Asian H5 highly pathogenic avian influenza viruses (HPAIVs) that possess the clade 2.3.4.4 HA gene have been identified in wild birds and poultry since late 2014 in both Europe and North America (N. America). Clade 2.3.4.4 H5 HPAIVs of the H5N8 subtype have been isolated in both regions, whereas reassortment viruses with NA N1 and N2 subtypes of the North American (N. American). avian lineage have only been identified in N. America. The HA genes of those isolates were closely related to genes of the HPAIVs that have caused massive outbreaks in poultry in Korea since January 2014. The outbreaks caused by those viruses and the genetic relatedness of their HA and NA genes are reviewed in this study. Although the illegal movement of poultry and poultry products cannot be ruled out as a cause of intercontinental and intracontinental dissemination of clade 2.3.4.4 H5 HPAIVs during the winter of 2014-2015, transmission of the viruses by infected migratory birds appears to be a more plausible mechanism for their dissemination. In particular, the involvement of migratory birds in HPAIV transmission between Asia and N. America is highly likely because of the reassortments between H5N8 HPAIV and the N. American lineage avian influenza viruses.

To elucidate the evolutionary pathway, we sequenced the entire genomes of 89 H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated in Japan during winter 2016-2017 and 117 AIV/HPAIVs isolated in Japan and Russia. Phylogenetic analysis showed that at least 5 distinct genotypes of H5N6 HPAIVs affected poultry and wild birds during that period. Japanese H5N6 isolates shared a common genetic ancestor in 6 of 8 genomic segments, and the PA and NS genes demonstrated 4 and 2 genetic origins, respectively. Six gene segments originated from a putative ancestral clade 2.3.4.4 H5N6 virus that was a possible genetic reassortant among Chinese clade 2.3.4.4 H5N6 HPAIVs. In addition, 2 NS clusters and a PA cluster in Japanese H5N6 HPAIVs originated from Chinese HPAIVs, whereas 3 distinct AIV-derived PA clusters were evident. These results suggest that migratory birds were important in the spread and genetic diversification of clade 2.3.4.4 H5 HPAIVs.

A highly pathogenic avian influenza virus (HPAIV) of subtype H5N8, A/chicken/Kumamoto/1-7/2014, was isolated from a Japanese chicken farm during an outbreak in April 2014. Phylogenetic analysis revealed that this virus belonged to HA clade 2.3.4.4. All eight genomic segments showed high sequence similarity to those of the H5N8 subtype HPAIVs A/broiler duck/Korea/Buan2/2014 and A/baikal teal/Korea/Donglim3/2014, which were isolated in Korea in January 2014. Intranasal experimental infection of chickens and ducks with A/chicken/Kumamoto/1-7/2014 was performed to assess the pathogenicity of the virus in chickens and the potential for waterfowl to act as a virus reservoir and carrier. A high-titer virus challenge (10(6) EID50 per animal) was lethal in chickens, but they were unaffected by lower virus doses (10(2) EID50 or 10(4) EID50 per animal). Virus challenge at all doses examined was found to result in asymptomatic infection of ducks. An HI assay revealed that A/chicken/Kumamoto/1-7/2014 possessed relatively low cross-reactivity with H5 viruses belonging to clades other than clade 2.3.4.4. These results suggest that waterfowl may be able to spread the virus even if they possess antibodies resulting from a previous infection with H5 HPAIV that was antigenically distinguishable from viruses belonging to clade 2.3.4.4.

H ighly pathogenic avian influenza (HPAI) viruses are known to have zoonotic potential (1). There-

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.

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.

Summary In Japan during the 2016–2017 winter season, clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIVs) of the H5N6 subtype caused 12 outbreaks in chicken and Muscovy duck farms. These viruses have been circulating in Vietnam and China since 2014. In this study, we evaluated the susceptibility of chicken, Pekin duck (Anas platyrhynchos domesticus) and Muscovy duck (Cairina moschata) to H5N6 HPAIVs that originated in Japan, Vietnam and China. The H5N6 HPAIVs examined in this study were highly lethal to chickens compared with their pathogenicity in Pekin duck and Muscovy duck. One of five chickens infected with A/Muscovy duck/Aomori/1‐3T/2016 (MusDk/Aomori) survived despite viral shedding, although all of the chickens infected with the other viruses died. The 50% chicken lethal dose differed among the Japanese strains that shared the same gene constellation indicating that gene constellation was not a major determinant of pathogenicity in chicken. MusDk/Aomori, A/chicken/Niigata/1‐1T/2016 (Ck/Niigata) and A/duck/Hyogo/1/2016 (Dk/Hyogo) infected all Muscovy ducks inoculated; Ck/Niigata killed 50% of the ducks it infected whereas the other two did not kill any ducks. A/chicken/Japan/AnimalQuarantine‐HE144/2016 (HE144) isolated from chicken meat that originated in China was highly pathogenic to Pekin duck: all of the ducks died within 3.75 days of inoculation. This study shows that the pathogenicity of the clade 2.3.4.4 H5N6 HPAIVs differs not only between hosts but also within the same host species.

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.