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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.

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.

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.

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.

Human infection by low-pathogenic avian influenza viruses of the H7N9 subtype was first reported in March 2013 in China. Subsequently, these viruses caused five outbreaks through September 2017. In the fifth outbreak, H7N9 virus possessing a multiple basic amino acid insertion in the cleavage site of hemagglutinin emerged and caused 4% of all human infections in that period. To date, H7N9 highly pathogenic avian influenza viruses (HPAIVs) have been isolated from poultry, mostly chickens, as well as the environment. To evaluate the relative infectivity of these viruses in poultry, chickens and ducks were subjected to experimental infection with two H7N9 HPAIVs isolated from humans, namely A/Guangdong/17SF003/2016 and A/Taiwan/1/2017. When chickens were inoculated with the HPAIVs at a dose of 106 50% egg infectious dose (EID50), all chickens died within 2-5 days after inoculation, and the viruses replicated in most of the internal organs examined. The 50% lethal doses of A/Guangdong/17SF003/2016 and A/Taiwan/1/2017 in chickens were calculated as 103.3 and 104.7 EID50, respectively. Conversely, none of the ducks inoculated with either virus displayed any clinical signs, and less-efficient virus replication and less shedding were observed in ducks compared to chickens. These findings indicate that chickens, but not ducks, are highly permissive hosts for emerging H7N9 HPAIVs.

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.

Surveillance studies of influenza A virus of swine (IAV-S) have accumulated information regarding IAVs-S circulating in Thailand, but how IAVs-S evolve within a farm remains unclear. In the present study, we isolated 82 A(H1N1)pdm09 and 87 H3N2 viruses from four farms from 2011 through 2017. We then phylogenetically and antigenically analyzed the isolates to elucidate their evolution within each farm. Phylogenetic analysis demonstrated multiple introductions of A(H1N1)pdm09 viruses that resembled epidemic A(H1N1)pdm09 strains in humans in Thailand, and they reassorted with H3N2 viruses as well as other A(H1N1)pdm09 viruses. Antigenic analysis revealed that the viruses had acquired antigenic diversity either by accumulating substitutions in the hemagglutinin protein or through the introduction of IAV-S strains with different antigenicity. Our results, obtained through continuous longitudinal surveillance, revealed that IAV-S can be maintained on a pig farm over several years through the generation of antigenic diversity due to the accumulation of mutations, introduction of new strains, and reassortment events.

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.

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.

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.

The viral protein N-pro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, N-pro suppresses type I IFN (IFN-alpha/beta) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the N-pro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of N-pro coordinating zinc by means of the amino acid residues 0112, 0134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-alpha/beta induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of N-pro of these four isolates were related to the lack of IRF-3-degrading activity. restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional N-pro contributed to higher stability of the reconstructed N-pro compared with the N-pro from the Thai isolate. This led to enhanced interaction of N-pro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of N-pro are involved in the stability of N-pro, in interaction of N-pro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-alpha/beta production.