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Summary: The transportation of poultry and related products for international trade contributes to transboundary pathogen spread and disease outbreaks worldwide. To prevent pathogen incursion through poultry products, many countries have regulations about animal health and poultry product quarantine. However, in Japan, animal products have been illegally introduced into the country in baggage and confiscated at the airport. Lately, the number of illegally imported poultry and the incursion risk of transboundary pathogens through poultry products have been increasing. In this study, we isolated avian influenza viruses (AIVs) from raw poultry products illegally imported to Japan by international passengers. Highly (H5N1 and H5N6) and low (H9N2 and H1N2) pathogenic AIVs were isolated from raw chicken and duck products carried by flight passengers. H5 and H9 isolates were phylogenetically closely related to viruses isolated from poultry in China, and haemagglutinin genes of H5N1 and H5N6 isolates belonged to clades 2.3.2.1c and 2.3.4.4, respectively. Experimental infections of H5 and H9 isolates in chickens and ducks demonstrated pathogenicity and tissue tropism to skeletal muscles. To prevent virus incursion by poultry products, it is important to encourage the phased cleaning based on the disease control and eradication and promote the reduction in contamination risk in animal products. [ABSTRACT FROM AUTHOR]

Abstract Background The pandemic 2009 (H1N1) influenza virus has spread throughout the world and is now causing seasonal influenza. To prepare for the emergence of pandemic influenza, we have established a library of virus strains isolated from birds, pigs, and humans in global surveillance studies. Methods Inactivated whole virus particle (WV) and ether-split (ES) vaccines were prepared from an influenza virus strain, A/swine/Hokkaido/2/1981 (H1N1), from the library and from A/Narita/1/2009 (H1N1) pandemic strain. Each of the vaccines was injected subcutaneously into mice and their potencies were evaluated by challenge with A/Narita/1/2009 (H1N1) virus strain in mice. Results A/swine/Hokkaido/2/81 (H1N1), which was isolated from the lung of a diseased piglet, was selected on the basis of their antigenicity and growth capacity in embryonated chicken eggs. Two injections of the WV vaccine induced an immune response in mice, decreasing the impact of disease caused by the challenge with A/Narita/1/2009 (H1N1), as did the vaccine prepared from the homologous strain. Conclusion The WV vaccine prepared from an influenza virus in the library is useful as an emergency vaccine in the early phase of pandemic influenza.

Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named "Vac2sub-P0", which is nonpathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs, and a virus with high intravenous pathogenicity, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 resulted in limited lethality in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the resultant virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages were responsible for the increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with those of the parental virus in vitro; the virus growth at 40 °C was faster, the binding affinity to a sialic acid receptor was lower, and the rate of release by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study demonstrates that viruses that are highly pathogenic when administered intranasally require additional adaptations for increased pathogenicity to be highly lethal to intranasally infected chickens., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

We isolated highly pathogenic avian influenza (HPAI) H5N5 and H5N1 viruses from crows in Hokkaido, Japan, during winter 2023-24. They shared genetic similarity with HPAI H5N5 viruses from northern Europe but differed from those in Asia. Continuous monitoring and rapid information sharing between countries are needed to prevent HPAI virus transmission.

Chimeric marker vaccine candidates, vGPE - /PAPeV E rns and vGPE - /PhoPeV E rns , have been generated and their efficacy and capability to differentiate infected from vaccinated animals were confirmed in previous studies. The safety profile of the two chimeric marker vaccine candidates, particularly in the potential reversion to virulence, was evaluated. Each virus was administered to pigs with a dose equivalent to the vaccination dose, and pooled tonsil homogenates were subsequently inoculated into further pigs. Chimeric virus vGPE - /PAPeV E rns displayed the most substantial attenuation, achieving this within only two passages, whereas vGPE - /PhoPeV E rns was detectable until the third passage and disappeared entirely by the fourth passage. The vGPE - strain, assessed alongside, consistently exhibited stable virus recovery across each passage without any signs of increased virulence in pigs. In vitro assays revealed that the type I interferon-inducing capacity of vGPE - /PAPeV E rns was significantly higher than that of vGPE - /PhoPeV E rns and vGPE - . In conclusion, the safety profile of the two chimeric marker vaccine candidates was affirmed. Further research is essential to ensure the stability of their attenuation and safety in diverse pig populations.

High pathogenicity avian influenza is an acute zoonotic disease with high mortality in birds caused by a high pathogenicity avian influenza virus (HPAIV). Recently, HPAIV has rapidly spread worldwide and has killed many wild birds, including endangered species. Baloxavir marboxil (BXM), an anti-influenza agent used for humans, was reported to reduce mortality and virus secretion from HPAIV-infected chickens ( Gallus domesticus , order Galliformes) at a dosage of ≥2.5 mg/kg when administered simultaneously with viral challenge. Application of this treatment to endangered birds requires further information on potential avian-specific toxicity caused by repeated exposure to BXM over the long term. To obtain information of potential avian-specific toxicity, a 4-wk oral repeated-dose study of BXM was conducted in chickens ( n = 6 or 7 per group), which are commonly used as laboratory avian species. The study was conducted in reference to the human pharmaceutical guidelines for nonclinical repeated-dose drug toxicity studies to evaluate systemic toxicity and exposure. No adverse changes were observed in any organs examined, and dose proportional increases in systemic exposure to active pharmaceutical ingredients were noted from 12.5 to 62.5 mg/kg per day. BXM showed no toxicity to chickens at doses of up to 62.5 mg/kg per day, at which systemic exposure was approximately 71 times higher than systemic exposure at 2.5 mg/kg, the reported efficacious dosage amount, in HPAIV-infected chickens. These results also suggest that BXM could be considered safe for treating HPAIV-infected endangered birds due to its high safety margin compared with the efficacy dose. The data in this study could contribute to the preservation of endangered birds by using BXM as a means of protecting biodiversity.

Background: It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting E rns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/E rns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed., Methods: Recombinant E2 or E rns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens . ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or E rns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and E rns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates., Results: E2 and E rns proteins were successfully expressed in N. benthamiana -produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and E rns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for E rns antibody detection. ICS confirmed the absence of CSFV E rns -specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates., Conclusion: E2 and E rns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/E rns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and E rns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates., Competing Interests: E-JS, YP, and JK were employed by BioApplications Inc. S-HH and H-NL were employed by Celltrix Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Huynh, Sohn, Park, Kim, Shimoda, Hiono, Isoda, Hong, Lee and Sakoda.)

The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (γ-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the γ-variant S protein; however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.

Pestiviruses are classified into two biotypes based on their cytopathogenicity. As the majority of pestivirus field isolates are noncytopathogenic, their titration requires alternative methods rather than direct observation of cytopathogenic effects, such as immunostaining using specific antibodies or interference with cytopathogenic strains. However, these methods require microscopic observation to assess virus growth, which is time- and labor-intensive, especially when handling several samples. In this study, we developed a novel luciferase-based pestivirus titration method using the superinfection exclusion phenomenon with recombinant reporter pestiviruses that possessed an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). In this method, swine kidney cells were inoculated with classical swine fever virus (CSFV) and superinfected with the reporter CSFV vGPE - /HiBiT 5 days postinoculation. Virus titer was determined based on virus growth measured in luminescence using the culture fluid 3 days after superinfection; the resultant virus titer was comparable to that obtained by immunoperoxidase staining. Furthermore, this method has proven to be applicable for the titration of border disease virus (BDV) by superinfection with both the homologous reporter BDV and heterologous reporter CSFV, suggesting that this novel virus titration method is a simple technique for automated virus detection based on the luciferase system.

Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology., Importance: The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research., Competing Interests: The authors declare no conflict of interest.

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.

A previous study proved that vGPE - mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE - vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE - vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein E rns of the GPE - vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE - /PAPeV E rns and vGPE - /PhoPeV E rns , were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE - vaccine strain. Vaccination at a dose of 10 4.0 TCID 50 with either vGPE - /PAPeV E rns or vGPE - /PhoPeV E rns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE - /PAPeV E rns and vGPE - /PhoPeV E rns affirmed their properties, as the vGPE - vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces , called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S -adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

The H9 and H6 subtypes of low pathogenicity avian influenza viruses (LPAIVs) cause substantial economic losses in poultry worldwide, including Vietnam. Herein, we characterized Vietnamese H9 and H6 LPAIVs to facilitate the control of avian influenza. The space-time representative viruses of each subtype were selected based on active surveillance from 2014 to 2018 in Vietnam. Phylogenetic analysis using hemagglutinin genes revealed that 54 H9 and 48 H6 Vietnamese LPAIVs were classified into the sublineages Y280/BJ94 and Group II, respectively. Gene constellation analysis indicated that 6 and 19 genotypes of the H9 and H6 subtypes, respectively, belonged to the representative viruses. The Vietnamese viruses are genetically related to the previous isolates and those in neighboring countries, indicating their circulation in poultry after being introduced into Vietnam. The antigenicity of these subtypes was different from that of viruses isolated from wild birds. Antigenicity was more conserved in the H9 viruses than in the H6 viruses. Furthermore, a representative H9 LPAIV exhibited systemic replication in chickens, which was enhanced by coinfection with avian pathogenic Escherichia coli O2. Although H9 and H6 were classified as LPAIVs, their characterization indicated that their silent spread might significantly affect the poultry industry.

In winter/spring 2021-2022, high pathogenicity avian influenza viruses (HPAIVs) that are genetically closely related to each other were detected worldwide. In a public garden in Sapporo, Hokkaido, Japan, a crow die-off by HPAIV infection occurred from March 29 to May 18, 2022. During the event, H5N1 HPAIVs were isolated from an Ezo red fox (Vulpes vulpes schrencki) and a tanuki (Nyctereutes procyonoides albus) found in the same garden. The fox showed viral meningoencephalitis and moderate virus replication in the upper respiratory tract, whereas the tanuki showed viral conjunctivitis and secondary bacterial infection in the eyes accompanied with visceral larva migrans. Viruses isolated from the fox and the tanuki were genetically closely related to those isolated from crows in the same garden. Various α2-3 sialosides were found in the respiratory tracts of these canid mammals, consistent with HPAIV infections in these animals. This study highlighted the importance of monitoring HPAIV infections in wild carnivore mammals to detect the potential virus spreading in nature., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Many high pathogenicity avian influenza (HPAI) cases in wild birds due to H5N1 HPAI virus (HPAIV) infection were reported in northern Japan in the winter of 2021-2022. To investigate the epidemiology of HPAIVs brought to Japan from surrounding areas, a genetic analysis of H5 HPAIVs isolated in northern Japan was performed, and the pathogenicity of the HPAIV in chickens was assessed by experimental infection. Based on the genetic analysis of the hemagglutinin gene, pathogenic viruses detected in northern Japan as well as one in Sakhalin, the eastern part of Russia, were classified into the same subgroup as viruses prevalent in Europe in the same season but distinct from those circulating in Asia in winter 2020-2021. High identities of all eight segment sequences of A/crow/Hokkaido/0103B065/2022 (H5N1) (Crow/Hok), the representative isolates in northern Japan in 2022, to European isolates in the same season could also certify the unlikeliness of causing gene reassortment between H5 HPAIVs and viruses locally circulating in Asia. According to intranasal challenge results in six-week-old chickens, 50% of the chicken-lethal dose of Crow/Hok was calculated as 10 4.5 times of the 50% egg-infectious dose. These results demonstrated that the currently prevalent H5 HPAIVs could spread widely from certain origins throughout the Eurasian continent, including Europe and the Far East, and implied a possibility that contagious viruses are gathered in lakes in the northern territory via bird migration. Active monitoring of wild birds at the global level is essential to estimate the geographical source and spread dynamics of HPAIVs., Competing Interests: The funders did not have any influence on the analysis or interpretation of the results in this study. Authors declare no conflict of interest.

Avian or human influenza A viruses bind preferentially to avian- or human-type sialic acid receptors, respectively, indicating that receptor tropism is an important factor for determining the viral host range. However, there are currently no reliable methods for analyzing receptor tropism biologically under physiological conditions. In this study, we established a novel system using MDCK cells with avian- or human-type sialic acid receptors and with both sialic acid receptors knocked out (KO). When we examined the replication of human and avian influenza viruses in these KO cells, we observed unique viral receptor tropism that could not be detected using a conventional solid-phase sialylglycan binding assay, which directly assesses physical binding between the virus and sialic acids. Furthermore, we serially passaged an engineered avian-derived H4N5 influenza virus, whose PB2 gene was deleted, in avian-type receptor KO cells stably expressing PB2 to select a mutant with enhanced replication in KO cells; however, its binding to human-type sialylglycan was undetectable using the solid-phase binding assay. These data indicate that a panel of sialic acid receptor KO cells could be a useful tool for determining the biological receptor tropism of influenza A viruses. Moreover, the PB2KO virus experimental system could help to safely and efficiently identify the mutations required for avian influenza viruses to adapt to human cells that could trigger a new influenza pandemic. IMPORTANCE The acquisition of mutations that allow avian influenza A virus hemagglutinins to recognize human-type receptors is mandatory for the transmission of avian viruses to humans, which could lead to a pandemic. In this study, we established a novel system using a set of genetically engineered MDCK cells with knocked out sialic acid receptors to biologically evaluate the receptor tropism for influenza A viruses. Using this system, we observed unique receptor tropism in several virus strains that was undetectable using conventional solid-phase binding assays that measure physical binding between the virus and artificially synthesized sialylglycans. This study contributes to elucidation of the relationship between the physical binding of virus and receptor and viral infectivity. Furthermore, the system using sialic acid knockout cells could provide a useful tool to explore the sialic acid-independent entry mechanism. In addition, our system could be safely used to identify mutations that could acquire human-type receptor tropism.

Avian influenza viruses (AIVs) circulating in wild ducks are rarely transmitted directly to chickens. Previous studies demonstrated that chickens possess fucosylated and/or sulfated α2,3 sialosides on their tracheal epithelia, whereas intestinal epithelia of ducks express canonical α2,3 sialosides. Turkeys, the third major poultry species in the world, are known to show broad susceptibility to various avian influenza viruses. To elucidate the molecular basis of the broad susceptibility of turkeys to duck and chicken AIVs, we characterized various receptors for AIVs on their tissues. The experimental infection of turkeys demonstrated their dual susceptibility to duck and chicken AIVs. Further, comprehensive histochemical analyses using lectins, anti-glycan antibodies, and recombinant hemagglutinins, combined with glycosidase digestions, identified the presence of fucosylated and/or sulfated in addition to canonical α2,3 sialosides on their respiratory epithelia. The receptor distributions in turkeys were consistent with their dual susceptibility to duck and chicken AIVs. Also, our findings suggested the potential roles of turkeys in interspecies transmission of AIVs from ducks to chickens., (Copyright © 2022. Published by Elsevier B.V.)

The impact of low pathogenicity avian influenza (LPAI) has been confirmed mainly in farms. Unlike apparent losses caused by the high pathogenicity avian influenza (HPAI), the LPAI impact has been hardly evaluated due to underestimating its spread and damage. In 2019, a questionnaire study was conducted in southern Vietnam to identify the specific risk factors of LPAI virus (LPAIV) circulation and to find associations between husbandry activities and LPAI prevalence. A multilevel regression analysis indicated that keeping Muscovy ducks during farming contributed to LPAIV positivity [Odds ratio=208.2 (95% confidence interval: 13.4-1.1 × 10 4 )]. In cluster analysis, farmers willing to report avian influenza (AI) events and who agreed with the local AI control policy had a slightly lower risk for LPAIV infection although there was no significance in the correlation between farmer characteristics and LPAI occurrence. These findings indicated that keeping Muscovy ducks without appropriate countermeasures might increase the risk of LPAIV infection. Furthermore, specific control measures at the local level are effective for LPAIV circulation, and the improvement of knowledge about biosecurity and attitude contributes to reducing LPAI damage.

The pathogenicity of the H5 subtype high pathogenicity avian influenza viruses (HPAIVs) in Ardeidae bird species has not been investigated yet , despite the increasing infections reported. Therefore, the present study aimed to examine the susceptibility of the Ardeidae species, which had already been reported to be susceptible to HPAIVs, to a clade 2.3.2.1 H5N1 HPAIV. Juvenile herons (four grey herons, one intermediate egret, two little egrets, and three black-crowned night herons) were intranasally inoculated with 10 6 50% egg infectious dose of the virus and observed for 10 days. Two of the four grey herons showed lethargy and conjunctivitis; among them, one died at 6 days post-inoculation (dpi). The viruses were transmitted to the other two cohoused naïve grey herons. Some little egrets and black-crowned night herons showing neurological disorders died at 4-5 dpi; these birds mainly shed the virus via the oral route. The viruses predominantly replicated in the brains of birds that died of infection. Seroconversion was observed in most surviving birds, except some black-crowned night herons. These results demonstrate that most Ardeidae species are susceptible to H5 HPAIVs, sometimes with lethal effects. Herons are mostly colonial and often share habitats with Anseriformes , natural hosts of influenza A viruses; therefore, the risks of cluster infection and contribution to viral dissemination should be continuously evaluated. RESEARCH HIGHLIGHTSClade 2.3.2.1 H5N1 HPAIV causes lethal infections in Ardeidae sp.Viruses are transmitted among grey herons.Some herons with HPAIV showed conjunctivitis or neurological symptoms.HPAIV systemically replicated in herons tissues.

The inhibitory effects of 5-aminolevulinic acid phosphate (5-ALA), an important amino acid for energy production in the host, against viral infections were previously reported. Here, the antiviral effects of 5-ALA against classical swine fever virus (CSFV) belonging to the genus Pestivirus in the Flaviviridae family and its possible mechanisms were investigated. CSFV replication was suppressed in swine cells supplemented with 5-ALA or its metabolite, protoporphyrin IX (PPIX). The infectivity titer of CSFV was decreased after mixing with PPIX extracellularly. In addition, the activities of the replication cycle were decreased in the presence of PPIX based on the CSFV replicon assay. These results showed that PPIX exerted antiviral effects by inactivating virus particles and inhibiting the replication cycle. To evaluate the in vivo efficacy of 5-ALA, pigs were supplemented daily with 5-ALA for 1 week before virus inoculation and then inoculated with a virulent CSFV strain at the 10 7.0 50% tissue culture infectious dose. The clinical scores of the supplemented group were significantly lower than those of the nonsupplemented group, whereas the virus growth was not. Taken together, 5-ALA showed antiviral effects against CSFV in vitro, and PPIX played a key role by inactivating virus particles extracellularly and inhibiting the replication cycle intracellularly.

It is well known that influenza viruses utilize host cell glycans for virus attachment factors via their major glycoprotein, hemagglutinin (HA), to initiate their invasion to host cells. Unlike well-known theories in human and avian influenza viruses, barriers laying between interspecies transmission of influenza viruses among bird species are not well understood. Recently, it was speculated that glycan binding of the HA to fucosylated Siaα2-3Gal is related to the expansion in the host range of the virus in avian species. Accordingly, the binding specificity of avian influenza viruses to fucosylated Siaα2-3Gal glycans should be monitored for the better control of avian influenza in both poultry and wild birds. Here, general methods and points for the glycan-binding assay that are specifically modified to target fucosylated Siaα2-3Gal glycans are provided., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recently, structural analyses on the glycans attached to viral surface proteins have been intensively conducted since previous studies demonstrated that glycoform of the viral glycoproteins is closely related to their immunogenicity as vaccine antigens. Although mass spectrometric approach is a gold standard for the glycoproteomic analysis of viral glycoproteins, lectin microarray (LMA) is regarded as an alternative method for analyzing glycan attached to viruses. The previous studies demonstrated that LMA provides highly sensitive and straightforward platforms for the glycoproteomic analyses of viral glycans. Here, two methods, antibody-overlay method, and direct-labeling method, for profiling glycoforms of viral glycoprotein using LMA are described., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Sporadic spreads of swine-origin influenza H3N2 variant (H3N2v) viruses were reported in humans, resulting in 437 human infections between 2011 and 2021 in the USA. Thus, an effective vaccine is needed to better control a potential pandemic for these antigenically distinct viruses from seasonal influenza. In this study, a candidate vaccine strain with efficient growth capacity in chicken embryos was established through serial blind passaging of A/Indiana/08/2011 (H3N2)v in mice and chicken embryos. Seven amino acid substitutions (M21I in PA; A138T, N165K, and V226A in HA; S312L in NP; T167I in M1; G62A in NS1 proteins) were found in the passaged viruses without a major change in the antigenicity. This mouse- and egg-adapted virus was used as a vaccine and challenge strain in mice to evaluate the efficacy of the H3N2v vaccine in different doses. Antibodies with high neutralizing titers were induced in mice immunized with 100 µg of inactivated whole-virus particles, and those mice were significantly protected from the challenge of homologous strain. The findings indicated that the established strain in the study was useful for vaccine study in mouse models.

The GPE - strain is a live attenuated vaccine for classical swine fever (CSF) developed in Japan. In the context of increasing attention for the differentiating infected from vaccinated animals (DIVA) concept, the achievement of CSF eradication with the GPE - proposes it as a preferable backbone for a recombinant CSF marker vaccine. While its infectious cDNA clone, vGPE - , is well characterized, 10 amino acid substitutions were recognized in the genome, compared to the original GPE - vaccine seed. To clarify the GPE - seed availability, this study aimed to generate and characterize a clone possessing the identical amino acid sequence to the GPE - seed. The attempt resulted in the loss of the infectious GPE - seed clone production due to the impaired replication by an amino acid substitution in the viral polymerase NS5B. Accordingly, replication-competent GPE - seed variant clones were produced. Although they were mostly restricted to propagate in the tonsils of pigs, similarly to vGPE - , their type I interferon-inducing capacity was significantly lower than that of vGPE - . Taken together, vGPE - mainly retains ideal properties for the CSF vaccine, compared with the seed variants, and is probably useful in the development of a CSF marker vaccine.

Glycans attached to the viruses regulate their pathogenicity, immunogenicity, and antigenicity. We have previously shown that lectin microarray provided an easy and highly sensitive platform for analyzing glycan profiles of hemagglutinin (HA) of influenza A viruses in culture supernatants. On the other hand, the system is not applicable for neuraminidase (NA), the other viral glycoprotein of influenza A viruses, due to the limited availability of specific antibodies used to detect NA in the lectin microarray. Accordingly, we established replication-competent viruses harboring the short peptide-tag sequence at the C-terminus of NA in this study. The generated viruses underwent normal proliferation cycles and showed similar properties to the wild-type viruses. Lectin microarray analyses of the tagged NA enriched from the viral particles showed that glycan profiles of NA were mostly occupied by mannose-type glycans. Interestingly, the profiles were distinct from those of HA separated from the same particle preparation, in which core-fucosylated complex-type N-glycans terminating with non-sialylated N-acetyllactosamine were dominant. Collectively, this study provides novel platforms for the analyses of the distinction between the glycan profiles of NA and HA, and contributes to a better understanding of later stages of the viral life cycles through analyzing the glycans attached to NA.

The circulation of highly pathogenic avian influenza viruses (HPAIVs) of various subtypes (e.g., H5N1, H5N6, H5N8, and H7N9) in poultry remains a global concern for animal and public health. Migratory waterfowls play important roles in the transmission of these viruses across countries. To monitor virus spread by wild birds, active surveillance for avian influenza in migratory waterfowl was conducted in Mongolia from 2015 to 2019. In total, 5000 fecal samples were collected from lakesides in central Mongolia, and 167 influenza A viruses were isolated. Two H5N3, four H7N3, and two H7N7 viruses were characterized in this study. The amino acid sequence at hemagglutinin (HA) cleavage site of those isolates suggested low pathogenicity in chickens. Phylogenetic analysis revealed that all H5 and H7 viruses were closely related to recent H5 and H7 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds in Asia and Europe. Antigenicity of H7Nx was similar to those of typical non-pathogenic avian influenza viruses (AIVs). While HPAIVs or A/Anhui/1/2013 (H7N9)-related LPAIVs were not detected in migratory waterfowl in Mongolia, sporadic introductions of AIVs including H5 and H7 viruses into Mongolia through the wild bird migration were identified. Thus, continued monitoring of H5 and H7 AIVs in both domestic and wild birds is needed for the early detection of HPAIVs spread into the country.

Avian influenza viruses (AIVs) recognize sialic acid linked α2,3 to galactose (SAα2,3Gal) glycans as receptors. In this study, the interactions between hemagglutinins (HAs) of AIVs and sulfated SAα2,3Gal glycans were analyzed to clarify the molecular basis of interspecies transmission of AIVs from ducks to chickens. It was revealed that E190V and N192D substitutions of the HA increased the recovery of viruses derived from an H6 duck virus isolate, A/duck/Hong Kong/960/1980 (H6N2), in chickens. Recombinant HAs from an H6 chicken virus, A/chicken/Tainan/V156/1999 (H6N1), bound to sulfated SAα2,3Gal glycans, whereas the HAs from an H6 duck virus did not. Binding preference of mutant HAs revealed that an E190V substitution is critical for the recognition of sulfated SAα2,3Gal glycans. These results suggest that the binding of the HA from H6 AIVs to sulfated SAα2,3Gal glycans explains a part of mechanisms of interspecies transmission of AIVs from ducks to chickens., (© 2020 The Societies and John Wiley & Sons Australia, Ltd.)

Recent discoveries of tick-borne pathogens have raised public health concerns on tick-borne infectious diseases and emphasize the need to assess potential risks of unrecognized tick-borne pathogens. First, to determine the existence of tick-borne phleboviruses (TBPVs), genetic surveillance of phleboviruses in ticks was conducted mainly in Hokkaido, the northernmost island in Japan from 2013 to 2015. Genes of two TBPVs, previously reported as Mukawa virus (MKWV) and a newly identified relative of MKWV, Kuriyama virus (KURV), were detected and the viruses were isolated from Ixodes persulcatus collected in Hokkaido, but not in I. persulcatus collected from other areas of Japan. These viruses were phylogenetically and antigenically similar to each other. Next, to investigate the infection of MKWV in mammals, serum samples from wildlife captured in Hokkaido from 2007 to 2011 were used for serological screening. Neutralizing antibodies against MKWV were detected in both Yezo-deer (Cervus nippon yesoensis) (2/50) and raccoons (Procyon lotor) (16/64). However, no infectious MKWV was recovered from laboratory mice in experimental infections, though viral RNAs were detected in their tissues. Thus, MKWV and KURV may maintain tick-mammalian life cycles in Hokkaido, suggesting their potential as causative agents of tick-borne diseases in mammals., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Glycan structures on hemagglutinin (HA) of influenza A viruses have been analyzed previously to understand their significance. However, the formerly established methods using mass spectrometry present disadvantages such as procedure complexity, sensitivity, and throughput. Our study has established a novel method for analyzing glycan profiles of HA using lectin microarray techniques. We successfully obtained glycan profiles of HA starting from 1 ml of the 10 6 TCID 50 samples through simple antigen enrichment using optimized immunoprecipitation. The profiles were reasonably consistent with known glycan structures of HA. Next, we compared glycan profiles of the HAs prepared from chicken embryos, MDCK, Vero, and A549 cells, and demonstrated the host cell-specific HA glycan profiles. Notably, the HA from MDCK cells was α1-3 galactosylated. Our method provides a highly sensitive and simple procedure for glycan profiling of the viral glycoproteins, thereby paving way for direct glycan analyses of human- and animal-derived virions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Among 16 haemagglutinin (HA) subtypes of avian influenza viruses (AIVs), H13 AIVs have rarely been isolated in wild waterfowl. H13 AIVs cause asymptomatic infection and are maintained mainly in gull and tern populations; however, the recorded antigenic information relating to the viruses has been limited. In this study, 2 H13 AIVs, A/duck/Hokkaido/W345/2012 (H13N2) and A/duck/Hokkaido/WZ68/2012 (H13N2), isolated from the same area in the same year in our surveillance, were genetically and antigenically analyzed with 10 representative H13 strains including a prototype strain, A/gull/Maryland/704/1977 (H13N6). The HA genes of H13 AIVs were phylogenetically divided into 3 groups (I, II, and III). A/duck/Hokkaido/W345/2012 (H13N2) was genetically classified into Group III. This virus was distinct from a prototype strain, A/gull/Maryland/704/1977 (H13N6), and the virus, A/duck/Hokkaido/WZ68/2012 (H13N2), both belonging to Group I. Antigenic analysis indicated that the viruses of Group I were antigenically closely related to those of Group II, but distinct from those of Group III, including A/duck/Hokkaido/W345/2012 (H13N2). In summary, our study indicates that H13 AIVs have undergone antigenic diversification in nature.

H2N2 influenza virus caused a pandemic starting in 1957 but has not been detected in humans since 1968. Thus, most people are immunologically naive to viruses of the H2 subtype. In contrast, H2 influenza viruses are continually isolated from wild birds, and H2N3 viruses were isolated from pigs in 2006. H2 influenza viruses could cause a pandemic if re-introduced into humans. In the present study, a vaccine against H2 influenza was prepared as an effective control measure against a future human pandemic. A/duck/Hokkaido/162/2013 (H2N1), which showed broad antigenic cross-reactivity, was selected from the candidate H2 influenza viruses recently isolated from wild birds in Asian countries. Sufficient neutralizing antibodies against homologous and heterologous viruses were induced in mice after two subcutaneous injections of the inactivated whole virus particle vaccine. The inactivated vaccine induced protective immunity sufficient to reduce the impact of challenges with A/swine/Missouri/2124514/2006 (H2N3). This study demonstrates that the inactivated whole virus particle vaccine prepared from an influenza virus library would be useful against a future H2 influenza pandemic.

Vaccination-primed immunity in poultry has been suggested for selection of antigenically drifted highly pathogenic avian influenza viruses (HPAIVs). In this study, we performed two consecutive passage studies of an H5N1 HPAIV in vaccinated chickens, namely, study-I and study-II, to select antigenic variants under immune pressure from the vaccination. In study-I, nine consecutive passages of a wild-type H5N1 HPAIV were carried out in chickens vaccinated with the homologous challenge strain. Antigenically drifted variants with mutations at position 179 in the hemagglutinin (HA) were selected after three passages. Similarly, in study-II, a vaccination-mediated antigenic variant isolated in study-I was used as the vaccine and challenge strain to confirm further antigenic drift after updating the vaccine; after the third passage, additional antigenic variants with a mutation at position 256 in the HA were selected. Thus, our study demonstrated the contribution of vaccination in the selection of antigenic variants of H5 HPAIVs in chickens., (Copyright © 2017 Elsevier Inc. All rights reserved.)

On 15 November 2016, a black swan that had died in a zoo in Akita prefecture, northern Japan, was strongly suspected to have highly pathogenic avian influenza (HPAI); an HPAI virus (HPAIV) belonging to the H5N6 subtype was isolated from specimens taken from the bird. After the initial report, 230 cases of HPAI caused by H5N6 viruses from wild birds, captive birds, and domestic poultry farms were reported throughout the country during the winter season. In the present study, 66 H5N6 HPAIVs isolated from northern Japan were further characterized. Phylogenetic analysis of the hemagglutinin gene showed that the H5N6 viruses isolated in northern Japan clustered into Group C of Clade 2.3.4.4 together with other isolates collected in Japan, Korea and Taiwan during the winter season of 2016-2017. The antigenicity of the Japanese H5N6 isolate differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with that of an H5N8 HPAIV isolate collected in Japan in 2014. Comprehensive analyses of Japanese isolates, including those from central, western, and southern Japan, as well as rapid publication of this information are essential for facilitating greater control of HPAIVs., (© 2017 The Societies and John Wiley & Sons Australia, Ltd.)

Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds.

Classical swine fever (CSF), a highly contagious viral disease affecting domestic and wild pigs in many developing countries, is now considered endemic in Mongolia, with 14 recent outbreaks in 2007, 2008, 2011, 2012, 2014, and 2015. For the first time, CSF viruses isolated from these 14 outbreaks were analyzed to assess their molecular epidemiology and pathogenicity in pigs. Based on the nucleotide sequences of their 5'-untranslated region, isolates were phylogenetically classified as either sub-genotypes 2.1b or 2.2, and the 2014 and 2015 isolates, which were classified as 2.1b, were closely related to isolates from China and Korea. In addition, at least three different viruses classified as 2.1b circulated in Mongolia. Experimental infection of the representative isolate in 2014 demonstrated moderate pathogenicity in 4-week-old pigs, with relatively mild clinical signs. Understanding the diversity of circulating CSF viruses gleans insight into disease dynamics and evolution, and may inform the design of effective CSF control strategies in Mongolia.

Transdermal vaccination using a microneedle (MN) confers enhanced immunity compared with subcutaneous (SC) vaccination. Here we developed a novel dissolving MN patch for the influenza vaccine. The potencies of split virion and whole virus particle (WVP) vaccines prepared from A/Puerto Rico/8/1934 (H1N1) and A/duck/Hokkaido/Vac-3/2007 (H5N1), respectively, were evaluated. MN vaccination induced higher neutralizing antibody responses than SC vaccination in mice. Moreover, MN vaccination with a lower dose of antigens conferred protective immunity against lethal challenges of influenza viruses than SC vaccination in mice. These results suggest that the WVP vaccines administered using MN are an effective combination for influenza vaccine to be further validated in humans., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

H5 highly pathogenic avian influenza viruses (HPAIV) have spread in both poultry and wild birds since late 2003. Continued circulation of HPAIV in poultry in several regions of the world has led to antigenic drift. In the present study, we analyzed the antigenic properties of H5 HPAIV isolated in Asia using four neutralizing mAbs recognizing hemagglutinin, which were established using A/chicken/Kumamoto/1-7/2014 (H5N8), belonging to clade 2.3.4.4 and also using polyclonal antibodies. Viruses of clades 1.1, 2.3.2.1, 2.3.4, and 2.3.4.4 had different reactivity patterns to the panel of mAbs, thereby indicating that the antigenicity of the viruses of clade 2.3.4.4 were similar but differed from the other clades. In particular, the antigenicity of the viruses of clade 2.3.4.4 differed from those of the viruses of clades 2.3.4 and 2.3.2.1, which suggests that the recent H5 HPAIV have further evolved antigenically divergent. In addition, reactivity of antiserum suggests that the antigenicity of viruses of clade 2.3.4.4 differed slightly among groups A, B, and C. Vaccines are still used in poultry in endemic countries, so the antigenicity of H5 HPAIV should be monitored continually to facilitate control of avian influenza. The panel of mAbs established in the present study will be useful for detecting antigenic drift in the H5 viruses that emerge from the current strains., (© 2017 The Societies and John Wiley & Sons Australia, Ltd.)

Highly pathogenic avian influenza viruses (HPAIVs) A(H5N6) were concurrently introduced into several distant regions of Japan in November 2016. These viruses were classified into the genetic clade 2.3.4.4c and were genetically closely related to H5N6 HPAIVs recently isolated in South Korea and China. In addition, these HPAIVs showed further antigenic drift.

Cattle are major reservoirs of the provisionally named influenza D virus, which is potentially involved in the bovine respiratory disease complex. Here, we conducted a serological survey for the influenza D virus in Japan, using archived bovine serum samples collected during 2010-2016 from several herds of apparently healthy cattle in various regions of the country. We found sero-positive cattle across all years and in all the prefectural regions tested, with a total positivity rate of 30.5%, although the positivity rates varied among regions (13.5-50.0%). There was no significant difference in positivity rates for Holstein and Japanese Black cattle. Positivity rates tended to increase with cattle age. The herds were clearly divided into two groups: those with a high positive rate and those with a low (or no) positive rate, indicating that horizontal transmission of the virus occurs readily within a herd. These data demonstrate that bovine influenza D viruses have been in circulation for at least 5 years countrywide, emphasizing its ubiquitous distribution in the cattle population of Japan., Competing Interests: The authors have declared that no competing interests exist.

Here, we report the complete genome sequence of the avian paramyxovirus serotype 5 strain APMV-5/budgerigar/Japan/TI/75, which was determined using the Illumina MiSeq platform. The determined sequence shares 97% homology and similar genetic features with the previously known genome sequence of avian paramyxovirus serotype 5 strain APMV-5/budgerigar/Japan/Kunitachi/74., (Copyright © 2016 Hiono et al.)

The diagnosis of influenza A virus infections in poultry or wild birds is difficult due to variations in the pathogenicity of the viruses in different avian hosts and also the antigenic and genetic diversity of the virus, particularly the recent H5 highly pathogenic avian influenza viruses. A classical standard laboratory technique is virus isolation prior to subtyping and pathotyping. This diagnostic technique is crucial for further virological analyses, particularly during an initial outbreak; however, delays in diagnosis have thwarted effective disease control in recent years. Recent developments in molecular biological techniques provide an accelerated diagnosis. Such technologies, which include real-time reverse transcriptase PCR, isothermal nucleic acid amplification, next-generation sequencing and immunochromatography, contribute to simpler and more rapid diagnosis. The advantages of each of these diagnostic techniques should be considered for effective control of avian influenza., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A total of 3,045 environmental samples and oropharyngeal and cloacal swabs from apparently healthy poultry have been collected at three live bird markets (LBMs) at which practices were applied to reduce avian influenza (AI) virus transmission (intervention LBMs) and six conventional LBMs (non-intervention LBMs) in Thua Thien Hue province in 2014 to evaluate the efficacy of the intervention LBMs. The 178 AI viruses, including H3 (19 viruses), H4 (2), H5 (8), H6 (30), H9 (114), and H11 (5), were isolated from domestic ducks, muscovy ducks, chickens, and the environment. The prevalence of AI viruses in intervention LBMs (6.1%; 95% CI: 5.0-7.5) was similar to that in non-intervention LBMs (5.6%; 95% CI: 4.5-6.8; χ(2)=0.532; df=1; P=0.53) in the study area. Eight H5N6 highly pathogenic avian influenza (HPAI) viruses were isolated from apparently healthy ducks, muscovy ducks, and an environmental sample in an intervention LBM. The hemagglutinin genes of the H5N6 HPAI viruses belonged to the genetic clade 2.3.4.4, and the antigenicity of the H5N6 HPAI viruses differed from the H5N1 HPAI viruses previously circulating in Vietnam. Phylogenetic and antigenic analyses of the H6 and H9 viruses isolated in both types of LBMs revealed that they were closely related to the viruses isolated from domestic birds in China, Group II of H6 viruses and Y280 lineage of H9 viruses. These results indicate that the interventions currently applied in LBMs are insufficient to control AI. A risk analysis should be conducted to identify the key factors contributing to AI virus prevalence in intervention LBMs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Influenza viruses isolated from wild ducks do not replicate in chickens. This fact is not explained solely by the receptor specificity of the hemagglutinin (HA) from such viruses for target host cells. To investigate this restriction in host range, the fusion activities of HA molecules from duck and chicken influenza viruses were examined. Influenza viruses A/duck/Mongolia/54/2001 (H5N2) (Dk/MNG) and A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR), which replicate only in their primary hosts, were used. The optimal pH for membrane fusion of Ck/IBR was 5.9, higher than that of Dk/MNG at 4.9. To assess the relationship between the optimal pH for fusion and the host range of avian influenza viruses, the optimal pH for fusion of 55 influenza virus strains isolated from ducks and chickens was examined. No correlation was found between the host range and optimal pH for membrane fusion by the viruses, and this finding applied also to the H5N1 highly pathogenic avian influenza viruses. The optimal pH for membrane fusion for avian influenza viruses was shown to not necessarily be correlated with their host range or pathogenicity in ducks and chickens.

Eye spray influenza vaccines for chickens are increasingly available; however, how to enhance cellular and antibody responses to them remains undetermined. Here, eye-drops containing the immune-enhancing adjuvants Pam2CSK4 or polyI:C were assessed in chickens. Application of these TLR agonists to chicken conjunctiva resulted in up-regulation of IL-1β, but not other cytokines, including IFN and IL-6, in the spleen, lung and Harderian gland. Thus, responses to adjuvant applied to the conjunctival mucosa of chickens differ from those expected from the responses to intra-nasal adjuvants in mammals. Identifying an appropriate delivery route for adjuvants is crucial for evoking immune responses in chickens., (© 2016 The Societies and John Wiley & Sons Australia, Ltd.)

Influenza viruses isolated from ducks are rarely able to infect chickens; it is therefore postulated that these viruses need to adapt in some way to be able to be transmitted to chickens in nature. Previous studies revealed that sialyl Lewis X (3'SLeX), which is fucosylated α2,3 sialoside, was predominantly detected on the epithelial cells of the chicken trachea, whereas this glycan structure is not found in the duck intestinal tract. To clarify the mechanisms of the interspecies transmission of influenza viruses between ducks and chickens, we compared the receptor specificity of low-pathogenic avian influenza viruses isolated from these two species. Glycan-binding analysis of the recombinant hemagglutinin (HA) of a chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2), revealed a binding preference to α1,3 fucosylated sialosides. On the other hand, the HA of a duck influenza virus, A/duck/Mongolia/54/2001 (H5N2) (Dk/MNG), particularly bound to non-fucosylated α2,3 sialosides such as 3'-sialyllactosamine (3'SLacNAc). Computational analysis along with binding analysis of the mutant HAs revealed that this glycan-binding specificity of the HA was determined by amino acid residues at positions 222 and 227. Inconsistent with the glycan-binding specificity of the recombinant HA protein, virions of Dk/MNG bound to both 3'SLacNAc and 3'SLeX. Glycan-binding analysis in the presence of a neuraminidase (NA) inhibitor revealed that the NA conferred binding to 3'SLeX to virions of Dk/MNG. The present results reveal the molecular basis of the interaction between fucosylated α2,3 sialosides and influenza viruses.

Highly pathogenic avian influenza viruses (HPAIVs) have spread in both poultry and wild birds. Determining transmission routes of these viruses during an outbreak is essential for the control of avian influenza. It has been widely postulated that migratory ducks play crucial roles in the widespread dissemination of HPAIVs in poultry by carrying viruses along with their migrations; however close contacts between wild migratory ducks and poultry are less likely in modern industrial poultry farming settings. Therefore, we conducted experimental infections of HPAIVs and low pathogenic avian influenza viruses (LPAIVs) to chickens, domestic ducks, tree sparrows, jungle crows, and black rats to evaluate their roles in virus transmission. The results showed that chickens, ducks, sparrows, and crows were highly susceptible to HPAIV infection. Significant titers of virus were recovered from the sparrows and crows infected with HPAIVs, which suggests that they potentially play roles of transmission of HPAIVs to poultry. In contrast, the growth of LPAIVs was limited in each of the animals tested compared with that of HPAIVs. The present results indicate that these common synanthropes play some roles in influenza virus transmission from wild birds to poultry., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Migratory water birds are the natural reservoir of influenza A viruses. H5 and H7 influenza viruses are isolated over the world and also circulate among poultry in Asia. In 2010, two H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from fecal samples of water birds on the flyway of migration from Siberia, Russia to the south in Hokkaido, Japan. H7N9 viruses are sporadically isolated from humans and circulate in poultry in China. To monitor whether these viruses have spread in the wild bird population, we conducted virological surveillance of avian influenza in migratory water birds in autumn from 2010 to 2014. A total of 8103 fecal samples from migratory water birds were collected in Japan and Mongolia, and 350 influenza viruses including 13 H5 and 19 H7 influenza viruses were isolated. A phylogenetic analysis revealed that all isolates are genetically closely related to viruses circulating among wild water birds. The results of the antigenic analysis indicated that the antigenicity of viruses in wild water birds is highly stable despite their nucleotide sequence diversity but is distinct from that of HPAIVs recently isolated in Asia. The present results suggest that HPAIVs and Chinese H7N9 viruses were not predominantly circulating in migratory water birds; however, continued monitoring of H5 and H7 influenza viruses both in domestic and wild birds is recommended for the control of avian influenza.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.

H7N9 influenza virus infection in humans was reported in China on March 31, 2013. Humans are immunologically naïve to the H7N9 subtype, for which the seasonal influenza vaccine is not effective. Thus, the development of an H7N9 influenza virus vaccine is an urgent issue. To prepare for the emergence of an influenza pandemic, we have established a library comprising more than 1300 influenza virus strains with 144 different combinations of 16 HA and 9 NA subtypes. An H7N9 virus strain isolated from a 35-year-old woman, A/Anhui/1/2013 (H7N9), was found to be antigenically similar to H7N9 influenza viruses isolated from migratory ducks. In the present study, the potency of an inactivated whole virus particle vaccine prepared from an H7N9 low pathogenic avian influenza virus, A/duck/Mongolia/119/2008 (H7N9), selected from the library, was assessed by a challenge with A/Anhui/1/2013 (H7N9). The results indicate that the test vaccine was potent enough to induce sufficient immunity to reduce the impact of disease caused by the challenge with A/Anhui/1/2013 (H7N9) in mice. The present results indicate that an inactivated whole virus particle vaccine prepared from an influenza virus strain stored in the library could be useful as a vaccine strain in case of an influenza pandemic., (Copyright © 2014. Published by Elsevier Ltd.)