Genetic diversity of H5N1 and H5N2 high pathogenicity avian influenza viruses isolated from poultry in Japan during the winter of 2022-2023.

High pathogenicity avian influenza viruses (HPAIVs) of the H5N1 and H5N2 subtypes were responsible for 84 HPAI outbreaks on poultry premises in Japan during October 2022-April 2023. The number of outbreaks during the winter of 2022-2023 is the largest ever reported in Japan. In this study, we performed phylogenetic analyses using the full genetic sequences of HPAIVs isolated in Japan during 2022-2023 and those obtained from a public database to identify their genetic origin. Based on the hemagglutinin genes, these HPAIVs were classified into the G2 group of clade 2.3.4.4b, whose ancestors were H5 HPAIVs that circulated in Europe in late 2020, and were then further divided into three subgroups (G2b, G2d, and G2c). Approximately one-third of these viruses were classified into the G2b and G2d groups, which also included H5N1 HPAIVs detected in Japan during 2021-2022. In contrast, the remaining two-thirds were classified into the G2c group, which originated from H5N1 HPAIVs isolated in Asian countries and Russia during the winter of 2021-2022. Unlike the G2b and G2d viruses, the G2c viruses were first detected in Japan in the fall of 2022. Importantly, G2c viruses caused the largest number of outbreaks throughout Japan over the longest period during the season. Phylogenetic analyses using eight segment genes revealed that G2b, G2d, and G2c viruses were divided into 2, 4, and 11 genotypes, respectively, because they have various internal genes closely related to those of avian influenza viruses detected in wild birds in recent years in Asia, Russia, and North America, respectively. These results suggest that HPAIVs were disseminated among migratory birds, which may have generated numerous reassortant viruses with various gene constellations, resulting in a considerable number of outbreaks during the winter of 2022-2023.

Sequence Analysis of the Malaysian Low Pathogenic Avian Influenza Virus Strain H5N2 from Duck.

The avian influenza viruses (AIV) of the H5 subtype have the ability to mutate from low pathogenic (LPAI) to highly pathogenic (HPAI), which can cause high mortality in poultry. Little is known about the pathogenic switching apart from the mutations at the haemagglutinin cleavage site, which significantly contributes to the virus virulence switching phenomenon. Therefore, this study aimed to compare the molecular markers in the haemagglutinin (HA), neuraminidase (NA), and matrix (M) genes of a locally isolated LPAI AIV strain H5N2 from Malaysia with the reference HPAI strains using bioinformatics approaches, emphasising the pathogenic properties of the viral genes. First, the H5N2 strain A/Duck/Malaysia/8443/2004 was propagated in SPF eggs. The viral presence was verified by haemagglutination assay, RT-PCR, and sequencing. Results showed successful amplifications of HA (1695 bp), NA (1410 bp), and M (1019 bp) genes. The genes were sequenced and the deduced amino acid sequences were analysed computationally using MEGA 11 and NetNGlyc software. Analysis of the HA protein showed the absence of the polybasic cleavage motif, but presence of two amino acid residues that are known to affect pathogenicity. There were also two glycosylation sites (glycosites) compared to the reference HPAI viruses, which had three or more at the HA globular head domain. No NA stalk deletion was detected but the haemadsorbing and active centres of the studied NA protein were relatively similar to the reference HPAI H5N2 isolates of duck but not chicken origins. Six NA glycosites were also identified. Finally, we observed a consistent M1 and M2 amino acid sequences between our LPAI isolate with the other HPAI H5N1 or H5N2 reference proteins. These data demonstrate distinct characteristics of the Malaysian LPAI H5N2, compared to HPAI H5N2 or H5N1 from ducks or chickens, potentially aiding the epidemiological research on genetic dynamics of circulating AIV in poultry.

Evolutionary Dynamics of Mexican Lineage H5N2 Avian Influenza Viruses.

We have demonstrated for the first time a comprehensive evolutionary analysis of the Mexican lineage H5N2 avian influenza virus (AIV) using complete genome sequences (n = 189), from its first isolation in 1993 until 2019. Our study showed that the Mexican lineage H5N2 AIV originated from the North American wild bird gene pool viruses around 1990 and is currently circulating in poultry populations of Mexico, the Dominican Republic, and Taiwan. Since the implementation of vaccination in 1995, the highly pathogenic AIV (HPAIV) H5N2 virus was eradicated from Mexican poultry in mid-1995. However, the low pathogenic AIV (LPAIV) H5N2 virus has continued to circulate in domestic poultry populations in Mexico, eventually evolving into five distinct clades. In the current study, we demonstrate that the evolution of Mexican lineage H5N2 AIVs involves gene reassortments and mutations gained over time. The current circulating Mexican lineage H5N2 AIVs are classified as LPAIV based on the amino acid sequences of the hemagglutinin (HA) protein cleavage site motif as well as the results of the intravenous pathogenicity index (IVPI). The immune pressure from vaccinations most likely has played a significant role in the positive selection of antigenic drift mutants within the Mexican H5N2 AIVs. Most of the identified substitutions in these viruses are located on the critical antigenic residues of the HA protein and as a result, might have contributed to vaccine failures. This study highlights and stresses the need for vaccine updates while emphasizing the importance of continued molecular monitoring of the HA protein for its antigenic changes compared to the vaccines used.

Phylogenetic analysis, molecular changes, and adaptation to chickens of Mexican lineage H5N2 low-pathogenic avian influenza viruses from 1994 to 2019.

The Mexican lineage H5N2 low pathogenic avian influenza viruses (LPAIVs) were first detected in 1994 and mutated to highly pathogenic avian influenza viruses (HPAIVs) in 1994-1995 causing widespread outbreaks in poultry. By using vaccination and other control measures, the HPAIVs were eradicated but the LPAIVs continued circulating in Mexico and spread to several other countries. To get better resolution of the phylogenetics of this virus, the full genome sequences of 44 H5N2 LPAIVs isolated from 1994 to 2011, and 6 detected in 2017 and 2019, were analysed. Phylogenetic incongruence demonstrated genetic reassortment between two separate groups of the Mexican lineage H5N2 viruses between 2005 and 2010. Moreover, the recent H5N2 viruses reassorted with previously unidentified avian influenza viruses. Bayesian phylogeographic results suggested that mechanical transmission involving human activity is the most probable cause of the virus spillover to Central American, Caribbean, and East Asian countries. Increased infectivity and transmission of a 2011 H5N2 LPAIV in chickens compared to a 1994 virus demonstrates improved adaptation to chickens, while low virus shedding, and limited contact transmission was observed in mallards with the same 2011 virus. The sporadic increase in basic amino acids in the HA cleavage site, changes in potential N-glycosylation sites in the HA, and truncations of PB1-F2 should be further examined in relation to the increased infectivity and transmission in poultry. The genetic changes that occur as this lineage of H5N2 LPAIVs continues circulating in poultry is concerning not only because of the effect of these changes on vaccination efficacy, but also because of the potential of the viruses to mutate to the highly pathogenic form. Continued vigilance and surveillance efforts, and the pathogenic and genetic characterization of circulating viruses, are required for the effective control of this virus.

Phylogenetic and phenotypic characterization of two novel clade 2.3.2.1 H5N2 subtype avian influenza viruses from chickens in China.

The extended co-circulation of H5 subtype highly pathogenic avian influenza (HPAI) viruses and H9N2 low pathogenic avian influenza (LPAI) viruses has inevitably facilitated gene reassortment between the two subtypes in fields. And, novel reassortant H5NX viruses harboring partial or even whole sets of H9N2 internal genes have continuously been detected, such as clade 2.3.4.4 H5N2 or H5N6 reassortants. Here, we report two novel H5N2 subtype HPAI isolates of HF9 and QY5 from chickens in live poultry markets during routine surveillance in 2018. Phylogenetic analysis showed that those two H5N2 strains both possessed the HA genes from clade 2.3.2.1e of H5N1 viruses but all the other seven gene segments consistently from the endemic S genotype of H9N2 subtype viruses. Further analysis revealed that HF9 and QY5 differed only in six sites including K353R, A588T and T661I in PB2, I682V and L704S in PB1 plus G631S in PA at the amino acid level. A chicken regression experiment confirmed that both HF9 and QY5 were lethal infection to all tested chickens via contact transmission. Moreover, those two isolates could immediately replicate in mice lungs without adaptation. However, mortality rate of those two variants were distinct in mice model, HF9 with 100% but QY5 with just 20% at the infection dosage of 106.0EID50 per mouse. We suppose that the phenotypic difference may probably be attributed to the amino acid substitutions in the polymerase genes between the two isolates that constitute of a subject of further ongoing research.

Genetic Characterization and Pathogenesis of Three Novel Reassortant H5N2 Viruses in South Korea, 2018.

The outbreaks of H5N2 avian influenza viruses have occasionally caused the death of thousands of birds in poultry farms. Surveillance during the 2018 winter season in South Korea revealed three H5N2 isolates in feces samples collected from wild birds (KNU18-28: A/Wild duck/South Korea/KNU18-28/2018, KNU18-86: A/Bean Goose/South Korea/KNU18-86/2018, and KNU18-93: A/Wild duck/South Korea/KNU18-93/2018). Phylogenetic tree analysis revealed that these viruses arose from reassortment events among various virus subtypes circulating in South Korea and other countries in the East Asia-Australasian Flyway. The NS gene of the KNU18-28 and KNU18-86 isolates was closely related to that of China's H10N3 strain, whereas the KNU18-93 strain originated from the H12N2 strain in Japan, showing two different reassortment events and different from a low pathogenic H5N3 (KNU18-91) virus which was isolated at the same day and same place with KNU18-86 and KNU18-93. These H5N2 isolates were characterized as low pathogenic avian influenza viruses. However, many amino acid changes in eight gene segments were identified to enhance polymerase activity and increase adaptation and virulence in mice and mammals. Experiments reveal that viral replication in MDCK cells was quite high after 12 hpi, showing the ability to replicate in mouse lungs. The hematoxylin and eosin-stained (H&E) lung sections indicated different degrees of pathogenicity of the three H5N2 isolates in mice compared with that of the control H1N1 strain. The continuing circulation of these H5N2 viruses may represent a potential threat to mammals and humans. Our findings highlight the need for intensive surveillance of avian influenza virus circulation in South Korea to prevent the risks posed by these reassortment viruses to animal and public health.

Evolutionary Dynamics of H5 Highly Pathogenic Avian Influenza Viruses (Clade 2.3.4.4B) Circulating in Bulgaria in 2019-2021.

The first detection of a Highly Pathogenic Avian Influenza (HPAI) H5N8 virus in Bulgaria dates back to December 2016. Since then, many outbreaks caused by HPAI H5 viruses from clade 2.3.4.4B have been reported in both domestic and wild birds in different regions of the country. In this study, we characterized the complete genome of sixteen H5 viruses collected in Bulgaria between 2019 and 2021. Phylogenetic analyses revealed a persistent circulation of the H5N8 strain for four consecutive years (December 2016-June 2020) and the emergence in 2020 of a novel reassortant H5N2 subtype, likely in a duck farm. Estimation of the time to the most recent common ancestor indicates that this reassortment event may have occurred between May 2019 and January 2020. At the beginning of 2021, Bulgaria experienced a new virus introduction in the poultry sector, namely a HPAI H5N8 that had been circulating in Europe since October 2020. The periodical identification in domestic birds of H5 viruses related to the 2016 epidemic as well as a reassortant strain might indicate undetected circulation of the virus in resident wild birds or in the poultry sector. To avoid the concealed circulation and evolution of viruses, and the risk of emergence of strains with pandemic potential, the implementation of control measures is of utmost importance, particularly in duck farms where birds display no clinical signs.

Rapid Emergence of the Reassortant 2.3.4.4b H5N2 Highly Pathogenic Avian Influenza Viruses in a Live Poultry Market in Xinjiang, Northwest China.

Live poultry markets (LPMs) play a key role in reassorting and spreading avian influenza viruses (AIVs). In 2018, four strains of H5N2 AIVs were isolated from domestic ducks (Anas platyrhynchos) during AIV surveillance from the LPM in Urumqi, Xinjiang, China. All gene segments of the isolates were amplified by reverse transcription-PCR and sequenced; then, the viral genetic mutations, reassortant, and origin were analyzed. Higher nucleotide identities were observed among each gene of the isolates, indicating a common ancestor. The hemagglutinin (HA) genes of the isolates all classified into the clade 2.3.4.4b; the HA, matrix protein (MP), and nonstructural protein (NS) genes were all clustered together with the local H5N6 highly pathogenic AIVs (HPAIVs) identified in the same LPM of Urumqi in July 2017; the neuraminidase albumen, polymerase basic proteins 1 and 2, polymerase acidic protein, and nucleocapsid protein genes (NA, PB1, PB2, PA, and NP) all had close phylogenetic relationships with the local H9N2 AIVs identified in the same LPM from September to October 2018. Multiple basic amino acids were present at the cleavage site of the HA protein, which was associated with HPAIVs. These results indicated that the reassortant clade 2.3.4.4b H5N2 HPAIVs were rapidly generated from reassortment between the H5N6 and H9N2 AIVs in the local LPM of Urumqi in 2018.

Rápida aparición de los virus de influenza aviar altamente patógenos H5N2 reacomodados 2.3.4.4b en un mercado de aves vivas en Xinjiang, en el noroeste de China. Los mercados de aves vivas desempeñan un papel clave en el reacomodo y en la propagación de los virus de la influenza aviar. En el año 2018, se aislaron cuatro cepas del virus de influenza aviar H5N2 de patos domésticos durante los procedimientos de vigilancia para influenza aviar en mercados de aves vivas en Urumqi, Xinjiang, China. Todos los segmentos de genes de los aislados se amplificaron mediante transcripción reversa y PCR y se secuenciaron; posteriormente, se analizaron las mutaciones genéticas virales, el reacomodamiento y el origen. Se observaron altas identidades de nucleótidos entre cada gene de los aislados, lo que indica un ancestro común. Todos los genes de hemaglutinina (HA) de los aislamientos se clasificaron en el clado 2.3.4.4b; los genes de la proteína HA, la proteína de matriz (MP) y la proteína no estructural (NS) se agruparon junto con los virus de influenza altamente patógenos locales H5N6 identificados en el mismo mercado de aves vivas de Urumqi en julio de 2017; la albúmina de la neuraminidasa, las proteínas básicas de la polimerasa 1 y 2, la proteína ácida de la polimerasa y los genes de la proteína de la nucleocápsida (NA, PB1, PB2, PA y NP) tenían relaciones filogenéticas cercanas con virus de influenza locales H9N2 identificados en el mismo mercado de aves vivas de septiembre a octubre del 2018. Hubo múltiples aminoácidos básicos presentes en el sitio de disociación de la proteína HA, que se asoció con virus de influenza de alta patogenicidad. Estos resultados indicaron que los virus de influenza de alta patogenicidad H5N2 del clado reacomodado 2.3.4.4b se generaron rápidamente a partir del reacomodo entre los virus de influenza H5N6 y H9N2 en el mercado de aves vivas local de Urumqi en el año 2018.

Low Pathogenicity Avian Influenza (H5N2) Viruses, Dominican Republic.

Low pathogenicity avian influenza (H5N2) virus was detected in poultry in the Dominican Republic in 2007 and re-emerged in 2017. Whole-genome sequencing and phylogenetic analysis show introduction of an H5N2 virus lineage from Mexico into poultry in the Dominican Republic, then divergence into 3 distinct genetic subgroups during 2007-2019.

Recombinant hemagglutinin glycoproteins provide insight into binding to host cells by H5 influenza viruses in wild and domestic birds.

Clade 2.3.4.4, H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have caused devastating effects across wild and domestic bird populations. We investigated differences in the intensity and distribution of the hemagglutinin (HA) glycoprotein binding of a clade 2.3.4.4 H5 HPAIV compared to a H5 low pathogenic avian influenza virus (LPAIV). Recombinant HA from gene sequences from a HPAIV, A/Northern pintail/Washington/40964/2014(H5N2) and a LPAIV, A/mallard/MN/410/2000(H5N2) were generated and, via protein histochemistry, HA binding in respiratory, intestinal and cloacal bursal tissue was quantified as median area of binding (MAB). Poultry species, shorebirds, ducks and terrestrial birds were used. Differences in MAB were observed between the HPAIV and LPAIV H5 HAs. We demonstrate that clade 2.3.4.4 HPAIV H5 HA has a broader host cell binding across a variety of bird species compared to the LPAIV H5 HA. These findings support published results from experimental trials, and outcomes of natural disease outbreaks with these viruses.

FluConvert and IniFlu: a suite of integrated software to identify novel signatures of emerging influenza viruses with increasing risk.

BACKGROUND: The pandemic threat of influenza has attracted great attention worldwide. To assist public health decision-makers, new suites of tools are needed to rapidly process and combine viral information retrieved from public-domain databases for a better risk assessment. RESULTS: Using our recently developed FluConvert and IniFlu software, we automatically processed and rearranged sequence data by standard viral nomenclature, determined the group-related consensus sequences, and identified group-specific polygenic signatures. The software possesses powerful ability to integrate viral, clinical, and epidemiological data. We demonstrated that both multiple basic amino acids at the cleavage site of the HA gene and also at least 11 more evidence-based viral amino acid substitutions present in global highly pathogenic avian influenza H5N2 viruses during the years 2009-2016 that are associated with viral virulence and human infection. CONCLUSIONS: FluConvert and IniFlu are useful to monitor and assess all subtypes of influenza viruses with pandemic potential. These programs are implemented through command-line and user-friendly graphical interfaces, and identify molecular signatures with virological, epidemiological and clinical significance. FluConvert and IniFlu are available at https://apps.flutures.com or https://github.com/chinrur/FluConvert_IniFlu.

Eco-Epidemiological Evidence of the Transmission of Avian and Human Influenza A Viruses in Wild Pigs in Campeche, Mexico.

Influenza, a zoonosis caused by various influenza A virus subtypes, affects a wide range of species, including humans. Pig cells express both sialyl-α-2,3-Gal and sialyl-α-2,6-Gal receptors, which make them susceptible to infection by avian and human viruses, respectively. To date, it is not known whether wild pigs in Mexico are affected by influenza virus subtypes, nor whether this would make them a potential risk of influenza transmission to humans. In this work, 61 hogs from two municipalities in Campeche, Mexico, were sampled. Hemagglutination inhibition assays were performed in 61 serum samples, and positive results were found for human H1N1 (11.47%), swine H1N1 (8.19%), and avian H5N2 (1.63%) virus variants. qRT-PCR assays were performed on the nasal swab, tracheal, and lung samples, and 19.67% of all hogs were positive to these assays. An avian H5N2 virus, first reported in 1994, was identified by sequencing. Our results demonstrate that wild pigs are participating in the exposure, transmission, maintenance, and possible diversification of influenza viruses in fragmented habitats, highlighting the synanthropic behavior of this species, which has been poorly studied in Mexico.

Genotyping and reassortment analysis of highly pathogenic avian influenza viruses H5N8 and H5N2 from Egypt reveals successive annual replacement of genotypes.

Highly pathogenic (HP) H5N1, clade 2.2.1, and low pathogenic avian influenza (LPAI) H9N2 viruses, G1-B lineage, are endemic in poultry in Egypt and have co-circulated for almost a decade. Surprisingly, no inter-subtypic reassortment events have been reported from the field during that time. After the introduction of HPAIV H5N8, clade 2.3.4.4b, in Egyptian poultry in 2016, suddenly HP H5N2 reassortants with H9N2 viruses emerged. The current analyses focussed on studying 32 duck flocks, 4 broiler chicken flocks, and 1 turkey flock, suffering from respiratory manifestations with moderate to high mortality reared in two Egyptian governorates during 2019. Real-time RT-PCR substantiated the presence of HP H5N8 in 21 of the 37 investigated flocks with mixed infection of H9N2 in two of them. HP H5N1 was not detected. Full hemagglutinin (HA) sequencing of 10 samples with full-genome sequencing of three of them revealed presence of a single genotype. Very few substituting mutations in the HA protein were detected versus previous Egyptian HA sequences of that clade. Interestingly, amino acid substitutions in the Matrix (M2) and the Neuraminidase (NA) proteins associated with conferring both Amantadine and Oseltamivir resistance were present. Systematic reassortment analysis of all publicly available Egyptian whole genome sequences of HP H5N8 (n = 23), reassortant HP H5N2 (n = 2) and LP H9N2 (n = 53) viruses revealed presence of at least seven different genotypes of HPAI H5Nx viruses of clade 2.3.4.4b in Egypt since 2016. For H9N2 viruses, at least three genotypes were distinguishable. Heat mapping and tanglegram analyses suggested that several internal gene segments in both HP H5Nx and H9N2 viruses originated from avian influenza viruses circulating in wild bird species in Egypt. Based on the limited set of whole genome sequences available, annual replacement patterns of HP H5Nx genotypes emerged and suggested selective advantages of certain genotypes since 2016.

The Impacts of Reassortant Avian Influenza H5N2 Virus NS1 Proteins on Viral Compatibility and Regulation of Immune Responses.

Avian influenza virus (AIV) can cause severe diseases in poultry worldwide. H6N1 AIV was the dominant enzootic subtype in 1985 in the chicken farms of Taiwan until the initial outbreak of a low pathogenic avian influenza (LPAI) H5N2 virus in 2003; thereafter, this and other LPAIs have been sporadically detected. In 2015, the outbreak of three novel H5Nx viruses of highly pathogenic avian influenza (HPAI) emerged and devastated Taiwanese chicken and waterfowl industries. The mechanism of variation in pathogenicity among these viruses is unclear; but, in light of the many biological functions of viral non-structural protein 1 (NS1), including interferon (IFN) antagonist and host range determinant, we hypothesized that NS genetic diversity contributes to AIV pathogenesis. To determine the impact of NS1 variants on viral infection dynamics, we established a reverse genetics system with the genetic backbone of the enzootic Taiwanese H6N1 for generation of reassortant AIVs carrying exogenous NS segments of three different Taiwanese H5N2 strains. We observed distinct cellular distributions of NS1 among the reassortant viruses. Moreover, exchange of the NS segment significantly influenced growth kinetics and induction of cytokines [IFN-α, IFN-ß, and tumor necrosis factor alpha (TNF-α)] in an NS1- and host-specific manner. The impact of NS1 variants on viral replication appears related to their synergic effects on viral RNA-dependent RNA polymerase activity and IFN response. With these approaches, we revealed that NS1 is a key factor responsible for the diverse characteristics of AIVs in Taiwan.

Protection of White Leghorn chickens by recombinant fowlpox vector vaccine with an updated H5 insert against Mexican H5N2 avian influenza viruses.

Despite decades of vaccination, surveillance, and biosecurity measures, H5N2 low pathogenicity avian influenza (LPAI) virus infections continue in Mexico and neighboring countries. One explanation for tenacity of H5N2 LPAI in Mexico is the antigenic divergence of circulating field viruses compared to licensed vaccines due to antigenic drift. Our phylogenetic analysis indicates that the H5N2 LPAI viruses circulating in Mexico and neighboring countries since 1994 have undergone antigenic drift away from vaccine seed strains. Here we evaluated the efficacy of a new recombinant fowlpox virus vector containing an updated H5 insert (rFPV-H5/2016), more relevant to the current strains circulating in Mexico. We tested the vaccine efficacy against a closely related subcluster 4 Mexican H5N2 LPAI (2010 H5/LP) virus and the historic H5N2 HPAI (1995 H5/HP) virus in White Leghorn chickens. The rFPV-H5/2016 vaccine provided hemagglutinin inhibition (HI) titers pre-challenge against viral antigens from both challenge viruses in almost 100% of the immunized birds, with no differences in number of birds seroconverting or HI titers among all tested doses (1.5, 2.0, and 3.1 log10 mean tissue culture infectious doses/bird). The vaccine conferred 100% clinical protection and a significant decrease in oral and cloacal virus shedding from 1995 H5/HP virus challenged birds when compared to the sham controls at all tested doses. Virus shedding titers from vaccinated 2010 H5/LP virus challenged birds significantly decreased compared to sham birds especially at earlier time points. Our results confirm the efficacy of the new rFPV-H5/2016 against antigenic drift of LPAI virus in Mexico and suggest that this vaccine would be a good candidate, likely as a primer in a prime-boost vaccination program.

Novel Reassortant Highly Pathogenic Avian Influenza A(H5N2) Virus in Broiler Chickens, Egypt.

We detected a novel reassortant highly pathogenic avian influenza A(H5N2) virus in 3 poultry farms in Egypt. The virus carried genome segments of a pigeon H9N2 influenza virus detected in 2014, a nucleoprotein segment of contemporary chicken H9N2 viruses from Egypt, and hemagglutinin derived from the 2.3.4.4b H5N8 virus clade.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 107.6 TCID50/mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 µg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.

Isolation of a Novel Reassortant Highly Pathogenic Avian Influenza (H5N2) Virus in Egypt.

Highly pathogenic avian influenza (HPAI) H5N1 and H5N8 have become endemic among domestic poultry in Egypt since 2006 and 2016, respectively. In parallel, the low pathogenic avian influenza H9N2 virus has been endemic since 2010. Despite the continuous circulation of these subtypes for several years, no natural reassortant has been detected so far among the domestic poultry population in Egypt. In this study, the HPAI (H5N2) virus was isolated from a commercial duck farm, giving evidence of the emergence of the first natural reassortment event in domestic poultry in Egypt. The virus was derived as a result of genetic reassortment between avian influenza viruses of H5N8 and H9N2 subtypes circulating in Egypt. The exchange of the neuraminidase segment and high number of acquired mutations might be associated with an alteration in the biological propensities of this virus.

Age-dependent pathogenesis of clade 2.3.4.4A H5N2 HPAIV in experimentally infected Broad Breasted White turkeys.

Highly pathogenic avian influenza (HPAI) is a viral disease with devastating consequences to the poultry industry as it results in high morbidity, mortality and international trade restrictions. In the present study, we characterized age-related differences in terms of pathology in commercial white broad breasted turkeys inoculated with A/turkey/Minnesota/12582/2015 (H5N2) HPAIV clade 2.3.4.4A, a virus from the largest HPAI poultry outbreak that affected the Unites States in 2014-2015. Turkeys infected at 6-weeks of age showed inapparent to little clinical signs with rapid disease progression, reaching 100% mortality at 3 days post infection (dpi). In contrast, turkeys infected at 16-weeks of age developed ataxia and lethargy and reached 100% mortality by 5 dpi. Infection in the 6-weeks old turkeys resulted in peracute lesions consistent of extensive hemorrhages, edema and necrosis, but inflammation was not prominent. In the 16-weeks old turkeys, necrosis and hemorrhages in tissues were accompanied by a more prominent subacute inflammatory infiltrate. Both age groups showed presence of avian influenza virus (AIV) nucleoprotein (NP) in multiple cell types including neurons, glial cells, ependymal cells, respiratory epithelial cells, air capillary epithelium and pulmonary macrophages, cardiac myocytes, smooth muscle fibers, pancreatic acini and ductal cells. Cells of the vascular walls stained strongly positive for viral antigens, but no positivity was found in the endothelial cells of any organs. These findings indicate that age is a determinant factor in the progression of the disease and delay of mortality during infection with the H5N2 clade 2.3.4.4A HPAI virus in naïve white broad breasted turkeys.

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.