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A free-range organic broiler ( Gallus gallus domesticus ) premises in Staffordshire was infected by high pathogenicity avian influenza virus (HPAIV) H5N8 during the 2020-2021 epizootic in the United Kingdom (UK). Following initial confirmation of the infection in poultry, multiple wild bird species were seen scavenging on chicken carcasses. Detected dead wild birds were subsequently demonstrated to have been infected and succumbed to HPAIV H5N8. Initially, scavenging species, magpie ( Pica pica ) and raven ( Corvus corax ) were found dead on the premises but over the following days, buzzards ( Buteo buteo ) were also found dead within the local area with positive detection of HPAIV in submitted carcasses. The subacute nature of microscopic lesions within a buzzard was consistent with the timeframe of infection. Finally, a considerable number of free-living pheasants ( Phasianus colchicus ) were also found dead in the surrounding area, with carcasses having higher viral antigen loads compared to infected chickens. Limited virus dissemination was observed in the carcasses of the magpie, raven, and buzzard. Further, an avirulent avian paramyxovirus type 1 (APMV-1) was detected within poultry samples as well as in the viscera of a magpie infected with HPAIV. Immunohistochemistry did not reveal colocalization of avian paramyxovirus antigens with lesions, supporting an avirulent APMV-1 infection. Overall, this case highlights scenarios in which bi-directional transmission of avian viral diseases between commercial and wild bird species may occur. It also underlines the importance of bio separation and reduced access when infection pressure from HPAIV is high.

Since its first isolation from migratory birds in Egypt in 2016, highly pathogenic avian influenza (HPAI) H5N8 has caused several outbreaks among domestic poultry in various areas of the country affecting poultry health and production systems. However, the genetic and biological properties of the H5N8 HPAI viruses have not been fully elucidated yet. In this study, we aimed to monitor the evolution of circulating H5N8 viruses and identify the pathogenicity and mammalian adaptation in vitro and in vivo. Three H5N8 HPAI viruses were used in this study and were isolated in 2021-2022 from poultry and wild birds during our routine surveillance. RNA extracts were subjected to full genome sequencing. Genetic, phylogenetic, and antigenic analyses were performed to assess viral characteristics and similarities to previously isolated viruses. Phylogenetic analysis showed that the hemagglutinin genes of the three isolates belonged to clade 2.3.4.4b and grouped with the 2019 viruses from G3 with high similarity to Russian and European lineages. Multiple basic amino acids were observed at cleavage sites in the hemagglutinin proteins of the H5N8 isolates, indicating high pathogenicity. In addition, several mutations associated with increased virulence and polymerase activity in mammals were observed. Growth kinetics assays showed that the H5N8 isolate is capable of replicating efficiently in mammalian cells lines. In vivo studies were conducted in SPF chickens (White Leghorn), mice, and hamsters to compare the virological characteristics of the 2022 H5N8 isolates with previous H5N8 viruses isolated in 2016 from the first introduction. The H5N8 viruses caused lethal infection in all tested chickens and transmitted by direct contact. However, we showed that the 2016 H5N8 virus causes a higher mortality in chickens compared to 2022 H5N8 virus. Moreover, the 2022 virus can replicate efficiently in hamsters and mice without preadaptation causing systemic infection. These findings underscore the need for continued surveillance of H5 viruses to identify circulating strains, determine the commercial vaccine's effectiveness, and identify zoonotic potential., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Moatasim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Avian influenza (AI) is an extremely contagious viral disease of domestic and wild birds that can spread rapidly among bird populations, inducing serious economic losses in the poultry industry. During the winter season 2021-2022, we isolated seventeen highly pathogenic avian influenza (HPAI) H5N8 viruses from outbreaks involving ducks in Egypt, occurring in both backyard and farm settings. The aim of this study was to pinpoint genetic key substitutions (KSs) that could heighten the risk of a human pandemic by influencing the virus's virulence, replication ability, host specificity, susceptibility to drugs, or transmissibility. To understand their evolution, origin, and potential risks for a human pandemic, whole-genome sequencing and phylogenetic analysis were conducted. Our analysis identified numerous distinctive mutations in the Egyptian H5N8 viruses, suggesting potential enhancements in virulence, resistance to antiviral drugs, and facilitation of transmission in mammals. In this study, at least five genotypes within one genome constellation of H5N8 viruses were identified, raising concerns about the potential emergence of novel viruses with altered characteristics through reassortment between different genotypes and distinct groups. These findings underscore the role of ducks in the virus's evolutionary process and emphasize the urgent need for enhanced biosecurity measures in domestic duck farms to mitigate pandemic risk.

Avian Alpha-influenza-virus (AIV) massively affects poultry, targeting mainly the respiratory tract for virus replication. Recently, two major H5N8 and H5N1 outbreaks caused tremendous losses in Algerian poultry. The clinical symptoms that had not been seen in the past didn't prompt a rapid reaction to control the epidemics. We report here the characteristics of these outbreaks and the epidemiological status of AIV in Algeria. Following autopsy observation samples from target organs were taken and analyzed by the classical real-time reverse transcription polymerase chain reaction (RRT-PCR). Specific PCR HA and NA identification was used for subtyping H5 and N1/N8 genes. Systemic damage was observed in the upper-respiratory tracts with hemorrhagic and congestive tracheas, lungs, proventriculus, gut, and cecal tonsils were bloody. Out of 77 positive cases 13 were H5N8, 8 H5N1, and 10 H5Nx strains. These findings raise questions about the strain's pathotype considering severe organ damage and high mortality., 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 © 2024 Elsevier Ltd. All rights reserved.)

In the 2021/22 winter, one H5N1 and nine H5N8 high pathogenicity avian influenza viruses (HPAIVs) of clade 2.3.3.4b were isolated from the water in crane roosts on the Izumi plain, Japan. Additionally, we isolated low pathogenicity avian influenza viruses (LPAIVs) of five subtypes: H1N1, H4N2, H4N6, H7N7, and H10N4. H5N8 HPAIVs belonging to the G2a group were isolated throughout winter, whereas H5N1 HPAIV belonging to the G2b group were isolated only in early winter. These findings suggest co-circulation of both G2a and G2b HPAIVs in early winter. Although two H7N7 LPAIVs were isolated from cranes' roost water collected on the same day, the gene constellations of the two isolates were clearly different, indicating the contemporary invasion of at least two different genotypes of H7N7 LPAIVs in the Izumi plain. This study underscores the importance of monitoring both HPAIVs and LPAIVs to understand avian influenza virus ecology in migratory waterfowl populations., 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 © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The recent concurrent emergence of H5N1, H5N6, and H5N8 avian influenza viruses (AIVs) has led to significant avian mortality globally. Since 2020, frequent human-animal interactions have been documented. To gain insight into the novel H5 subtype AIVs (i.e., H5N1, H5N6 and H5N8), we collected 6102 samples from various regions of China between January 2021 and September 2022, and identified 41 H5Nx strains. Comparative analyses on the evolution and biological properties of these isolates were conducted. Phylogenetic analysis revealed that the 41 H5Nx strains belonged to clade 2.3.4.4b, with 13 related to H5N1, 19 to H5N6, and 9 to H5N8. Analysis based on global 2.3.4.4b viruses showed that all the viruses described in this study were likely originated from H5N8, exhibiting a heterogeneous evolutionary history between H5N1 and H5N6 during 2015-2022 worldwide. H5N1 showed a higher rate of evolution in 2021-2022 and more sites under positive selection pressure in 2015-2022. The antigenic profiles of the novel H5N1 and H5N6 exhibited notable variations. Further hemagglutination inhibition assay suggested that some A(H5N1) viruses may be antigenically distinct from the circulating H5N6 and H5N8 strains. Mammalian challenge assays demonstrated that the H5N8 virus (21GD001_H5N8) displayed the highest pathogenicity in mice, followed by the H5N1 virus (B1557_H5N1) and then the H5N6 virus (220086_H5N6), suggesting a heterogeneous virulence profile of H5 AIVs in the mammalian hosts. Based on the above results, we speculate that A(H5N1) viruses have a higher risk of emergence in the future. Collectively, these findings unveil a new landscape of different evolutionary history and biological characteristics of novel H5 AIVs in clade 2.3.4.4b, contributing to a better understanding of designing more effective strategies for the prevention and control of novel H5 AIVs., Competing Interests: Conflict of interest All authors declare that there are no competing interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Highly pathogenic influenza A(H5N8) viruses have caused several worldwide outbreaks in birds and are able cross the species barrier to infect humans, posing a substantial threat to public health. After the first detection of H5N8 viruses in deceased swans in Inner Mongolia, we performed early warning and active monitoring along swan migration routes in central China. We isolated and sequenced 42 avian influenza viruses, including 40 H5N8 viruses, 1 H5N2 virus, and 1 H9N2 virus, in central China. Our H5N8 viruses isolated in swan stopover sites and wintering grounds showed high nucleotide homologies in the whole genome, revealing a common evolutionary source. Phylogenetic analysis revealed that the H5 viruses of clade 2.3.4.4b prevalent in 2020 have further diverged into two sub-clades: b1 and b2. The phylogeographic analysis also showed that the viruses of sub-clade b2 most likely originated from poultry in Russia. Notably, whooper swans were found to be responsible for the introduction of sub-clade b2 viruses in central China; whooper and tundra swans play a role in viral spread in the Yellow River Basin and the Yangtze River Basin, respectively. Our findings highlight swans as an indicator species for transborder spreading and monitoring of the H5N8 virus.

Highly pathogenic avian influenza (HPAI) outbreaks have become increasingly frequent in wild bird populations and have caused mass mortality in many wild bird species. The 2020/2021 epizootic was the largest and most deadly ever reported in Europe, and many new bird species tested positive for HPAI virus for the first time. This study investigated the tropism of HPAI virus in wild birds. We tested the pattern of virus attachment of 2020 H5N8 virus to intestinal and respiratory tissues of key bird species; and characterized pathology of naturally infected Eurasian wigeons ( Mareca penelope ) and barnacle geese ( Branta leucopsis ). This study determined that 2020 H5N8 virus had a high level of attachment to the intestinal epithelium (enterotropism) of dabbling ducks and geese and retained attachment to airway epithelium (respirotropism). Natural HPAI 2020 H5 virus infection in Eurasian wigeons and barnacle geese also showed a high level of neurotropism, as both species presented with brain lesions that co-localized with virus antigen expression. We concluded that the combination of respirotropism, neurotropism, and possibly enterotropism, contributed to the successful adaptation of 2020/2021 HPAI H5 viruses to wild waterbird populations.

Background: Rigorous testing is a prerequisite to prove freedom of notifiable influenza A virus infections in commercially farmed ostriches, as is the isolation and identification of circulating strains. Pooling 5 ostrich tracheal swabs in a 50 % v/v phosphate-buffered saline (PBS): glycerol transport medium (without antibiotics) is the current standard practice to increase reverse transcription real time PCR (RT-rtPCR) testing throughput and simultaneously reduce the test costs. In this study we investigated whether doubling ostrich tracheal swabs to 10 per pool would affect the sensitivity of detection of H5N8 high pathogenicity avian influenza virus (HPAIV) and H7N1 low pathogenicity avian influenza virus (LPAIV) by quantitative RT-rtPCR, and we also compared the effect of a protein-rich, brain heart infusion broth (BHI) virus transport media containing broad spectrum antimicrobials (VTM) on the efficacy of isolating the H5N8 and H7N1 viruses from ostrich tracheas, since the historical isolation success rate from these birds has been poor., Results: Increasing the ostrich swabs from 5 to 10 per pool in 3 mls of transport medium had no detrimental effect on the sensitivity of the RT-rtPCR assay in detecting H5N8 HPAIV or H7N1 LPAIV; and doubling of the swab pool size even seemed to improve the sensitivity of virus detection at levels that were statistically significant (p less than or equal to 0.05) in medium and low doses of spiked H5N8 HPAIV and at high levels of spiked H7N1 LPAIV. On virus isolation, more samples were positive when swabs were stored in a protein-rich viral transport medium supplemented with antimicrobials in PBS: glycerol (10/18 vs. 7/18 for H5N8 HPAI); although the differences were not statistically significant, overall higher virus titres were detected (10 6.7 - 10 3.0 vs. 10 6.6 - 10 3.1 EID 50 for H5N8 HPAIV and 10 5.5 - 10 1.4 vs. 10 5.1 - 10 1.3 EID 50 for H7N1 LPAIV); and fewer passages were required with less filtration for both H5N8 HPAI and H7N1 LPAI strains., Conclusion: Ostrich tracheal swab pool size could be increased from 5 to 10 in 3mls of VTM with no loss in sensitivity of the RT-rtPCR assay in detecting HPAI or LPAI viruses, and HPAI virus could be isolated from a greater proportion of swabs stored in VTM compared to PBS: glycerol without antibiotics., (© 2022. The Author(s).)

Highly pathogenic avian influenza (HPAI) in wild birds is a major emerging disease, and a cause of increased mortality during outbreaks. The Common buzzard (Buteo buteo) has a considerable chance of acquiring the infection and therefore may function as bio-sentinel for the presence of virus in wildlife. This study aimed to determine the virus distribution and associated pathological changes in the tissues of Common buzzards that died with HPAI H5 virus infection during the 2020-2021 epizootic. Eleven freshly dead, HPAI H5 virus-positive Common buzzards were necropsied. Based on RT-PCR, all birds were systemically infected with HPAI H5N8 virus, as viral RNA was detected in cloacal and pharyngeal swabs and in all 10 selected tissues of the birds, with mean Ct values per tissue ranging from 22 for heart to 32 for jejunum. Based on histology and immunohistochemistry, the most common virus-associated pathological changes were necrotizing encephalitis (9/11 birds) and necrotizing myocarditis (7/11 birds). The proventriculus of two birds showed virus-associated necrosis, indicating tropism of this virus for the digestive tract. Our advice is to collect at least a miniset of samples including brain, heart, liver, and spleen, as these tissues were positive both by RT-PCR and for virus-antigen-associated lesions., (© 2022. The Author(s).)

During the 2020-2021 winter season, an outbreak of clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) virus occurred in South Korea. Here, we evaluated the pathogenicity and transmissibility of A/mandarin duck/Korea/H242/2020 (H5N8) (H242/20(H5N8)) first isolated from this outbreak in specific pathogen-free (SPF) chickens and commercial ducks in comparison with those of A/duck/Korea/HD1/2017(H5N6) (HD1/17(H5N6)) from a previous HPAI outbreak in 2017-2018. In chickens, the 50% chicken lethal dose and mean death time of H242/20(H5N8) group were 10 4.5 EID 50 and 4.3 days, respectively, which indicate less virulent than those of HD1/17(H5N6) (10 3.6 EID 50 and 2.2 days). Whereas, chickens inoculated with H242/20(H5N8) survived longer and had a higher titer of viral shedding than those inoculated with HD1/17(H5N6), which may increase the risk of viral contamination on farms. All ducks infected with either HPAI virus survived without clinical symptoms. In addition, they exhibited a longer virus shedding period and a higher transmission rate, indicating that ducks may play an important role as a silent carrier of both HPAI viruses. These results suggest that the pathogenic characteristics of HPAI viruses in chickens and ducks need to be considered to effectively control HPAI outbreaks in the field.

The highly pathogenic (HPAI) avian influenza A(H5N1) viruses have undergone reassortment with multiple non-N1-subtype neuraminidase genes since 2008, leading to the emergence of H5Nx viruses. H5Nx viruses established themselves quickly in birds and disseminated from China to Africa, the Middle East, Europe and North America. Multiple genetic clades have successively evolved through frequent mutations and reassortment, posing a continuous threat to domestic poultry and causing substantial economic losses. Live bird markets are recognized as major sources of avian-to-human infection and for the emergence of zoonotic influenza. In Pakistan, the A(H5N1) virus was first reported in domestic birds in 2007; however, avian influenza surveillance is limited and there is a lack of knowledge on the evolution and transmission of the A(H5) virus in the country. We collected oropharyngeal swabs from domestic poultry and environmental samples from six different live bird markets during 2018-2019. We detected and sequenced HPAI A(H5N8) viruses from two chickens, one quail and one environmental sample in two markets. Temporal phylogenetics indicated that all novel HPAI A(H5N8) viruses belonged to clade 2.3.4.4b, with all eight genes of Pakistan A(H5N8) viruses most closely related to 2017 Saudi Arabia A(H5N8) viruses, which were likely introduced via cross-border transmission from neighboring regions approximately three months prior to virus detection into domestic poultry. Our data further revealed that clade 2.3.4.4b viruses underwent rapid lineage expansion in 2017 and acquired significant amino acid mutations, including mutations associated with increased haemagglutinin affinity to human α-2,6 receptors, prior to the first human A(H5N8) infection in Russian poultry workers in 2020. These results highlight the need for systematic avian influenza surveillance in live bird markets in Pakistan to monitor for potential A(H5Nx) variants that may arise from poultry populations.

Background: Several outbreaks of highly pathogenic avian influenza (HPAI) caused by influenza A virus of subtype H5N8 have been reported in wild birds and poultry in Europe during autumn 2020. Norway is one of the few countries in Europe that had not previously detected HPAI virus, despite widespread active monitoring of both domestic and wild birds since 2005., Results: We report detection of HPAI virus subtype H5N8 in a wild pink-footed goose (Anser brachyrhynchus), and several other geese, ducks and a gull, from south-western Norway in November and December 2020. Despite previous reports of low pathogenic avian influenza (LPAI), this constitutes the first detections of HPAI in Norway., Conclusions: The mode of introduction is unclear, but a northward migration of infected geese or gulls from Denmark or the Netherlands during the autumn of 2020 is currently our main hypothesis for the introduction of HPAI to Norway. The presence of HPAI in wild birds constitutes a new, and ongoing, threat to the Norwegian poultry industry, and compliance with the improved biosecurity measures on poultry farms should therefore be ensured. [MK1]Finally, although HPAI of subtype H5N8 has been reported to have very low zoonotic potential, this is a reminder that HPAI with greater zoonotic potential in wild birds may pose a threat in the future. [MK1]Updated with a sentence emphasizing the risk HPAI pose to poultry farms, both in the Abstract and in the Conclusion-section in main text, as suggested by Reviewer 1 (#7).

Background: Influenza viruses are a continuous threat to avian and mammalian species, causing epidemics and pandemics. After the circulation of H5N1 in 2006, 2015, and 2016 in Iraq, an H5N8 influenza virus emerged in domestic geese in Sulaymaniyah Province, Iraq. This study analyzed the genetic characteristics of the Iraqi H5N8 viruses., Results: An HPAI virus subtype H5N8 was identified from domestic backyard geese in the Kurdistan Region, north Iraq. Phylogenic analyses of the hemagglutinin (HA) and neuraminidase (NA) genes indicated that Iraq H5N8 viruses belonged to clade 2.3.4.4 group B and clustered with isolates from Iran, Israel, and Belgium. Genetic analysis of the HA gene indicated molecular markers for avian-type receptors. Characterization of the NA gene showed that the virus had sensitive molecular markers for antiviral drugs., Conclusions: This is the first study ever on H5N8 in Iraq, and it is crucial to understand the epidemiology of the viruses in Iraq and the Middle East. The results suggest a possible role of migratory birds in the introduction of HPAI subtype H5N8 into Iraq.

On 5 November 2020, a confirmed outbreak due to an H5N8 highly pathogenic avian influenza virus (HPAIV) occurred at an egg-hen farm in Kagawa prefecture (western Japan). This virus, A/chicken/Kagawa/11C/2020 (Kagawa11C2020), was the first HPAI poultry isolate in Japan in 2020 and had multiple basic amino acids-a motif conferring high pathogenicity to chickens-at the hemagglutinin cleavage site. Mortality of chickens was 100% through intravenous inoculation tests performed according to World Organization for Animal Health criteria. Phylogenetic analysis showed that the hemagglutinin of Kagawa11C2020 belongs to clade 2.3.4.4B of the H5 Goose/Guangdong lineage and clusters with H5N8 HPAIVs isolated from wild bird feces collected in Hokkaido (Japan) and Korea in October 2020. These H5N8 HPAIVs are closely related to H5N8 HPAIVs isolated in European countries during the winter of 2019-2020. Intranasal inoculation of chickens with 10 6 fifty-percent egg infectious doses of Kagawa11C2020 revealed that the 50% chicken lethal dose was 10 4.63 and the mean time to death was 134.4 h. All infected chickens demonstrated viral shedding beginning on 2 dpi-before clinical signs were observed. These results suggest that affected chickens could transmit Kagawa11C2020 to surrounding chickens in the absence of clinical signs for several days before they died.

During October 2020-January 2021, we isolated a total of 67 highly pathogenic avian influenza (HPAI) H5N8 viruses from wild birds and outbreaks in poultry in South Korea. We sequenced the isolates and performed phylogenetic analysis of complete genome sequences to determine the origin, evolution, and spread patterns of these viruses. Phylogenetic analysis of the hemagglutinin (HA) gene showed that all the isolates belong to H5 clade 2.3.4.4 subgroup B (2.3.4.4b) and form two distinct genetic clusters, G1 and G2. The cluster G1 was closely related to the 2.3.4.4b H5N8 HPAI viruses detected in Europe in early 2020, while the cluster G2 had a close genetic relationship with the 2.3.4.4b H5N8 viruses that circulated in Europe in late 2020. A total of seven distinct genotypes were identified, including five novel reassortants carrying internal genes of low pathogenic avian influenza viruses. Our Bayesian discrete trait phylodynamic analysis between host types suggests that the viruses initially disseminated from migratory waterfowl to domestic duck farms in South Korea. Subsequently, domestic duck farms most likely contributed to the transmission of HPAI viruses to chicken and minor poultry farms, highlighting the need for enhanced, high levels of biosecurity measures at domestic duck farms to effectively prevent the introduction and spread of HPAI.

In October 2020, a highly pathogenic avian influenza (HPAI) subtype H5N8 virus was identified from a fecal sample of a wild mandarin duck ( Aix galericulata ) in South Korea. We sequenced all eight genome segments of the virus, designated as A/Mandarin duck/Korea/K20-551-4/2020(H5N8), and conducted genetic characterization and comparative phylogenetic analysis to track its origin. Genome sequencing and phylogenetic analysis show that the hemagglutinin gene belongs to H5 clade 2.3.4.4 subgroup B. All genes share high levels of nucleotide identity with H5N8 HPAI viruses identified from Europe during early 2020. Enhanced active surveillance in wild and domestic birds is needed to monitor the introduction and spread of HPAI via wild birds and to inform the design of improved prevention and control strategies.

Lethal infection of wild birds with different subtypes of H5 viruses continuously occur. To investigate the genetic evolution and pathogenicity of H5 viruses in wild birds, we performed a detailed genetic and biologic analysis of 27 viruses, including H5N1, H5N2, H5N6, and H5N8 subtypes, that were responsible for avian influenza outbreaks in wild birds in China over the past decade. We found that these 27 viruses, bearing different clades/subclades of HA, were complicated reassortants and formed 12 different genotypes. Ten of the viruses tested were highly pathogenic in chickens, but showed distinct pathotypes in ducks and mice. Five of these 10 viruses, which were all from clade2.3.4.4, could bind human-type receptors. Our findings reveal the diversity of the genetic and biologic properties of H5 viruses circulating in wild birds and highlight the need to carefully monitor and evaluate the risks these viruses pose to animal and public health.

Infections by A/H5 and A/H7 avian influenza viruses (AIVs) can cause acute disease and are therefore notifiable in poultry and wild birds. During winter 2015-2016, several cases of infection caused by highly pathogenic (HP) AIVs belonging to the A/H5N1, A/H5N2, and A/H5N9 subtypes were detected in southwestern France. Throughout winter 2016-2017, several cases of infections caused mainly by A/H5N8 HP AIV (A/goose/GD/1/1996, clade 2.3.4.4) were detected across Europe. On both occasions, the viruses were widely detected on palmiped farms in France. This study was designed to evaluate the persistence of A/H5 HP AIV in slurry from various duck productions. This was achieved (i) in the laboratory setting by artificially spiking four AIV-free slurry samples with known amounts of A/H5N9 HP AIV and monitoring virus infectivity, with or without lime treatment to achieve pH 10 or pH 12, and (ii) by sampling slurry tanks on five naturally A/H5N8 HP-contaminated farms. Experimental results in artificially spiked slurry suggested virus survival for 4 weeks in slurry from Muscovy or Pekin duck breeders and for 2 weeks in slurry from ducks for foie gras production during the assisted-feeding period, without lime treatment. Persistence of infectious A/H5N9 HP AIV in all slurry samples after lime treatment at pH 10 or pH 12 was less than 1 week. The A/H5N8 HP AIV persisted in naturally contaminated untreated slurry for 7 weeks. The results obtained provide experimental support for the 60-day storage period without treatment or the 7-day interval after lime treatment defined in French regulations for slurry sanitization. IMPORTANCE From November 2015 to July 2017, two successive episodes of H5 highly pathogenic avian influenza viruses (HP AIVs) infections occurred on poultry farms in France, mostly in domestic ducks raised for foie gras production in southwestern France. During the two epizootics, epidemiological investigations were carried out on infected farms and control and biosafety measures were implemented in association with surveillance in order to stop the spread of the viruses. Effluents are known to be an important factor in environmental dissemination of viruses, and suitable effluent management is needed to help prevent the spread of epizootics to other farms or pathogen persistence at the farm level. The present study was therefore designed to assess how long infectious A/H5 HP AIVs can persist in naturally or experimentally contaminated fecal slurry samples from ducks, with or without sanitization by lime treatment., (Copyright © 2020 Schmitz et al.)

Emergence and intercontinental spread of highly pathogenic avian influenza A (HPAI) H5Nx virus clade 2.3.4.4 has resulted in substantial economic losses to the poultry industry in Asia, Europe, and North America. The long-distance migratory birds have been suggested to play a major role in the global spread of avian influenza viruses during this wave of panzootic outbreaks since 2013. Poultry farm epidemics caused by multiple introduction of different HPAI novel subtypes of clade 2.3.4.4 viruses also occurred in Taiwan between 2015 and 2017. The mandatory and active surveillance detected H5N3 and H5N6 circulation in 2015 and 2017, respectively, while H5N2 and H5N8 were persistently identified in poultry farms since their first arrival in 2015. This study intended to assess the importance of various ecological factors contributed to the persistence of HPAI during three consecutive years. We used satellite technology to identify the location of waterfowl flocks. Four risk factors consistently showed strong association with the spatial clustering of H5N2 and H5N8 circulations during 2015 and 2017, including high poultry farm density (aOR:17.46, 95%CI: 5.91-74.86 and 8.23, 95% CI: 2.12-54.86 in 2015 and 2017, respectively), poultry heterogeneity index (aOR of 12.28, 95%CI: 5.02-31.14 and 2.79, 95%CI: 1.00-7.69, in 2015 and 2017, respectively), non-registered waterfowl flock density (aOR: 6.8, 95%CI: 3.41-14.46 and 9.17, 95%CI: 3.73-26.20, in 2015 and 2017, respectively) and higher percentage of cropping land coverage (aOR of 1.36, 95%CI: 1.10-1.69 and 1.04, 95%CI: 1.02-1.07, in 2015 and 2017, respectively). Our study highlights the application of remote sensing and clustering analysis for the identification and characterization of environmental factors in facilitating and contributing to the persistent circulation of certain subtypes of H5Nx in poultry farms in Taiwan., Competing Interests: The authors have declared that no competing interests exist.

Avian influenza (AI) virus (H9N2 and H5 subtypes) infections in birds cause major concerns around the world. The majority of the avian species, such as domestic, pet, and wild birds, are natural and experimental hosts of avian influenza viruses. There are global concerns about members of the Columbidae family, namely pigeons or doves, for their role as the potential interspecies bridge in influenza A viruses ecology. The acquired scientific data in this regard is still not clear since there are doubts about whether or not they transmit viruses between susceptible populations, and spread viruses among farms during outbreaks. To monitor H5 and H9 influenza virus infection status in the rural, backyard, and domestic birds, an annual active surveillance program was performed from September to October 2016. In December 2016, an outbreak of highly pathogenic avian influenza (HPAI) virus subtype H5N8 was detected in a layer farm in Tehran province, Iran. The present research was conducted to study H9N2 or H5 infections in pigeons within HPAI H5N8 2016 outbreaks and annual national AI surveillance in Iran. For this purpose, cloacal swabs and tissue samples (trachea, lung, brain, liver, heart, pancreas, and cecal tonsil) were collected and examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) method and virus isolation. Results of the tests performed on the swab and tissue samples were negative for H5 nor H9N2 viruses. The samples in real-time RT-PCR that after three passages still showed negative results in HA and molecular tests were considered negative. Moreover, the Newcastle disease virus was isolated in most of the samples taken from dead pigeons, after inoculation in embryonated chicken eggs., (Copyright © 2020, Archives of Razi Institute. Published by Kowsar.)

Asian-origin H5N8 highly pathogenic avian influenza (HPAI) viruses of the H5 Goose/Guangdong/96 lineage, clade 2.3.4.4 group B, reached South Africa by June 2017. By the end of that year, 5.4 million layers and broiler chickens died or were culled, with total losses in the poultry industry estimated at US$ 140 million, and thousands of exotic birds in zoological collections, endangered endemic species and backyard poultry and pet birds also perished. The 2017 H5N8 HPAI outbreaks were characterized by two distinct spatial clusters, each associated with specific reassortant viral genotypes. Genotypes 1, 2, 3 and 5 were restricted to the northern regions, spanning the provinces of Limpopo, Gauteng, North West, Mpumalanga, KwaZulu-Natal and Free State. The second, much larger cluster of outbreaks was in the south, in the Western and Eastern Cape provinces, wherein 2017 and 2018 outbreaks were caused solely by genotype 4. The last confirmed case of H5N8 HPAI in the northern region in 2017 was in early October, and the viruses seemed to disappear over the summer. However, starting in mid-February 2018, H5N8 HPAI outbreaks resurged in the north. Viruses from two of the eight outbreaks were sequenced, one from an outbreak in quails (Coturnix japonica) in the North West Province, and another from commercial pullets in the Gauteng province. Phylogenetic analysis identified the viruses as a distinct sixth genotype that was most likely a new introduction to South Africa in early 2018., (© 2019 Blackwell Verlag GmbH.)

Background: In June 2017, an outbreak of the highly pathogenic avian influenza A(H5N8) was detected in commercial poultry farms in South Africa, which rapidly spread to all nine South African provinces., Objectives: We conducted active surveillance for the transmission of influenza A(H5N8) to humans working with infected birds during the South African outbreak., Methods: Influenza A(H5N8)-positive veterinary specimens were used to evaluate the ability of real-time PCR-based assays to detect contemporary avian influenza A(H5N8) strains. Whole genome sequences were generated from these specimens by next-generation sequencing for phylogenetic characterization and screening for mammalian-adaptive mutations., Results: Human respiratory samples from 74 individuals meeting our case definition, all tested negative for avian influenza A(H5) by real-time PCR, but 2 (3%) were positive for human influenza A(H3N2). 54% (40/74) reported wearing personal protective equipment including overalls, boots, gloves, masks, and goggles. 94% (59/63) of veterinary specimens positive for H5N8 were detected on an influenza A(H5) assay for human diagnostics. A commercial H5N8 assay detected H5 in only 6% (3/48) and N8 in 92% (44/48). Thirteen (13/25; 52%) A(H5N8) genomes generated from veterinary specimens clustered in a single monophyletic clade. These sequences contained the NS (P42S) and PB2 (L89V) mutations noted as markers of mammalian adaptation., Conclusions: Diagnostic assays were able to detect and characterize influenza A(H5N8) viruses, but poor performance is reported for a commercial assay. Absence of influenza A(H5N8) in humans with occupational exposure and no clear impression of molecular adaptation for mammalian infection suggest that this avian pathogen continues to be low-risk human pathogen., (© 2020 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Avian influenza viruses (AIV) affect a wide range of birds and mammals, cause severe economic damage to the poultry industry, and pose a serious threat to humans. Highly pathogenic avian influenza viruses (HPAI) H5N1 were first identified in Southeast Asia in 1996 and spread to four continents over the following years. The viruses have caused high mortality in chickens and various bird species and deadly infections in humans. Multiple conventional methods have been so far introduced for the detection and identification of avian influenza viruses. Traditional virus isolation methods are gold standard protocol in AI detection; nonetheless, virus isolation in embryonating chicken eggs (ECE) is not a rapid method for the detection of influenza viruses since it is time-consuming and labor-intensive. Furthermore, the isolation of highly pathogenic viruses, such as H5, needs BSL3 laboratories. Real-Time Reverse Transcription-Polymerase Chain Reaction (RRT-PCR) is a sensitive and specific method for the detection of influenza viruses. The application of these nucleic acid-based techniques has increased our ability to identify and perform influenza virus care programs, especially in surveillance programs. The current study aimed to detect H5 subtype of avian influenza (AI) virus using fast, specific, and sensitive TaqMan RRT-PCR. Notably, single step RRT-PCR was used to prevent possible laboratory contamination. The specificity of this test was evaluated using nucleic acid extracted from several poultry pathogenic microorganisms and negative clinical specimens from AI-uninfected birds. The sensitivity analysis of the RRT-PCR assay was performed using in vitro-transcribed RNA copy and 10-fold serial dilution of standard AI virus with specific titer. The results indicated the high sensitivity of this method and the lowest detectable dilution of this method based on RNA copies and 1:10 serial dilutions of the standard virus was 10 1.9 EID50 /100., (Copyright © 2020, Archives of Razi Institute. Published by Kowsar.)

The relative roles that movement and proximity networks play in the spread of highly pathogenic avian influenza (HPAI) viruses are often unknown during an epidemic, preventing effective control. We used network analysis to explore the devastating epidemic of HPAI A(H5N8) among poultry, in particular ducks, in France during 2016-2017 and to estimate the likely contribution of live-duck movements. Approximately 0.2% of live-duck movements could have been responsible for between-farm transmission events, mostly early during the epidemic. Results also suggest a transmission risk of 35.5% when an infected holding moves flocks to another holding within 14 days before detection. Finally, we found that densely connected groups of holdings with sparse connections between groups overlapped farmer organizations, which represents important knowledge for surveillance design. This study highlights the importance of movement bans in zones affected by HPAI and of understanding transmission routes to develop appropriate HPAI control strategies.

In May 2017, high mortality of chickens and Muscovy ducks due to the H5N8 highly pathogenic avian influenza virus (HPAIV) was reported in the Democratic Republic of Congo (DR Congo). In this study, we assessed the molecular, antigenic, and pathogenic features in poultry of the H5N8 HPAIV from the 2017 Congolese outbreaks. Phylogenetic analysis of the eight viral gene segments revealed that all 12 DR Congo isolates clustered in clade 2.3.4.4B together with other H5N8 HPAIVs isolated in Africa and Eurasia, suggesting a possible common origin of these viruses. Antigenically, a slight difference was observed between the Congolese isolates and a representative virus from group C in the same clade. After intranasal inoculation with a representative DR Congo virus, high pathogenicity was observed in chickens and Muscovy ducks but not in Pekin ducks. Viral replication was higher in chickens than in Muscovy duck and Pekin duck organs; however, neurotropism was pronounced in Muscovy ducks. Our data confirmed the high pathogenicity of the DR Congo virus in chickens and Muscovy ducks, as observed in the field. National awareness and strengthening surveillance in the region are needed to better control HPAIVs.

We detected a highly pathogenic avian influenza A(H5N8) virus in lung samples of 2 gray seals (Halichoerus grypus) stranded on the Baltic coast of Poland in 2016 and 2017. This virus, clade 2.3.4.4 B, was closely related to avian H5N8 viruses circulating in Europe at the time.

Highly pathogenic avian influenza H5N8 clade 2.3.4.4 virus caused outbreaks in poultry and unusually high mortality in wild birds in 2016-2017. The pathobiology of one of these viruses was examined in mallards and chickens. High mortality and transmission to direct contacts were observed in mallards inoculated with medium and high doses of the virus. However, in chickens, high mortality occurred only when birds are given the high virus dose and no transmission was observed, indicating that the virus was better adapted to mallards. In comparison with the virus inoculum, viral sequences obtained from the chickens had a higher number of nucleotide changes but lower intra-host genomic diversity than viral sequences obtained from the mallards. These observations are consistent with population bottlenecks occurring when viruses infect and replicate in a host that it is not well adapted to. Whether these observations apply to influenza viruses in general remains to be determined., (Published by Elsevier Inc.)

The surveillance and virological characterization of H5N8 avian influenza viruses are important in order to assess their zoonotic potential. The genetic analyses of the Egyptian H5N8 viruses isolated through active surveillance in wild birds and domestic poultry in the winter of 2016/2017 showed multiple introductions of reassortant viruses. In this study, we investigated and compared the growth kinetics, infectivity, and pathogenicity of the three reassortant forms of H5N8 viruses detected in wild birds and domestic poultry in Egypt during the first introduction wave in the winter of 2016/2017. Three representative H5N8 viruses (abbreviated as 813, 871, and 13666) were selected. The 871/H5N8 virus showed enhanced growth properties in vitro in Madin Darby canine kidney (MDCK) and A549 cells. Interestingly, all viruses replicated well in mice without prior adaptation. Infected C57BL/6 mice showed 20% mortality for 813/H5N8 and 60% mortality for 871/H5N8 and 13666/H5N8, which could be attributed to the genetic differences among the viruses. Studies on the pathogenicity in experimentally infected ducks revealed a range of pathogenic effects, with mortality rate ranging from 0% for 813/H5N8 and 13666/H5N8 to 28% for 871/H5N8. No significant differences were observed among the three compared viruses in infected chickens. Overall, different H5N8 viruses had variable biological characteristics, indicating a continuous need for surveillance and virus characterization efforts.

Highly pathogenic avian influenza H5N6 viruses have been circulating in poultry in Asia since 2013 and producing serious diseases in chickens. Here, we analyzed the genetic properties of 10 H5N6 subtypes AIVs from geese in 2015-2016 in Guangdong province. Phylogenic analysis showed that all HA genes of the 10 viruses belonged to clade 2.3.4.4, and their genes including HA, PA, PB1, M, NP, and NS all derived from Mix-like 1 (CH, VN, LS). Their PB2 genes come from Mix-like 2 (CH, VN, JP). The NA genes were classified into a Eurasian lineage. Therefore, the 10 viruses likely originate from the same ancestor and were all recombinant viruses between different genotypes. We selected A/Goose/Guangdong/GS144/2015(H5N6) (GS144) and A/Goose/Guangdong/GS148/2016(H5N6) (GS148) viruses to inoculate 5-week-old chickens intranasally with 10 4 EID 50 /0.1 mL dose intranasally to assess their pathogenicity and transmissibility. Inoculated chickens showed that the GS144 virus caused systematic infection with a lethality of 100%, but the lethality of GS148 virus was 0%. The two viruses were efficiently transmitted to contact chickens. The lethality of GS144 and GS148 virus in contact with chickens was 87.5% and 0%, respectively, which suggests that the transmissibility of GS144 virus was stronger than GS148 virus in chickens. Thus, different H5N6 viruses from the same waterfowl can show different pathogenicity and transmissibility in chickens. Continued surveillance and characteristic analysis of the H5N6 viruses will help us to keep abreast of evolution and variation in avian influenza viruses in the future.

During 2014-2017 Clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses (HPAIVs) have spread worldwide. In 2016, an epidemic of HPAIV H5N8 in Iran caused mass deaths among wild birds, and several commercial poultry farms and captive bird holdings were affected and continue to experience problems. Several outbreaks were reported in 2017. One of them is related to Hooded crow (Corvus cornix) in a national park in Esfahan province in 2017. Whole genome sequencing and characterization have been done on the detected H5N8 sample. Based on HA sequencing results, it belongs to 2.3.4.4 clade, and the cleavage site is (PLREKRRKR/G). Phylogenetic analysis of the HA gene showed that the Iran 2017 H5N8 virus clustered within subgroup Russia 2016 2.3.4.4 b of group B in H5 clade 2.3.4.4 HPAIV. On the other hand, the NA gene of the virus is placed in group C of Eurasian lineage. Complete genome characterization of this virus revealed probable reassortment of the virus with East-Asian low-pathogenic influenza viruses. Furthermore, the virus possessed some phenotypic markers related to the increased potential for transmission and pathogenicity to mammals at internal segments. This study is the first full genome characterization H5N8 HPAIV in Iran. The data complete the puzzle of molecular epidemiology of H5N8 HPAIV in Iran and the region. Our study provides evidence for fast and continuing reassortment of H5 clade 2.3.4.4 viruses, that might lead to changes in virus structural and functional characteristics such as the route and method of transmission of the virus and virus infective, pathogenic and zoonotic potential., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the Netherlands, three commercial poultry farms and two hobby holdings were infected with highly pathogenic avian influenza (HPAI) H5N6 virus in the winter of 2017-2018. This H5N6 virus is a reassortant of HPAI H5N8 clade 2.3.4.4 group B viruses detected in Eurasia in 2016. H5N6 viruses were also detected in several dead wild birds during the winter. However, wild bird mortality was limited compared to the caused by the H5N8 group B virus in 2016-2017. H5N6 virus was not detected in wild birds after March, but in late summer infected wild birds were found again. In this study, the complete genome sequences of poultry and wild bird viruses were determined to study their genetic relationship. Genetic analysis showed that the outbreaks in poultry were not the result of farm-to-farm transmissions, but rather resulted from separate introductions from wild birds. Wild birds infected with viruses related to the first outbreak in poultry were found at short distances from the farm, within a short time frame. However, no wild bird viruses related to outbreaks 2 and 3 were detected. The H5N6 virus isolated in summer shares a common ancestor with the virus detected in outbreak 1. This suggests long-term circulation of H5N6 virus in the local wild bird population. In addition, the pathogenicity of H5N6 virus in ducks was determined, and compared to that of H5N8 viruses detected in 2014 and 2016. A similar high pathogenicity was measured for H5N6 and H5N8 group B viruses, suggesting that biological or ecological factors in the wild bird population may have affected the mortality rates during the H5N6 epidemic. These observations suggest different infection dynamics for the H5N6 and H5N8 group B viruses in the wild bird population., (© 2019 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

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., (© 2019 Blackwell Verlag GmbH.)

In winter 2016-2017, Highly Pathogenic Avian Influenza (HPAI) H5N8 virus spread across Europe, causing unprecedented epizootics. France was massively affected, resulting in the culling of over 6 million poultry. Boosted regression tree (BRT) models were used to quantify the association between spatial risk factors and HPAI H5N8 infection in poultry holdings and to generate predictive maps for HPAI infection. Three datasets were combined to build the model: a dataset of the reported outbreaks in poultry, a dataset of the poultry holdings where the virus has not been reported and a set of relevant spatial risk factors, including poultry production and trade, and water bird habitat. Results identified key associations between the 'foie gras' production systems and HPAI H5N8 risk of occurrence and indicate that strengthening surveillance of fattening duck production systems and making the transportation of fattening ducks more secure would be key priority options for HPAI prevention and control.

Reported mass mortalities in wild pigeons and doves during the 2017/2018 Clade 2.3.4.4 HPAI H5N8 outbreaks in South Africa necessitated an investigation of healthy racing pigeons for their susceptibility and ability to transmit a Clade 2.3.4.4 sub-group B virus of South African origin. Pigeons challenged with medium (10 4.5 EID 50 ) and high doses (10 6 EID 50 ) but not a low dose (10 3 EID 50 ) of virus, shed virus in low levels of <10 3 EID 50 /ml from the oropharynx and cloaca for up to eight days, with peak shedding around 4 days post challenge. Challenged pigeons were able to transmit the virus to contact pigeons, but not contact chickens. Neither pigeons nor chickens presented clinical disease, and only two pigeons in the group that received the high challenge dose developed influenza A-virus specific antibodies. The levels of virus shed by the racing pigeons were well below the published bird infectious dose 50 values for most poultry, especially chickens, therefore the risk that racing pigeons could act as propagators and disseminators through excretion of Clade 2.3.4.4 HPAI H5N8 strains remains negligible., (Copyright © 2018 Elsevier B.V. All rights reserved.)

American robins (Turdus migratorius) are commonly associated with farmsteads in the United States and have shown previous evidence of exposure to an H5 avian influenza A virus (IAV) near a poultry production facility affected by a highly pathogenic (HP) H5 virus in Iowa, USA during 2015. We experimentally infected American robins with three clade 2.3.4.4 HP H5 viruses (H5N2 and H5N8). A total of 22/24 American robins shed virus, and all three strains were represented. The highest virus titres shed were 10 4.3 , 10 4.3 and 10 4.8 PFU/ml, associated respectively with viruses isolated from poultry, a captive gyrfalcon (Falco rusticolus), and a Northern pintail (Anas acuta). Of those birds that shed, viral shedding was initiated 1 or 2 days post-infection (DPI) and shedding ceased in all birds by 7 DPI. This study adds an additional synanthropic wildlife species to a growing list of animals that can successfully replicate and shed IAVs., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Sixty-five poultry outbreaks and sixty-eight events in wild birds were reported during the highly pathogenic H5N8/H5N5 avian influenza epidemic in Poland in 2016-2017. The analysis of all gene segment sequences of selected strains revealed cocirculation of at least four different genome configurations (genotypes) generated through reassortment of clade 2.3.4.4 H5N8 viruses detected in Russia and China in mid-2016. The geographical and temporal distribution of three H5N8 genotypes indicates separate introductions. Additionally, an H5N5 virus with a different gene configuration was detected in wild birds. The compilation of the results with those from studies on the virus' diversity in Germany, Italy and the Netherlands revealed that Europe was affected by at least eight different H5N8/H5N5 reassortants. Analysis of the HA gene sequence of a larger subset of samples showed its diversification corresponding to the genotype classification. The close relationship between poultry and wild bird strains from the same locations observed in several cases points to wild birds as the primary source of the outbreaks in poultry., (© 2018 Blackwell Verlag GmbH.)

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.

Circulation of highly pathogenic avian influenza (HPAI) viruses poses a continuous threat to animal and public health. After the 2005-2006 H5N1 and the 2014-2015 H5N8 epidemics, another H5N8 is currently affecting Europe. Up to August 2017, 1,112 outbreaks in domestic and 955 in wild birds in 30 European countries have been reported, the largest epidemic by a HPAI virus in the continent. Here, the main epidemiological findings are described. While some similarities with previous HPAI virus epidemics were observed, for example in the pattern of emergence, significant differences were also patent, in particular the size and extent of the epidemic. Even though no human infections have been reported to date, the fact that A/H5N8 has affected so far 1,112 domestic holdings, increases the risk of exposure of humans and therefore represents a concern. Understanding the epidemiology of HPAI viruses is essential for the planning future surveillance and control activities., (© 2018 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.)

In contrast to previous incursions of highly pathogenic avian influenza (HPAIV) H5 viruses, H5N8 clade 2.3.4.4b viruses caused numerous cases of lethal infections in white-tailed sea eagles ( Haliaeetus albicilla ) affecting mainly young eagles (younger than five years of age) in Germany during winter 2016/2017. Until April 2017, 17 HPAIV H5N8-positive white-tailed sea eagles had been detected (three found alive and 14 carcasses) by real-time RT-PCR and partial nucleotide sequence analyses. Severe neurological clinical signs were noticed which were corroborated by immunohistopathology revealing mild to moderate, oligo- to multifocal necrotizing virus-induced polioencephalitis. Lethal lead (Pb) concentrations, a main factor of mortality in sea eagles in previous years, could be ruled out by atomic absorption spectrometry. HPAIV H5 clade 2.3.4.4b reportedly is the first highly pathogenic influenza virus known to induce fatal disease in European white-tailed see eagles. This virus strain may become a new health threat to a highly protected species across its distribution range in Eurasia. Positive cloacal swabs suggest that eagles can spread the virus with their faeces.

Here, we present a comprehensive analysis of the H5N8/H5N5 highly pathogenic avian influenza (HPAI) virus strains detected in the Czech Republic during an outbreak in 2017. Network analysis of the H5 Hemagglutinin (HA) from 99% of the outbreak localities suggested that the diversity of the Czech H5N8/H5N5 viruses was influenced by two basic forces: local microevolution and independent incursions. The geographical occurrence of the central node H5 HA sequences revealed three eco-regions, which apparently played an important role in the origin and further spread of the local H5N8/HPAI variants across the country. A plausible explanation for the observed pattern of diversity is also provided.

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.

A H5N6 highly pathogenic avian influenza virus (HPAIV) was detected in a black-faced spoonbill (Platalea minor) found dead in Taiwan during December 2017. Genome sequencing and phylogenetic analyses suggest the hemagglutinin gene belongs to H5 clade 2.3.4.4 Group B. All genes except neuraminidase gene shared high levels of nucleotide identity with H5N8 HPAIV identified from Europe during 2016-2017. Genetically similar H5N6 HPAIV was also identified from Japan during November 2017. Enhanced surveillance is required in this region., (Copyright © 2018 Elsevier B.V. All rights reserved.)

IntroductionFrance is one of Europe's foremost poultry producers and the world's fifth largest producer of poultry meat. In November 2016, highly pathogenic avian influenza (HPAI) virus subtype H5N8 emerged in poultry in the country. As of 23 March 2017, a total of 484 confirmed outbreaks were reported, with consequences on animal health and socio-economic impacts for producers. Methods: We examined the spatio-temporal distribution of outbreaks that occurred in France between November 2016 and March 2017, using the space-time K-function and space-time permutation model of the scan statistic test. Results : Most outbreaks affected duck flocks in south-west France. A significant space-time interaction of outbreaks was present at the beginning of the epidemic within a window of 8 km and 13 days. This interaction disappeared towards the epidemic end. Five spatio-temporal outbreak clusters were identified in the main poultry producing areas, moving sequentially from east to west. The average spread rate of the epidemic front wave was estimated to be 5.5 km/week. It increased from February 2017 and was negatively associated with the duck holding density. Conclusion : HPAI-H5N8 infections varied over time and space in France. Intense transmission events occurred at the early stages of the epidemic, followed by long-range jumps in the disease spread towards its end. Findings support strict control strategies in poultry production as well as the maintenance of high biosecurity standards for poultry holdings. Factors and mechanisms driving HPAI spread need to be further investigated.

Highly pathogenic avian influenza (HPAI) is essentially a poultry disease. Wild birds have traditionally not been involved in its spread, but the epidemiology of HPAI has changed in recent years. After its emergence in southeastern Asia in 1996, H5 HPAI virus of the Goose/Guangdong lineage has evolved into several sub-lineages, some of which have spread over thousands of kilometers via long-distance migration of wild waterbirds. In order to determine whether the virus is adapting to wild waterbirds, we experimentally inoculated the HPAI H5N8 virus clade 2.3.4.4 group A from 2014 into four key waterbird species-Eurasian wigeon (Anas penelope), common teal (Anas crecca), mallard (Anas platyrhynchos), and common pochard (Aythya ferina)-and compared virus excretion and disease severity with historical data of the HPAI H5N1 virus infection from 2005 in the same four species. Our results showed that excretion was highest in Eurasian wigeons for the 2014 virus, whereas excretion was highest in common pochards and mallards for the 2005 virus. The 2014 virus infection was subclinical in all four waterbird species, while the 2005 virus caused clinical disease and pathological changes in over 50% of the common pochards. In chickens, the 2014 virus infection caused systemic disease and high mortality, similar to the 2005 virus. In conclusion, the evidence was strongest for Eurasian wigeons as long-distance vectors for HPAI H5N8 virus from 2014. The implications of the switch in species-specific virus excretion and decreased disease severity may be that the HPAI H5 virus more easily spreads in the wild-waterbird population.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)