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.

Genetics of the rabbit haemorrhagic disease virus strains responsible for rabbit haemorrhagic disease outbreaks in Japan between 2000 and 2020.

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease in rabbits caused by the rabbit haemorrhagic disease virus (RHDV), which includes two genotypes, RHDV-GI.1 and RHDV2-GI.2. RHDVs tend to recombine among different strains, resulting in significant genetic evolution. This study evaluated the genetics of Japanese RHDV strains associated with six outbreaks between 2000 and 2020 using whole-genome sequencing, genomic recombination and phylogenetic analyses. Genomic recombination analysis using near-complete genomic sequences revealed that two Japanese strains detected in 2000 and 2002 were non-recombinant GI.1 (variant RHDVa-GI.1a) strains of different origins, most closely related to strains identified in PR China in 1997 and the USA in 2001, respectively. In contrast, four recent Japanese GI.2 strains detected between 2019 and 2020 were recombinant viruses harbouring structural protein (SP) genes from GI.2 strains and non-SP (NSP) genes from a benign rabbit calicivirus (RCV) strain of genotype RCV-E1-GI.3 (GI.3P-GI.2) or an RHDV G1-GI.1b variant (GI.1bP-GI.2). Phylogenetic analysis based on SP and NSP regions revealed that the GI.1bP-GI.2 recombinant virus detected in Ehime prefecture and the GI.3P-GI.2 recombinant viruses detected in Ibaraki, Tochigi and Chiba prefectures were most closely related to recombinant viruses identified in Australia in 2017 and Germany in 2017, respectively. These results suggested that past RHD outbreaks in Japan did not result from the evolution of domestic RHDVs but rather represented incursions of foreign RHDV strains, implying that Japan is constantly at risk of RHDV incursion from other countries.

Recombinant fiber-1 protein of fowl adenovirus serotype 4 induces high levels of neutralizing antibodies in immunized chickens.

Virulent fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium syndrome (HPS) with high mortality in chickens, leading to significant economic losses to the poultry industry. The development of an effective vaccine is essential for successful disease control. Here, we produced recombinant fiber-1 protein of FAdV-4, isolated from a Japanese HPS outbreak strain, JP/LVP-1/96, using a baculovirus expression system and evaluated its immunogenicity and protective efficacy. Recombinant fiber-1 protein induced high levels of neutralizing antibodies in immunized chickens, which were maintained for a minimum of 10 weeks. After being challenged with the virulent FAdV-4 strain JP/LVP-1/96, the immunized chickens did not exhibit clinical signs of infection or histopathological changes, there was a significant reduction in the viral load in various organs and total serum proteins, and albumin levels did not decline. These results suggest that the recombinant fiber-1 protein produced in this study can serve as a subunit vaccine to control HPS in chickens.

Ubiquitin-specific protease 18 in mallard (Anas platyrhynchos) interferes with type I interferon-mediated inhibition of high pathogenicity avian influenza virus replication.

Ubiquitin-specific protease 18 (USP18) is a well-established innate immune factor in vertebrates. Although Anatidae birds rarely exhibit distinctive clinical signs during high pathogenicity avian influenza virus (HPAIV) infections, some virus strains cause deadly diseases. Here, we investigated the association between USP18 expression and pathogenicity during HPAIV infections in the Anatidae mallard Anas platyrhynchos. First, mallard USP18 gene (duUSP18) was cloned, and its transcriptional variants, with three different open reading frames, were characterized. Experimental infections with two different pathogenic strains, Miyazaki and Takeo, demonstrated an early induction of duUSP18 mRNA upon HPAIV infection in a bird's whole body in vivo and in primary duck cells in vitro, which was positively associated with pathogenicity in mallards. In addition, duUSP18 knockdown under interferon-ß stimulation attenuated viral replication, regardless of pathogenicity. These results indicate a role for duUSP18 in favoring viral replication and virus resistance to type I interferon immunity in mallards.

Detection of Gyrovirus galga 1 in Cryopreserved Organs from Two Commercial Broiler Flocks in Japan.

Gyrovirus galga 1 (GyVg1, previously recognized as avian gyrovirus 2), which was first reported in chicken in 2011, is a new member of the genus Gyrovirus. The presence of GyVg1 has also been confirmed in different regions of Europe, South America, Africa, and Asia, indicating its global distribution. However, because there are no reports of examining the distribution of GyVg1 in animals in Japan, the epidemiology of this virus is unknown. In this study, we attempted to retrospectively detect GyVg1 in cryopreserved chicken materials derived from different two commercial broiler flocks in 1997. The GyVg1 genome was detected in organ materials derived from both flocks by PCR. GyVg1 detected in both flocks was classified into four genetic groups by analyzing the nucleotide sequences of the detected PCR products. These results suggest that diverse GyVg1 strains were present in commercial chicken flocks as early as 1997 in Japan.

Comparative susceptibility of mallard (Anas platyrhynchos) to infection with high pathogenicity avian influenza virus strains (Gs/Gd lineage) isolated in Japan in 2004-2017.

The Goose/Guangdong-lineage (Gs/Gd) H5 high pathogenicity avian influenza viruses (HPAIVs) spread among poultry and wild birds worldwide; an association has been identified between the migration of wild birds and spread of HPAIVs. Every autumn-spring season, the mallard (Anas platyrhynchos) migrates to Japan in substantial numbers for overwintering; however, to the best of our knowledge, no virological studies have focused on mallards' susceptibility to the HPAIVs in Japan. To evaluate the susceptibility of mallards to infection with Gs/Gd H5 HPAIVs isolated during previous outbreaks in Japan, we experimentally infected the birds with various virus strains: A/chicken/Yamaguchi/7/2004 (H5N1) (clade 2.5), A/chicken/Miyazaki/K11/2007 (H5N1) (clade 2.2), A/whooper swan/Akita/1/2008 (H5N1) (clade 2.3.2), A/mandarin duck/Miyazaki/22M-765/2011 (H5N1) (clade 2.3.2.1c), A/duck/Chiba/26-372-48/2014 (H5N8) (clade 2.3.4.4c), A/duck/Hyogo/1/2016 (H5N6) (clade 2.3.4.4e) and A/mute swan/Shimane/3211A002/2017 (H5N6) (clade 2.3.4.4b). The birds exhibited high tracheal shedding for a prolonged period, particularly those infected with A/duck/Hyogo/1/2016 (H5N6). Various clinical manifestations ranging from asymptomatic to mild (corneal opacity) infections to neurological disorders accompanied by mortality were noted depending on the virus strain. Furthermore, virus-infected mallards contaminated both cohoused mallards and water in their surroundings. Thus, mallards may disseminate viruses in the environment, thereby influencing HPAI outbreaks in Japan. Therefore, mallards represent an important migratory bird species that spread HPAIVs in Japan.

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.

Comparative susceptibility of the common teal (Anas crecca) to infection with high pathogenic avian influenza virus strains isolated in Japan in 2004-2017.

High pathogenic avian influenza viruses (HPAIVs) of the H5 subtype have spread in poultry and wild birds worldwide. Current studies have highlighted the association between the migration of wild birds and the spread of HPAIVs. However, virological studies examining responsible species of migratory birds to spread HPAIVs are limited. In Japan, the common teal (Anas crecca) arrives in great numbers for overwintering every autumn-spring season; therefore, we performed experimental infection using six H5 HPAIVs isolated in past outbreaks in Japan (A/chicken/Yamaguchi/7/2004 (H5N1), A/whooper swan/Akita/1/2008 (H5N1), A/mandarin duck/Miyazaki/22M-765/2011 (H5N1), A/duck/Chiba/26-372-48/2014 (H5N8), A/duck/Hyogo/1/2016 (H5N6) and A/mute swan/Shimane/3211A002/2017 (H5N6)) to evaluate the susceptibility of the species to HPAIV infection. The results illustrated that most birds in all experimental groups were infected by the strains, and they shed viruses for a prolonged period, in trachea than cloaca, without displaying distinctive clinical signs. In addition, comparative analysis using calculation value of total viral shedding during the experiment revealed that the birds shed viruses at above a certain level regardless of the differences of strains. These results suggested that the common teal could be a migratory bird species that disseminates viruses in the environment, thereby influencing HPAI outbreaks in wild birds in Japan.

Different Sensitivity of Japanese Native-Bred Chickens to H5 Subtypes of Highly Pathogenic Avian Influenza Viruses.

The aim of this study was to investigate the sensitivity of three breeds of Japanese native chickens, commercial broilers, and specific-pathogen-free (SPF) white leghorns to three strains of the H5 subtype of highly pathogenic avian influenza viruses (HPAIVs). Chickens were experimentally inoculated with doses of 102, 104, and 106 50% egg infective dose of A/mandarin duck/Miyazaki/22M-765/2011 (duck-11), A/chicken/Miyazaki/7/2014 (chicken-14), and A/chicken/Kumamoto/1-2C/2016 (chicken-16). The 50% chicken lethal dose of each virus, mean death time, and viral shedding patterns were compared. The Japanese native chickens showed varied susceptibility to the three H5 HPAIV isolates. Although two of the breeds showed some degree of resistance to duck-11 and chicken-14, all three were more sensitive to chicken-16 than commercial broiler chickens. We have shown that Japanese native chickens do not necessarily have resistance to HPAIV and that the pathogenic characteristics of HPAIVs are quite different between native and commercial chickens.

Nota de investigación­Diferente sensibilidad de pollos nativos de Japón a subtipos del virus de influenza aviar H5 altamente patógenos. El objetivo de este estudio fue investigar la sensibilidad de tres razas de pollos nativos de Japón, de pollos de engorde comerciales y de aves Leghorn blancas libres de patógenos específicos (SPF) a tres cepas altamente patógenas del virus de influenza aviar (HPAIV) del subtipo H5. Los pollos se inocularon experimentalmente con dosis de 102, 104 y 106 dosis infecciosas para embrión de pollo 50% del virus A/pato mandarín/Miyazaki/22M-765/2011 (pato-11), del virus A/pollo/Miyazaki/7/2014 (pollo-14) y A/ pollo/Kumamoto/1-2C-2016 (pollo-16). Se comparó la dosis letal de pollo 50% de cada virus, el tiempo medio de muerte y los patrones de diseminación viral. Los pollos nativos japoneses mostraron una susceptibilidad variada a los tres aislados del virus de influenza aviar altamente patógeno H5. Aunque dos de las razas mostraron cierto grado de resistencia al virus de pato-11 y al pollo-14, las tres eran más sensibles al virus del pollo-16 que los pollos de engorde comerciales. Se demostró que los pollos nativos japoneses no necesariamente tienen resistencia al virus de influenza aviar altamente patógeno y que las características patógenas de los virus de influenza aviar altamente patógenos son bastante diferentes entre pollos nativos y comerciales.

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.

Improvement of a recombinant avian avulavirus serotype 10 vectored vaccine by the addition of untranslated regions.

BACKGROUND: We have previously developed a recombinant avian avulavirus serotype 10 (rAAvV-10/HA) expressing the hemagglutinin (HA) gene of a highly pathogenic avian influenza virus (HPAIV) as an emergency vaccine for poultry. rAAvV-10/HA can overcome the activity of the anti-AAvV-1 (Newcastle disease virus) antibody acquired by commercial chickens upon routine vaccination. Most chickens do not have the anti-AAvV-10 antibody, which could interfere with the vaccine efficacy. However, the vaccine efficacy of rAAvV-10/HA is not satisfactory in chickens even though it affords protection against an HPAIV challenge. In the present study, we improved the rAAvV-10/HA vaccine by enhancing the expression of the exogenous HA protein. METHODS: The 5' and 3' untranslated regions (UTR) of each AAvV-10 gene were flanked with the exogenous HA gene cassette to modify rAAvV-10/HA, yielding different rAAv10-UTRs. As a control, rAAv10-nonUTR that did not contain any UTRs was generated. The effects of UTRs on mRNA transcription, HA protein expression, and vaccine efficacy were then examined using embryonated chicken eggs and white leghorn chickens. RESULTS: The proportion of the HA gene mRNA among the vector-derived mRNAs (1.55-1.84-fold increase vs. the control) and HA protein levels (148-1151-fold increase vs. the control) in cells infected with rAAv10-UTRs were higher than in those infected with rAAv10-nonUTR. In vivo, vaccination of chickens with rAAv10-UTRs resulted in 100% protection against an HPAIV challenge. No chickens vaccinated with rAAv10-NP-UTR, rAAv10-F-UTR, or rAAv10-HN-UTR shed the virus in the throat and cloaca swabs. By contrast, rAAv10-nonUTR vaccination offered 70% protection, with 50% of chickens shedding the virus in the cloaca or throat swabs after the challenge. We conclude that the AAvV-10 UTRs can enhance the expression of the exogenous HA gene, resulting in improved efficacy of the rAAvV-10/HA vector vaccine. This improvement aids in the protection of flocks worldwide from the highly pathogenic avian influenza.

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.

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.

Comparative pathogenicity of H5N6 subtype highly pathogenic avian influenza viruses in chicken, Pekin duck and Muscovy duck.

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.

Genetic and antigenic dynamics of influenza A viruses of swine on pig farms in Thailand.

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.

Pathogenicity of two novel human-origin H7N9 highly pathogenic avian influenza viruses in chickens and ducks.

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.

Spatial transmission of H5N6 highly pathogenic avian influenza viruses among wild birds in Ibaraki Prefecture, Japan, 2016-2017.

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.

Recombinant Avian Paramyxovirus Serotypes 2, 6, and 10 as Vaccine Vectors for Highly Pathogenic Avian Influenza in Chickens with Antibodies Against Newcastle Disease Virus.

Recombinant Newcastle disease virus (rNDV) expressing the hemagglutinin of highly pathogenic avian influenza virus (HPAIV HA) induces protective immunity against HPAIV in chickens. However, the efficacy of rNDV vectors is hampered when chickens are pre-immune to NDV, and most commercial chickens are routinely vaccinated against NDV. We recently showed that avian paramyxovirus serotypes 2, 6, and 10 (APMV-2, APMV-6, and APMV-10), which belong to the same genus as NDV, have low cross-reactivity with anti-NDV antisera. Here, we used reverse genetics to generate recombinant APMV-2, APMV-6, and APMV-10 (rAPMV-2/HA, rAPMV-6/HA, and rAPMV-10/HA) that expressed an HA protein derived of subtype H5N1 HPAIV, A/chicken/Yamaguchi/7/2004. Chickens pre-immunized against NDV (age, 7 wk) were vaccinated with rAPMV/HAs; 14 days after vaccination, chickens were challenged with a lethal dose of HPAIV. Immunization of chickens pre-immunized against NDV with rAPMV-2/HA, rAPMV-6/HA, or rAPMV-10/HA protected 50%, 50%, and 25%, respectively, in groups of chickens given an rAPMV/HA with 106 median embryo infectious dose (EID50) or 50%, 50%, and 90%, respectively, in those with 107 EID50; in contrast, rNDV/HA protected none of the chicken vaccinated with 106 EID50 and induced only partial protection even with 107 EID50. Therefore, the presence of anti-NDV antibodies did not hamper the efficacy of rAPMV-2/HA, rAPMV-6/HA, or rAPMV-10/HA. These results suggest that rAPMV-2, rAPMV-6, and rAPMV-10 are potential vaccine vectors, especially for commercial chickens, which are routinely vaccinated against NDV.