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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.

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.

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.

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.

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.

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.

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., 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.

From 29 November 2016 to 24 January 2017, sixty-three cases of H5N6 highly pathogenic avian influenza virus (HPAIV) infections were detected in wild birds in Ibaraki Prefecture, Japan. Here, we analyzed the genetic, temporal, and geographic correlations of these 63 HPAIVs to elucidate their dissemination throughout the prefecture. Full-genome sequence analysis of the Ibaraki isolates showed that 7 segments (PB2, PB1, PA, HA, NP, NA, NS) were derived from G1.1.9 strains while the M segment was from G1.1 strains; both groups of strains circulated in south China. Pathological studies revealed severe systemic infection in dead swans (the majority of dead birds and the only species necropsied), thus indicating high susceptibility to H5N6 HPAIVs. Coalescent phylogenetic analysis using the 7 G1.1.9-derived segments enabled detailed analysis of the short-term evolution of these highly homologous HPAIVs. This analysis revealed that the H5N6 HPAIVs isolated from wild birds in Ibaraki Prefecture were divided into 7 groups. Spatial analysis demonstrated that most of the cases concentrated around Senba Lake originated from a single source, and progeny viruses were transmitted to other locations after the infection expanded in mute swans. In contrast, within just a 5-km radius of the area in which cases were concentrated, three different intrusions of H5N6 HPAIVs were evident. Multi-segment analysis of short-term evolution showed that not only was the invading virus spread throughout Ibaraki Prefecture but also that, despite the small size of this region, multiple invasions had occurred during winter 2016-2017.

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.

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.

In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5′-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-1a (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.