Long-Term Protective Effect of Serial Infections with H5N8 Highly Pathogenic Avian Influenza Virus in Wild Ducks.

Highly pathogenic avian influenza viruses (HPAIVs) of the Goose/Guangdong (Gs/Gd) lineage are an emerging threat to wild birds. In the 2016-2017 H5N8 outbreak, unexplained variability was observed in susceptible species, with some reports of infected birds dying in high numbers and other reports of apparently subclinical infections. This experimental study was devised to test the hypothesis that previous infection with a less-virulent HPAIV (i.e., 2014 H5N8) provides long-term immunity against subsequent infection with a more-virulent HPAIV (i.e., 2016 H5N8). Therefore, two species of wild ducks-the more-susceptible tufted duck (Aythya fuligula) and the more-resistant mallard (Anas platyrhynchos)-were serially inoculated, first with 2014 H5N8 and after 9 months with 2016 H5N8. For both species, a control group of birds was first sham inoculated and after 9 months inoculated with 2016 H5N8. Subsequent infection with the more-virulent 2016 H5N8 caused no clinical signs in tufted ducks that had previously been infected with 2014 H5N8 (n = 6) but caused one death in tufted ducks that had been sham inoculated (n = 7). In mallards, 2016 H5N8 infection caused significant body weight loss in previously sham-inoculated birds (n = 8) but not in previously infected birds (n = 7). IMPORTANCE This study showed that ducks infected with a less-virulent HPAIV developed immunity that was protective against a subsequent infection with a more-virulent HPAIV 9 months later. Following 2014 H5N8 infection, the proportion of birds with detectable influenza nucleoprotein antibody declined from 100% (8/8) in tufted ducks and 78% (7/9) in mallards after 1 month to 33% (2/6) in tufted ducks and 29% (2/7) in mallards after 9 months. This finding helps predict the expected impact that an HPAIV outbreak may have on wild bird populations, depending on whether they are immunologically naive or have survived previous infection with HPAIV.

The C-terminus of non-structural protein 1 (NS1) in H5N8 clade 2.3.4.4 avian influenza virus affects virus fitness in human cells and virulence in mice.

Avian influenza viruses (AIV) H5N8 clade 2.3.4.4 pose a public health threat but the viral factors relevant for its potential adaptation to mammals are largely unknown. The non-structural protein 1 (NS1) of influenza viruses is an essential interferon antagonist. It commonly consists of 230 amino acids, but variations in the disordered C-terminus resulted in truncation or extension of NS1 with a possible impact on virus fitness in mammals. Here, we analysed NS1 sequences from 1902 to 2020 representing human influenza viruses (hIAV) as well as AIV in birds, humans and other mammals and with an emphasis on the panzootic AIV subtype H5N8 clade 2.3.4.4A (H5N8-A) from 2013 to 2015 and clade 2.3.4.4B (H5N8-B) since 2016. We found a high degree of prevalence for short NS1 sequences among hIAV, zoonotic AIV and H5N8-B, while AIV and H5N8-A had longer NS1 sequences. We assessed the fitness of recombinant H5N8-A and H5N8-B viruses carrying NS1 proteins with different lengths in human cells and in mice. H5N8-B with a short NS1, similar to hIAV or AIV from a human or other mammal-origins, was more efficient at blocking apoptosis and interferon-induction without a significant impact on virus replication in human cells. In mice, shortening of the NS1 of H5N8-A increased virus virulence, while the extension of NS1 of H5N8-B reduced virus virulence and replication. Taken together, we have described the biological impact of variation in the NS1 C-terminus in hIAV and AIV and shown that this affects virus fitness in vitro and in vivo.

Infectivity and pathobiology of H7N1 and H5N8 high pathogenicity avian influenza viruses for pigeons (Columba livia var. domestica).

Avian influenza (AI) is one of the most important viral diseases in poultry, wildlife and humans. Available data indicate that pigeons play a minimum role in the epidemiology of AI. However, a degree of variation exists in the susceptibility of pigeons to highly pathogenic AI viruses (HPAIVs), especially since the emergence of the goose/Guangdong H5 lineage. Here, the pathogenesis of H5N8 HPAIV in comparison with a H7N1 HPAIV and the role of pigeons in the epidemiology of these viruses were evaluated. Local and urban pigeons (Columba livia var. domestica) were intranasally inoculated with 105 ELD50 of A/goose/Spain/IA17CR02699/2017 (H5N8) or A/Chicken/Italy/5093/1999 (H7N1) and monitored during 14 days. Several pigeons inoculated with H5N8 or H7N1 seroconverted. However, clinical signs, mortality, microscopic lesions and viral antigen were only detected in a local pigeon inoculated with H5N8 HPAIV. This pigeon presented prostration and neurological signs that correlated with the presence of large areas of necrosis and widespread AIV antigen in the central nervous system, indicating that the fatal outcome was associated with neurological dysfunction. Viral RNA in swabs was detected in some pigeons inoculated with H7N1 and H5N8, but it was inconsistent, short-term and at low titres. The present study demonstrates that the majority of pigeons were resistant to H5N8 and H7N1 HPAIVs, despite several pigeons developing asymptomatic infections. The limited viral shedding indicates a minimum role of pigeons as amplifiers of HPAIVs, regardless of the viral lineage, and suggests that this species may represent a low risk for environmental contamination. RESEARCH HIGHLIGHTS H7N1 and H5N8 HPAIVs can produce subclinical infections in pigeons. The mortality caused by H5N8 HPAIV in one pigeon was associated with neurological dysfunction. Pigeons represent a low risk for environmental contamination by HPAIVs.

Genetic and antigenic characterization of H5 and H7 avian influenza viruses isolated from migratory waterfowl in Mongolia from 2017 to 2019.

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.

Outbreak Severity of Highly Pathogenic Avian Influenza A(H5N8) Viruses Is Inversely Correlated to Polymerase Complex Activity and Interferon Induction.

Highly pathogenic avian influenza A(H5N8) viruses first emerged in China in 2010 and in 2014 spread throughout Asia and to Europe and the United States via migrating birds. Influenza A(H5N8) viruses were first detected in the Netherlands in 2014 and caused five outbreaks in poultry farms but were infrequently detected in wild birds. In 2016, influenza A(H5N8) viruses were reintroduced into the Netherlands, resulting in eight poultry farm outbreaks. This outbreak resulted in numerous dead wild birds with severe pathology. Phylogenetic analysis showed that the polymerase genes of these viruses had undergone extensive reassortment between outbreaks. Here, we investigated the differences in virulence between the 2014-15 and the 2016-17 outbreaks by characterizing the polymerase complex of influenza A(H5N8) viruses from both outbreaks. We found that viruses from the 2014-15 outbreak had significantly higher polymerase complex activity in both human and avian cell lines than did those from the 2016-17 outbreak. No apparent differences in the balance between transcription and replication of the viral genome were observed. Interestingly, the 2014-15 polymerase complexes induced significantly higher levels of interferon beta (IFN-ß) than the polymerase complexes of the 2016-17 outbreak viruses, mediated via retinoic acid-inducible gene I (RIG-I). Inoculation of primary duck cells with recombinant influenza A(H5N8) viruses, including viruses with reassorted polymerase complexes, showed that the polymerase complexes from the 2014-15 outbreak induced higher levels of IFN-ß despite relatively minor differences in replication capacity. Together, these data suggest that despite the lower levels of polymerase activity, the higher 2016-17 influenza A(H5N8) virus virulence may be attributed to the lower level of activation of the innate immune system.IMPORTANCE Compared to the 2014-15 outbreak, the 2016-17 outbreak of influenza A(H5N8) viruses in the Netherlands and Europe was more virulent; the number of dead or diseased wild birds found and the severity of pathological changes were higher during the 2016-17 outbreak. The polymerase complex plays an important role in influenza virus virulence, and the gene segments of influenza A(H5N8) viruses reassorted extensively between the outbreaks. In this study, the 2014-15 polymerase complexes were found to be more active, which is counterintuitive with the observed higher virulence of the 2016-17 outbreak viruses. Interestingly, the 2014-15 polymerase complexes also induced higher levels of IFN-ß. These findings suggest that the higher virulence of influenza A(H5N8) viruses from the 2016-17 outbreak may be related to the lower induction of IFN-ß. An attenuated interferon response could lead to increased dissemination, pathology, and mortality, as observed in (wild) birds infected during the 2016-2017 outbreak.

Transmission dynamics between infected waterfowl and terrestrial poultry: Differences between the transmission and tropism of H5N8 highly pathogenic avian influenza virus (clade 2.3.4.4a) among ducks, chickens and turkeys.

H5N8 highly-pathogenic avian influenza viruses (HPAIVs, clade 2.3.4.4) have spread globally via migratory waterfowl. Pekin ducks infected with a UK virus (H5N8-2014) served as the donors of infection in three separate cohousing experiments to attempt onward transmission chains to sequentially introduced groups of contact ducks, chickens and turkeys. Efficient transmission occurred among ducks and turkeys up to the third contact stage, with all (100%) birds becoming infected. Introduction of an additional fourth contact group of ducks to the turkey transmission chain demonstrated retention of H5N8-2014's waterfowl-competent adaptation. However, onward transmission ceased in chickens at the second contact stage where only 13% became infected. Analysis of viral progeny at this contact stage revealed no emergent polymorphisms in the intra-species (duck) transmission chain, but both terrestrial species included changes in the polymerase and accessory genes. Typical HPAIV pathogenesis and mortality occurred in infected chickens and turkeys, contrasting with 5% mortality among ducks.

Amino acid substitutions in antigenic region B of hemagglutinin play a critical role in the antigenic drift of subclade 2.3.4.4 highly pathogenic H5NX influenza viruses.

As one of the important control strategies for highly pathogenic avian influenza (HPAI) in China, vaccination has been implemented compulsively in poultry flocks since 2004. However, the emergence and dominance of the circulating antigenic variants require the update of vaccines periodically. In order to investigate the key molecular sites responsible for the antigenic drift, a total of 13 amino acid positions divergent between clade 2.3.4 H5 viruses and their descendent subclade 2.3.4.4 variants in or around the recognized antigenic epitopes A-E were initially identified through inspecting a comprehensive HA sequence alignment of the H5 subtype HPAI viruses. Subsequently, a panel of single-site or multi-site HA mutants was constructed by reverse genetics with two H5N1 viruses of S (clade 2.3.4) and QD1 (subclade 2.3.4.4) as the HA backbone to study their antigenic variations, respectively. The hemagglutination-inhibition assay revealed an evident impact of mutations at sites 88, 156, 205, 208, 239 and 289 to the HA antigenicity and highlighted that the amino acid substitutions located in the antigenic region B, especially the combined mutations at sites 205 and 208, were the major antigenic determinant which was also consistent with results from flow cytometry and antigenic mapping. Our findings provided more insights into the molecular mechanism of antigenic drift of the H5 subtype HPAI virus, which would be helpful for the selection of vaccine candidates and accordingly for the prevention and control of this devastating viral agent.

No evidence of avian influenza antibodies in two species of raptor nestlings inhabiting Norway.

BACKGROUND: Since 2016, incursions of highly pathogenic avian influenza virus (HPAIV) H5N8 clade 2.3.4.4b have caused unprecedented clinical signs and mortality in white-tailed eagles (WTE; Haliaeetus albicilla) across Europe and have been found to be infecting other raptor species, such as the northern goshawk (NG; Accipiter gentilis). Before this study, no screening of Norwegian raptors had been undertaken. RESULTS: Plasma samples from 43 white-tailed eagle and 29 northern goshawk nestlings, from several locations across Norway were screened for antibodies to avian influenza viruses. No antibodies, and thus, no evidence of AIV exposure, were found in these Norwegian raptors. No clinical signs of AIV were observed in 43 white tailed eagles and 29 northern goshawks. CONCLUSIONS: There are currently no indications that white-tailed eagles and northern goshawks inhabiting Norway are threatened by the recent HPAIV outbreaks in other areas of Europe. Ongoing monitoring should, however, be maintained to detect potential future outbreaks.

Bioengineering a highly productive vaccine strain in embryonated chicken eggs and mammals from a non-pathogenic clade 2·3·4·4 H5N8 strain.

The clade 2·3·4·4 H5Nx is a highly pathogenic avian influenza (HPAI) virus, which first appeared in China and has spread worldwide since then, including Korea. It is divided into subclades a - d, but the PR8-derived recombinant clade 2·3·4·4 a viruses replicate inefficiently in embryonated chicken eggs (ECEs). High virus titer in ECEs and no mammalian pathogenicity are the most important prerequisites of efficacious and safer vaccine strains against HPAI. In this study, we have synthesized hemagglutinin (HA) and neuraminidase (NA) genes based on the consensus amino acid sequences of the clade 2·3·4·4a and b H5N8 HPAIVs, using the GISAID database. We generated PR8-derived H5N8 recombinant viruses with single point mutations in HA and NA, which are related to efficient replication in ECEs. The H103Y mutation in HA increased mammalian pathogenicity as well as virus titer in ECEs, by 10-fold. We also successfully eradicated mammalian pathogenicity in H103Y-bearing H5N8 recombinant virus by exchanging PB2 genes of PR8 and 01310 (Korean H9N2 vaccine strain). The final optimized H5N8 vaccine strain completely protected against a heterologous clade 2·3·4·4c H5N6 HPAIV in chickens, and induced hemagglutination inhibition (HI) antibody in ducks. However, the antibody titer of ducks showed age-dependent results. Thus, H103Y and 01310PB2 gene have been successfully applied to generate a highly productive, safe, and efficacious clade 2·3·4·4 H5N8 vaccine strain in ECEs.

Development of an effective contemporary trivalent avian influenza vaccine against circulating H5N1, H5N8, and H9N2 in Egypt.

Low pathogenicity avian influenza (LPAI) H9N2, highly pathogenic avian influenza (HPAI) H5N1, and H5N8 circulate in Egyptian poultry and cause veterinary and public health burdens. In response, AIV vaccines are commonly used. The main objective of this study was to develop a broad, cross-protective, trivalent vaccine based on circulating AIVs in Egypt. We generated highly replicating avirulent AIVs, H5N1, and H5N8, to be used in combination with H9N2 strain for the generation of an inactivated vaccine. Immunogenicity and protective efficacy of this vaccine were tested. Results showed that a single immunization dose enhanced humoral immune responses giving full protection against challenges with LPAI H9N2, HPAI H5N1, and H5N8 viruses. This efficacious vaccine will reduce the cost of vaccination for poultry growers and is expected to be effective in the field as it is based on contemporary viruses currently in circulation among Egyptian poultry.

Shedding of clade 2.3.4.4 H5N8 and H5N2 highly pathogenic avian influenza viruses in peridomestic wild birds in the U.S.

European starlings (Sturnus vulgaris), house sparrows (Passer domesticus) and rock pigeons (Columba livia) are all wild birds commonly found in large numbers in and around human dwellings and domestic livestock operations. This study evaluated the susceptibility of these species to three strains of highly pathogenic avian influenza virus (HP AIV) clade 2.3.4.4 isolated in the U.S.. Experimental infection of European starlings and rock pigeons did not result in any overt signs attributable to AIV infection and no virus shedding was detected from the oral and cloacal routes. House sparrows shed by the oral route and exhibited limited mortality. Individuals from all three species seroconverted following infection. These data suggest that none of these birds are a likely potential bridge host for future HP AIV outbreaks but that their seroconversion may be a useful surveillance tool for detection of circulating H5 HP AIV.

Co-infections, genetic, and antigenic relatedness of avian influenza H5N8 and H5N1 viruses in domestic and wild birds in Egypt.

A total of 50 poultry farms of commercial broilers (N = 39) and commercial layers (N = 11) suffered from respiratory problems and mortality during the period from January 2016 to December 2017 were investigated. Also, samples were collected from quail (N = 4), Bluebird (Sialis, N = 1), and Greenfinch (Chloris chloris, N = 1) for analysis. Respiratory viral pathogens were screened by PCR and positive samples were subjected to virus isolation and genetic identification. Antigenic relatedness of isolated avian influenza (AI) H5 subtype was evaluated using cross-hemagglutination inhibition. Results revealed that the incidence of single virus infections in commercial broilers was 64.1% (25/39), with the highest incidence for ND (33.3%) and H9N2 (20.5%), followed by H5N1 (7.7%) and H5N8 (2.7). Meanwhile, H9N2/ND mixed infection was the most observed case (7.7%). Other mixed infections H5N1/ND, H5N1/H9N2/ND, H5N1/H9N2/ND/IB, H9N2/IB, and H9N2/ILT were also observed (2.6% each). In commercial layers, H5N1 and ILT were the only detected single infections (18.1% each). Mixed H9N2/ND was the most predominant infection in layers (27.3%). Other mixed infections of H9N2/IB, H5N1/H5N8/H9N2, and H9N2/ND/IB were observed in 3 separate farms (9.1% each). The H5N8 virus was detected in one quail farm and 2 out of 3 wild bird's samples. Partial HA gene sequence analysis showed the clustering of the selected AI H5N8 within the 2.3.4.4 clade, while H5N1 clustered with the clade 2.2.1.2. Interestingly, the H5N8 isolated from chickens possessed 6 amino acids substitutions at HA1 compared to those isolated from wild birds with low antigenic relatedness to AI H5N1 clades 2.2.1 or 2.2.1.2. In conclusion, mixed viral infections were observed in both broiler and layer chickens in Egypt. The detected triple H5N1, H9N2, and H5N8 influenza co-infection raises the concern of potential AI epidemic strain emergence. The low genetic and antigenic relatedness between AI H5N1 and H5N8 viruses suggest the need for modification of vaccination strategies of avian influenza in Egypt along with strict biosecurity measures.

Development and comparison of two H5N8 influenza A vaccine candidate strains.

Avian influenza viruses circulating in birds have caused outbreaks of infection in poultry and humans, thereby threatening public health. Recently, a highly pathogenic avian influenza (HPAI) virus (H5N8) of clade 2.3.4.4 emerged in Korea and other countries and caused multiple outbreaks in domestic and wild birds, with concerns for human infection. To combat HPAI viral infections, novel vaccines are likely to be the most effective approach. Therefore, in this study, we generated H5N8 vaccine candidate viruses based on a Korean isolate (A/broiler duck/Korea/Buan2/2014). The vaccine candidate viruses were 2:6 reassortants expressing the two surface glycoproteins of A/broiler duck/Korea/Buan2/2014 on an A/Puerto Rico/8/34 (PR8) backbone generated by using an eight-plasmid-based reverse genetics system with or without replacement of the multi-basic amino acid cleavage motif (MBCM, a crucial pathogenic factor in HPAI virus) with a bi-basic amino acid cleavage motif (BBCM) in their HA. An H5N8 vaccine candidate virus containing the BBCM showed attenuated pathogenesis in embryonated eggs and exhibited less virulence in the infected mice compared with the wild H5N8 virus containing an MBCM. Vaccination with an inactivated preparation of the vaccine candidate virus protected mice from lethal H5N8 viral challenge. This is the first report of the development and evaluation of H5N8 vaccine strains (with an MBCM or BBCM) of HA clade 2.3.4.4 as vaccine candidates. Our findings suggest that H5N8 strains with a BBCM instead of an MBCM might be considered for H5N8 vaccine seed virus development or as a reference vaccine against H5N8 viral strains.

Preclinical evaluation of the efficacy of an H5N8 vaccine candidate (IDCDC-RG43A) in mouse and ferret models for pandemic preparedness.

Because H5N1 influenza viruses continuously threaten the public health, the WHO has prepared various clades of H5N1 mock-up vaccines as one of the measures for pandemic preparedness. The recent worldwide outbreak of H5Nx virus which belongs to clade 2.3.4.4 and of which H5N6 subtype belongs and already caused human infection also increases the need of pandemic vaccine for such novel emerging viruses. In this study, we evaluated the protective efficacy and immunogenicity of an egg-based and inactivated whole-virus H5N8 (IDCDC-RG43A) developed by CDC containing HA and NA gene of the parent virus A/gyrfalcon/Washington/41088-6/2014. Mice vaccinated two times elicited low to moderate antibody titer in varying amount of antigen doses against the homologous H5N8 vaccine virus and heterologous intra-clade 2.3.4.4 H5N6 (A/Sichuan/26221/2014) virus. Mice immunized with at least 3.0 µg/dose of IDCDC-RG43A with aluminum hydroxide adjuvant were completely protected from lethal challenge with the mouse-adapted H5N8 (A/Environment/Korea/ma468/2015, maH5N8) as well as cleared the viral replication in tissues including lung, brain, spleen, and kidney. Vaccinated ferrets induced high antibody titers against clade 2.3.4.4 H5N8/H5N6 viruses and the antibody showed high cross-reactivity to clade 2.2 H5N1 but not to clade 1 and 2.3.4 viruses as measured by hemagglutinin inhibition and serum neutralization assays. Furthermore, administration of the vaccine in ferrets resulted in attenuation of clinical disease signs and virus spread to peripheral organs including lung, spleen, and kidney from high dose challenge with maH5N8 virus. The protective and immunogenic characteristic of the candidate vaccine are essential attributes to be considered for further clinical trials as a pre-pandemic vaccine for a potential pandemic virus.

Humoral immunity to influenza in an at-risk population and severe influenza cases in Russia in 2016-2017.

This work aimed to analyze the herd immunity to influenza among a Russian population living in regions with an increased risk of emergence of viruses with pandemic potential, and to isolate and investigate virus strains from severe influenza cases, including fatal cases, during the 2016-2017 epidemic season. In November 2016 - March 2017 highly pathogenic influenza outbreaks were registered in Russia among wild birds and poultry. No cases of human infection were registered. Analysis of 760 sera from people who had contact with infected or perished birds revealed the presence of antibodies to A(H5N1) virus of clade 2.3.2.1c and A(H5N8) virus of clade 2.3.4.4. The 2016-2017 influenza epidemic season in Russia began in weeks 46-47 of 2016 with predominant circulation of influenza A(H3N2) viruses. Strains isolated from severe influenza cases mainly belonged to 3C.2a.2 and 3C.2a.3 genetic groups. Up to the 8th week of 2017 severe influenza cases were often caused by influenza B viruses which belonged to 1A genetic group with antigenic properties similar to B/Brisbane/60/2008. All influenza A and B virus strains isolated in the 2016-2017 epidemic season were sensitive to oseltamivir and zanamivir.

Characterization of highly pathogenic avian influenza H5N8 virus from Egyptian domestic waterfowl in 2017.

In 2016, the highly pathogenic avian influenza (HPAI) H5N8 virus was detected in wild birds for the first time in Egypt. In the present study, we identified the HPAI virus H5N8 of clade 2.3.4.4 from domestic waterfowl in Egypt, suggesting its transmission to the domestic poultry from the migratory birds. Based on partial haemagglutinin gene sequence, this virus has a close genetic relationship with subtype H5N8 viruses circulating in Asia and Europe. Pathologically, H5N8 virus in hybrid duck induced nervous signs accompanied by encephalomalacia, haemorrhages, nonsuppurative encephalitis and nonsuppurative vasculitis. The granular layer of cerebellum showed multifocal areas of hydropic degeneration and the Purkinje cell neurons were necrotized or lost. Additionally, the lung, kidney and spleen were congested, and necrotizing pancreatitis was also observed. The co-circulation of both HPAI H5N1 and H5N8 subtypes with the low pathogenic avian influenza H9N2 subtype complicate the control of avian influenza in Egypt with the possibility of emergence of new reassortant viruses. Therefore, continuous monitoring with implementation of strict control measures is required. Research highlights HPAI H5N8 virus clade 2.3.4.4 was detected in domestic ducks and geese in Egypt in 2017. Phylogenetically, the virus was closely related to HPAI H5N8 viruses identified in Asia and Europe Nonsuppurative encephalitis was widely observed in HPAI H5N8 virus-infected ducks. Degeneration of the cerebellar granular layer was found in most of the brain tissues examined.

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections.

Serological evidence of H5-subtype influenza A virus infection in indigenous avian and mammalian species in Korea.

In Korea, H5-subtype highly pathogenic avian influenza (HPAI) has caused huge economic losses in poultry farms through outbreaks of H5N1 since 2003, H5N8 since 2013 and H5N6 since 2016. Although it was reported that long-distance migratory birds may play a major role in the global spread of avian influenza viruses (AIVs), transmission from such birds to poultry has not been confirmed. Intermediate hosts in the wild also may be a potential factor in viral transmission. Therefore, a total of 367 serum samples from wild animals were collected near major migratory bird habitats from 2011 to 2016 and tested by AIV-specific blocking ELISA and hemagglutination inhibition (HI) test. Two mammalian and eight avian species were seropositive according to the ELISA test. Among these, two mammalian (Hydropotes inermis and Prionailurus bengalensis) and three avian (Aegypius monachus, Cygnus cygnus, and Bubo bubo) species showed high HI titres (> 1,280) against one or two H5-subtype AIVs. As H. inermis (water deer), P. bengalensis (leopard cat), and B. bubo (Eurasian eagle owl) are indigenous animals in Korea, evidence of H5-subtype AIV in these animals implies that continuous monitoring of indigenous animals should be followed to understand interspecies transmission ecology of H5-subtype influenza viruses.

Experimental infection of highly pathogenic avian influenza viruses, Clade 2.3.4.4 H5N6 and H5N8, in Mandarin ducks from South Korea.

Outbreaks of highly pathogenic avian influenza (HPAI) have been reported worldwide. Wild waterfowl play a major role in the maintenance and transmission of HPAI. Highly pathogenic avian influenza subtype H5N6 and H5N8 viruses simultaneously emerged in South Korea. In this study, the comparative pathogenicity and infectivity of Clade 2.3.4.4 Group B H5N8 and Group C H5N6 viruses were evaluated in Mandarin duck (Aix galericulata). None of the ducks infected with H5N6 or H5N8 viruses showed clinical signs or mortality. Serological assays revealed that the HA antigenicity of H5N8 and H5N6 viruses was similar to each other. Moreover, both the viruses did not replicate after cross-challenging with H5N8 and H5N6 viruses, respectively, as the second infection. Although both the viruses replicated in most of the internal organs of the ducks, viral replication and shedding through cloaca were higher in H5N8-infected ducks than in H5N6-infected ducks. The findings of this study provide preliminary information to help estimate the risks involved in further evolution and dissemination of Clade 2.3.4.4 HPAI viruses among wild birds.

Intracellular delivery of HA1 subunit antigen through attenuated Salmonella Gallinarum act as a bivalent vaccine against fowl typhoid and low pathogenic H5N3 virus.

Introduction of novel inactivated oil-emulsion vaccines against different strains of prevailing and emerging low pathogenic avian influenza (LPAI) viruses is not an economically viable option for poultry. Engineering attenuated Salmonella Gallinarum (S. Gallinarum) vaccine delivering H5 LPAI antigens can be employed as a bivalent vaccine against fowl typhoid and LPAI viruses, while still offering economic viability and sero-surveillance capacity. In this study, we developed a JOL1814 bivalent vaccine candidate against LPAI virus infection and fowl typhoid by engineering the attenuated S. Gallinarum to deliver the globular head (HA1) domain of hemagglutinin protein from H5 LPAI virus through pMMP65 constitutive expression plasmid. The important feature of the developed JOL1814 was the delivery of the HA1 antigen to cytosol of peritoneal macrophages. Immunization of chickens with JOL1814 produced significant level of humoral, mucosal, cellular and IL-2, IL-4, IL-17 and IFN-γ cytokine immune response against H5 HA1 and S. Gallinarum antigens in the immunized chickens. Post-challenge, only the JOL1814 immunized chicken showed significantly faster clearance of H5N3 virus in oropharyngeal and cloacal swabs, and 90% survival rate against lethal challenge with a wild type S. Gallinarum. Furthermore, the JOL1814 immunized were differentiated from the H5N3 LPAI virus infected chickens by matrix (M2) gene-specific real-time PCR. In conclusion, the data from the present showed that the JOL1814 can be an effective bivalent vaccine candidate against H5N3 LPAI and fowl typhoid infection in poultry while still offering sero-surveillance property against H5 avian influenza virus.