 days). Viral titers of the trachea and cloaca of Iwate7T-infected chicken were the highest detected. The transmission rate of the Akita7C strain (100%) was markedly higher than those of the two strains (<50%). These data suggest that the infectivity and transmissibility of the Akita7C strain (H5N8) in chickens are higher than those of H5N1 viruses, providing fundamental information needed for formulating effective prevention and control strategies for HPAI outbreaks.

Current research and future directions for realizing the ideal One-Health approach: A summary of key-informant interviews in Japan and a literature review.

The COVID-19 pandemic has highlighted the importance of the One Health (OH) approach, which considers the health of humans, animals, and the environment in preventing future pandemics. A wide range of sustainable interdisciplinary collaborations are required to truly fulfill the purpose of the OH approach. It is well-recognized, however, that such collaborations are challenging. In this study, we undertook key-informant interviews with a panel of stakeholders from Japan to identify their perceived needs and challenges related to OH research. This panel included scientists, government officials, journalists, and industry stakeholders. By combining a thematic analysis of these interviews and a literature review, we summarized two key themes pertinent to the effective implementation of OH research: types of required research and systems to support that research. As a technological issue, interviewees suggested the importance of research and development of methodologies that can promote the integration and collaboration of research fields that are currently fragmented. An example of such a methodology would allow researchers to obtain high-resolution metadata (e.g. ecological and wildlife data) with high throughput and then maximize the use of the obtained metadata in research, such as in environmental DNA analysis, database construction, or the use of computational algorithms to find novel viral genomes. In terms of systems surrounding OH research, some interviewees stressed the importance of creating a sustainable research system, such as one that has continuous budget support and allows researchers to pursue their academic careers and interests. These perceptions and challenges held by Japanese stakeholders may be common to others around the world. We hope this review will encourage more researchers and others to work together to create a resilient society against future pandemics.

Genetically Diverse Highly Pathogenic Avian Influenza A(H5N1/H5N8) Viruses among Wild Waterfowl and Domestic Poultry, Japan, 2021.

Genetic analyses of highly pathogenic avian influenza H5 subtype viruses isolated from the Izumi Plain, Japan, revealed cocirculation of 2 genetic groups of clade 2.3.4.4b viruses among migratory waterfowl. Our findings demonstrate that both continuous surveillance and timely information sharing of avian influenza viruses are valuable for rapid risk assessment.

Genetic Diversity of the Hemagglutinin Genes of Influenza a Virus in Asian Swine Populations.

Swine influenza (SI) is a major respiratory disease of swine; SI is due to the influenza A virus of swine (IAV-S), a highly contagious virus with zoonotic potential. The intensity of IAV-S surveillance varies among countries because it is not a reportable disease and causes limited mortality in swine. Although Asia accounts for half of all pig production worldwide, SI is not well managed in those countries. Rigorously managing SI on pig farms could markedly reduce the economic losses, the likelihood of novel reassortants among IAV-S, and the zoonotic IAV-S infections in humans. Vaccination of pigs is a key control measure for SI, but its efficacy relies on the optimal antigenic matching of vaccine strains with the viral strains circulating in the field. Here, we phylogenetically reviewed the genetic diversity of the hemagglutinin gene among IAVs-S that have circulated in Asia during the last decade. This analysis revealed the existence of country-specific clades in both the H1 and H3 subtypes and cross-border transmission of IAVs-S. Our findings underscore the importance of choosing vaccine antigens for each geographic region according to both genetic and antigenic analyses of the circulating IAV-S to effectively manage SI in Asia.

Genetics of Japanese H5N8 high pathogenicity avian influenza viruses isolated in winter 2020-2021 and their genetic relationship with avian influenza viruses in Siberia.

In winter 2020-2021, Japan experienced multiple serious outbreaks of H5N8 high pathogenicity avian influenza (HPAI)-52 outbreaks at poultry farms and 58 cases in wild birds or the environment-that occurred simultaneously with outbreaks in Europe. Here, we examined how the H5N8 HPAI viruses (HPAIVs) emerged and spread through Japan and across the Eurasian continent. Phylogenetic and phylogeographic analyses were performed using full genetic sequences of the viruses that caused 52 outbreaks at poultry farms or were isolated from 11 infected wild birds. Genetically, the viruses showed five genotypes (E1, E2, E3, E5 and E7) that have already been reported in Korea. The viruses showing the E3 genotype were found to have caused most of the HPAI outbreaks at poultry farms and were detected over the longest period of time. The internal genes of the viruses were genetically related to those of AIVs isolated through avian influenza surveillance activities in regions of Siberia including Buryatia, Yakutia and Amur regions, suggesting that the Japanese viruses emerged via reassortment events with AIVs genetically related to Siberian AIVs. In addition, H5N2 and H5N8 HPAIVs were isolated from wild birds during surveillance activities conducted in the Novosibirsk region of Siberia in summer 2020. Phylogenetic analyses revealed that these viruses possessed haemagglutinin genes that were related to those of H5N8 HPAIVs that were circulating in Europe in winter 2020-2021. These results suggest that the viruses in wild birds during summer in Siberia most likely spread in both Asia and Europe the following winter. Together, the present results emphasize the importance of continual monitoring of AIVs in Siberia for forecasting outbreaks not only in Asia but also further away in Europe.

Antiviral Susceptibilities of Avian Influenza A(H5), A(H7), and A(H9) Viruses Isolated in Japan.

The circulation of avian influenza A viruses in poultry is a public health concern due to the potential transmissibility and severity of these viral infections. Monitoring the susceptibility of these viruses to antivirals is important for developing measures to strengthen the level of preparedness against influenza pandemics. However, drug susceptibility information on these viruses is limited. Here, we determined the susceptibilities of avian influenza A(H5N1), A(H5N2), A(H5N8), A(H7N7), A(H7N9), A(H9N1), and A(H9N2) viruses isolated in Japan to the antivirals approved for use there: an M2 inhibitor (amantadine), neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, and laninamivir) and RNA polymerase inhibitors (baloxavir and favipiravir). Genotypic methods that detect amino acid substitutions associated with antiviral resistance and phenotypic methods that assess phenotypic viral susceptibility to drugs have revealed that these avian influenza A viruses are susceptible to neuraminidase and RNA polymerase inhibitors. These results suggest that neuraminidase and RNA polymerase inhibitors currently approved in Japan could be a treatment option against influenza A virus infections in humans.

Highly Pathogenic Avian Influenza A(H5N8) Virus Clade 2.3.4.4b, Western Siberia, Russia, 2020.

Two variants of highly pathogenic avian influenza A(H5N8) virus were detected in dead poultry in Western Siberia, Russia, during August and September 2020. One variant was represented by viruses of clade 2.3.4.4b and the other by a novel reassortant between clade 2.3.4.4b and Eurasian low pathogenicity avian influenza viruses circulating in wild birds.

First Outbreak of an H5N8 Highly Pathogenic Avian Influenza Virus on a Chicken Farm in Japan in 2020.

On 5 November 2020, a confirmed outbreak due to an H5N8 highly pathogenic avian influenza virus (HPAIV) occurred at an egg-hen farm in Kagawa prefecture (western Japan). This virus, A/chicken/Kagawa/11C/2020 (Kagawa11C2020), was the first HPAI poultry isolate in Japan in 2020 and had multiple basic amino acids-a motif conferring high pathogenicity to chickens-at the hemagglutinin cleavage site. Mortality of chickens was 100% through intravenous inoculation tests performed according to World Organization for Animal Health criteria. Phylogenetic analysis showed that the hemagglutinin of Kagawa11C2020 belongs to clade 2.3.4.4B of the H5 Goose/Guangdong lineage and clusters with H5N8 HPAIVs isolated from wild bird feces collected in Hokkaido (Japan) and Korea in October 2020. These H5N8 HPAIVs are closely related to H5N8 HPAIVs isolated in European countries during the winter of 2019-2020. Intranasal inoculation of chickens with 106 fifty-percent egg infectious doses of Kagawa11C2020 revealed that the 50% chicken lethal dose was 104.63 and the mean time to death was 134.4 h. All infected chickens demonstrated viral shedding beginning on 2 dpi-before clinical signs were observed. These results suggest that affected chickens could transmit Kagawa11C2020 to surrounding chickens in the absence of clinical signs for several days before they died.

Genetic Characterization of Influenza A Viruses in Japanese Swine in 2015 to 2019.

To assess the current status of influenza A viruses of swine (IAVs-S) throughout Japan and to investigate how these viruses persisted and evolve on pig farms, we genetically characterized IAVs-S isolated during 2015 to 2019. Nasal swab samples collected through active surveillance and lung tissue samples collected for diagnosis yielded 424 IAVs-S, comprising 78 H1N1, 331 H1N2, and 15 H3N2 viruses, from farms in 21 sampled prefectures in Japan. Phylogenetic analyses of surface genes revealed that the 1A.1 classical swine H1 lineage has evolved uniquely since the late 1970s among pig populations in Japan. During 2015 to 2019, A(H1N1)pdm09 viruses repeatedly became introduced into farms and reassorted with endemic H1N2 and H3N2 IAVs-S. H3N2 IAVs-S isolated during 2015 to 2019 formed a clade that originated from 1999-2000 human seasonal influenza viruses; this situation differs from previous reports, in which H3N2 IAVs-S derived from human seasonal influenza viruses were transmitted sporadically from humans to swine but then disappeared without becoming established within the pig population. At farms where IAVs-S were frequently isolated for at least 3 years, multiple introductions of IAVs-S with phylogenetically distinct hemagglutinin (HA) genes occurred. In addition, at one farm, IAVs-S derived from a single introduction persisted for at least 3 years and carried no mutations at the deduced antigenic sites of the hemagglutinin protein, except for one at the antigenic site (Sa). Our results extend our understanding regarding the status of IAVs-S currently circulating in Japan and how they genetically evolve at the farm level.IMPORTANCE Understanding the current status of influenza A viruses of swine (IAVs-S) and their evolution at the farm level is important for controlling these pathogens. Efforts to monitor IAVs-S during 2015 to 2019 yielded H1N1, H1N2, and H3N2 viruses. H1 genes in Japanese swine formed a unique clade in the classical swine H1 lineage of 1A.1, and H3 genes originating from 1999-2000 human seasonal influenza viruses appear to have become established among Japanese swine. A(H1N1)pdm09-derived H1 genes became introduced repeatedly and reassorted with endemic IAVs-S, resulting in various combinations of surface and internal genes among pig populations in Japan. At the farm level, multiple introductions of IAVs-S with phylogenetically distinct HA sequences occurred, or IAVs-S derived from a single introduction have persisted for at least 3 years with only a single mutation at the antigenic site of the HA protein. Continued monitoring of IAVs-S is necessary to update and maximize control strategies.

Characterization and Phylodynamics of Reassortant H12Nx Viruses in Northern Eurasia.

Wild waterfowl birds are known to be the main reservoir for a variety of avian influenza viruses of different subtypes. Some subtypes, such as H2Nx, H8Nx, H12Nx, and H14Nx, occur relatively rarely in nature. During 10-year long-term surveillance, we isolated five rare H12N5 and one H12N2 viruses in three different distinct geographic regions of Northern Eurasia and studied their characteristics. H12N2 from the Far East region was a double reassortant containing hemagglutinin (HA), non-structural (NS) and nucleoprotein (NP) segments of the American lineage and others from the classical Eurasian avian-like lineage. H12N5 viruses contain Eurasian lineage segments. We suggest a phylogeographical scheme for reassortment events associated with geographical groups of aquatic birds and their migration flyways. The H12N2 virus is of particular interest as this subtype has been found in common teal in the Russian Far East region, and it has a strong relation to North American avian influenza virus lineages, clearly showing that viral exchange of segments between the two continents does occur. Our results emphasize the importance of Avian Influenza Virus (AIV) surveillance in Northern Eurasia for the annual screening of virus characteristics, including the genetic constellation of rare virus subtypes, to understand the evolutionary ecology of AIV.

Novel reassortant of H1N1 swine influenza virus detected in pig population in Russia.

Pigs play an important role in interspecies transmission of the influenza virus, particularly as "mixing vessels" for reassortment. Two influenza A/H1N1 virus strains, A/swine/Siberia/1sw/2016 and A/swine/Siberia/4sw/2017, were isolated during a surveillance of pigs from private farms in Russia from 2016 to 2017. There was a 10% identity difference between the HA and NA nucleotide sequences of isolated strains and the most phylogenetically related sequences (human influenza viruses of 1980s). Simultaneously, genome segments encoding internal proteins were found to be phylogenetically related to the A/H1N1pdm09 influenza virus. In addition, two amino acids (129-130) were deleted in the HA of A/swine/Siberia/4sw/2017 compared to that of A/swine/Siberia/1sw/2016 HA.

A novel H7N3 reassortant originating from the zoonotic H7N9 highly pathogenic avian influenza viruses that has adapted to ducks.

The first human case of zoonotic H7N9 avian influenza virus (AIV) infection was reported in March 2013 in China. This virus continues to circulate in poultry in China while mutating to highly pathogenic AIVs (HPAIVs). Through monitoring at airports in Japan, a novel H7N3 reassortant of the zoonotic H7N9 HPAIVs, A/duck/Japan/AQ-HE30-1/2018 (HE30-1), was detected in a poultry meat product illegally brought by a passenger from China into Japan. We analysed the genetic, pathogenic and antigenic characteristics of HE30-1 by comparing it with previous zoonotic H7N9 AIVs and their reassortants. Phylogenetic analysis of the entire HE30-1 genomic sequence revealed that it comprised at least three different sources; the HA (H7), PB1, PA, NP, M and NS segments of HE30-1 were directly derived from H7N9 AIVs, whereas the NA (N3) and PB2 segments of HE30-1 were unrelated to zoonotic H7N9. Experimental infection revealed that HE30-1 was lethal in chickens but not in domestic or mallard ducks. HE30-1 was shed from and replicated in domestic and mallard ducks and chickens, whereas previous zoonotic H7N9 AIVs have not adapted well to ducks. This finding suggests the possibility that HE30-1 may disseminate to remote area by wild bird migration once it establishes in wild bird population. A haemagglutination-inhibition assay indicated that antigenic drift has occurred among the reassortants of zoonotic H7N9 AIVs; HE30-1 showed similar antigenicity to some of those H7N9 AIVs, suggesting it might be prevented by the H5/H7 inactivated vaccine that was introduced in China in 2017. Our study reports the emergence of a new reassortant of zoonotic H7N9 AIVs with novel viral characteristics and warns of the challenge we still face to control the zoonotic H7N9 AIVs and their reassortants.

Isolation of highly pathogenic H5N6 avian influenza virus in Southern Vietnam with genetic similarity to those infecting humans in China.

Since 2013, H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for outbreaks in poultry and wild birds around Asia. H5N6 HPAIV is also a public concern due to sporadic human infections being reported in China. In the current study, we isolated an H5N6 HPAIV strain (A/Muscovy duck/Long An/AI470/2018; AI470) from an outbreak at a Muscovy duck farm in Long An Province in Southern Vietnam in July 2018 and genetically characterized it. Basic Local Alignment Search Tool (BLAST) analysis revealed that the eight genomic segments of AI470 were most closely related (99.6%-99.9%) to A/common gull/Saratov/1676/2018 (H5N6), which was isolated in October 2018 in Russia. Furthermore, AI470 also shared 99.4%-99.9% homology with A/Guangxi/32797/2018, an H5N6 HPAIV strain that infected humans in China in 2018. Phylogenetic analyses of the entire genome showed that AI470 was directly derived from H5N6 HPAIVs that were in South China from 2015 to 2018 and clustered with four H5N6 HPAIV strains of human origin in South China from 2017 to 2018. This indicated that AI470 was introduced into Vietnam from China. In addition, molecular characteristics related to mammalian adaptation among the recent human H5N6 HPAIV viruses, except PB2 E627K, were shared by AI470. These findings are cause for concern since H5N6 HPAIV strains that possess a risk of human infection have crossed the Chinese border.

Phylogeographic evidence for the inter- and intracontinental dissemination of avian influenza viruses via migration flyways.

Genetically related highly pathogenic avian influenza viruses (HPAIVs) of H5N6 subtype caused outbreaks simultaneously in East Asia and Europe-geographically distinct regions-during winter 2017-2018. This situation prompted us to consider whether the application of phylogeographic analysis to a particular gene segment of AIVs could provide clues for understanding how AIV had been disseminated across the continent. Here, the N6 NA genes of influenza viruses isolated across the world were subjected to phylogeographic analysis to illustrate the inter- and intracontinental dissemination of AIVs. Those isolated in East Asia during winter and in Mongolia/Siberia during summer were comingled within particular clades of the phylogeographic tree. For AIVs in one clade, their dissemination in eastern Eurasia extended from Yakutia, Russia, in the north to East Asia in the south. AIVs in western Asia, Europe, and Mongolia were also comingled within other clades, indicating that Mongolia/Siberia plays an important role in the dissemination of AIVs across the Eurasian continent. Mongolia/Siberia may therefore have played a role in the simultaneous outbreaks of H5N6 HPAIVs in Europe and East Asia during the winter of 2017-2018. In addition to the long-distance intracontinental disseminations described above, intercontinental disseminations of AIVs between Eurasia and Africa and between Eurasia and North America were also observed. Integrating these results and known migration flyways suggested that the migration of wild birds and the overlap of flyways, such as that observed in Mongolia/Siberia and along the Alaskan Peninsula, contributed to the long-distance intra- and intercontinental dissemination of AIVs. These findings highlight the importance of understanding the movement of migratory birds and the dynamics of AIVs in breeding areas-especially where several migration flyways overlap-in forecasting outbreaks caused by HPAIVs.

Genetics and pathogenicity of H5N6 highly pathogenic avian influenza viruses isolated from wild birds and a chicken in Japan during 2017-2018.

An H5N6 highly pathogenic avian influenza virus (HPAIV) outbreak occurred in poultry in Japan during January 2018, and H5N6 HPAIVs killed several wild birds in 3 prefectures during Winter 2017-2018. Time-measured phylogenetic analyses demonstrated that the Hemagglutinin (HA) and internal genes of these isolates were genetically similar to clade 2.3.4.4.B H5N8 HPAIVs in Europe during Winter 2016-2017, and Neuraminidase (NA) genes of the poultry and wild bird isolates were gained through distinct reassortments with AIVs that were estimated to have circulated possibly in Siberia during Summer 2017 and Summer 2016, respectively. Lethal infectious dose to chickens was similar between the poultry and wild-bird isolates. H5N6 HPAIVs during Winter 2017-2018 in Japan had higher 50% chicken lethal doses and lower transmission efficiency than the H5Nx HPAIVs that caused previous outbreaks in Japan, thus explaining in part why cases during the 2017-2018 outbreak were sporadic.