Caught Right on the Spot: Isolation and Characterization of Clade 2.3.4.4b H5N8 High Pathogenicity Avian Influenza Virus from a Common Pochard (Aythya ferina) Being Attacked by a Peregrine Falcon (Falco peregrinus).

We isolated a high pathogenicity avian influenza (HPAI) virus from a common pochard (Aythya ferina) that was being attacked by a bird of prey in South Korea in December 2020. Genetic analyses indicated that the isolate was closely related to the clade 2.3.4.4b H5N8 HPAI viruses found in South Korea and Japan during the winter season of 2020-2021. The histopathological examination revealed multifocal necrotizing inflammation in the liver, kidney, and spleen. Viral antigens were detected in the liver, kidney, spleen, trachea, intestine, and pancreas, indicating the HPAI virus caused a systemic infection. The presence of immunoreactivity for the viral antigen was observed in the cells involved in multifocal necrotic inflammation. Notably, epitheliotropic-positive patterns were identified in the epithelial cells of the trachea, mucosal epithelium of the intestine, and ductular epithelium of the pancreas. These findings provide direct evidence supporting the possibility of HPAI transmission from infected waterfowl to predators.

Detectado en el acto: Aislamiento y caracterización de un virus de la influenza aviar de alta patogenicidad del clado 2.3.4.4b H5N8 de un porrón común (Aythya ferina) atacado por un halcón peregrino (Falco peregrinus). Se aisló un virus de la influenza aviar (HPAI) de alta patogenicidad de un porrón común (Aythya ferina) que estaba siendo atacado por un ave rapaz en Corea del Sur en diciembre de 2020. Los análisis genéticos indicaron que el aislado estaba estrechamente relacionado con virus de influenza aviar de alta patogenicidad H5N8, clado 2.3.4.4 b encontrados en Corea del Sur y Japón durante la temporada de invierno de 2020­2021. El examen histopatológico reveló inflamación necrotizante multifocal en hígado, riñón y bazo. Se detectaron antígenos virales en el hígado, el riñón, el bazo, la tráquea, el intestino y el páncreas, lo que indica que este virus de alta patogenicidad causó una infección sistémica. Se observó la presencia de inmunorreactividad para el antígeno viral en las células involucradas en la inflamación necrótica multifocal. En particular, se identificaron patrones epiteliotrópicos positivos en las células epiteliales de la tráquea, el epitelio mucoso del intestino y el epitelio ductular del páncreas. Estos hallazgos proporcionan evidencia directa que respalda la posibilidad de transmisión de HPAI de aves acuáticas infectadas a especies depredadoras.

Spatiotemporal genotype replacement of H5N8 avian influenza viruses contributed to H5N1 emergence in 2021/2022 panzootic.

Since 2020, clade 2.3.4.4b highly pathogenic avian influenza H5N8 and H5N1 viruses have swept through continents, posing serious threats to the world. Through comprehensive analyses of epidemiological, genetic, and bird migration data, we found that the dominant genotype replacement of the H5N8 viruses in 2020 contributed to the H5N1 outbreak in the 2021/2022 wave. The 2020 outbreak of the H5N8 G1 genotype instead of the G0 genotype produced reassortment opportunities and led to the emergence of a new H5N1 virus with G1's HA and MP genes. Despite extensive reassortments in the 2021/2022 wave, the H5N1 virus retained the HA and MP genes, causing a significant outbreak in Europe and North America. Furtherly, through the wild bird migration flyways investigation, we found that the temporal-spatial coincidence between the outbreak of the H5N8 G1 virus and the bird autumn migration may have expanded the H5 viral spread, which may be one of the main drivers of the emergence of the 2020-2022 H5 panzootic.IMPORTANCESince 2020, highly pathogenic avian influenza (HPAI) H5 subtype variants of clade 2.3.4.4b have spread across continents, posing unprecedented threats globally. However, the factors promoting the genesis and spread of H5 HPAI viruses remain unclear. Here, we found that the spatiotemporal genotype replacement of H5N8 HPAI viruses contributed to the emergence of the H5N1 variant that caused the 2021/2022 panzootic, and the viral evolution in poultry of Egypt and surrounding area and autumn bird migration from the Russia-Kazakhstan region to Europe are important drivers of the emergence of the 2020-2022 H5 panzootic. These findings provide important targets for early warning and could help control the current and future HPAI epidemics.

In Vitro and In Vivo Characterization of H5N8 High-Pathogenicity Avian Influenza Virus Neurotropism in Ducks and Chickens.

H5N8 high-pathogenicity avian influenza virus (HPAIV) of clade 2.3.4.4B, which circulated during the 2016 epizootics in Europe, was notable for causing different clinical signs in ducks and chickens. The clinical signs preceding death were predominantly neurological in ducks versus respiratory in chickens. To investigate the determinants for the predominant neurological signs observed in ducks, we infected duck and chicken primary cortical neurons. Viral replication was identical in neuronal cultures from both species. In addition, we did not detect any major difference in the immune and inflammatory responses. These results suggest that the predominant neurological involvement of H5N8 HPAIV infection in ducks could not be recapitulated in primary neuronal cultures. In vivo, H5N8 HPAIV replication in ducks peaked soon after infection and led to an early colonization of the central nervous system. In contrast, viral replication was delayed in chickens but ultimately burst in the lungs of chickens, and the chickens died of respiratory distress before brain damage became significant. Consequently, the immune and inflammatory responses in the brain were significantly higher in duck brains than those in chickens. Our study thus suggests that early colonization of the central nervous system associated with prolonged survival after the onset of virus replication is the likely primary cause of the sustained inflammatory response and subsequent neurological disorders observed in H5N8 HPAIV-infected ducks. IMPORTANCE The severity of high-pathogenicity avian influenza virus (HPAIV) infection has been linked to its ability to replicate systemically and cause lesions in a variety of tissues. However, the symptomatology depends on the host species. The H5N8 virus of clade 2.3.4.4B had a pronounced neurotropism in ducks, leading to severe neurological disorders. In contrast, neurological signs were rarely observed in chickens, which suffered mostly from respiratory distress. Here, we investigated the determinants of H5N8 HPAIV neurotropism. We provide evidence that the difference in clinical signs was not due to a difference in neurotropism. Our results rather indicate that chickens died of respiratory distress due to intense viral replication in the lungs before viral replication in the brain could produce significant lesions. In contrast, ducks better controlled virus replication in the lungs, thus allowing the virus to replicate for a sufficient duration in the brain, to reach high levels, and to cause significant lesions.

Tropism of Highly Pathogenic Avian Influenza H5 Viruses from the 2020/2021 Epizootic in Wild Ducks and Geese.

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.

Opposite Outcomes of the Within-Host Competition between High- and Low-Pathogenic H5N8 Avian Influenza Viruses in Chickens Compared to Ducks.

Highly pathogenic avian influenza viruses (HPAIV) emerge from low-pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse genetics-engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8LP increased H5N8HP replication and pathogenesis. In contrast, the H5N8LP antagonized H5N8HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8LP, which correlated with H5N8HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variant interactions in viral evolution. IMPORTANCE Highly pathogenic avian influenza viruses represent a threat to poultry production systems and to human health because of their impact on food security and because of their zoonotic potential. It is therefore crucial to better understand how these viruses emerge. Using a within-host competition model between high- and low-pathogenic avian influenza viruses, we provide evidence that highly pathogenic avian influenza viruses could be more likely to emerge in chickens than in ducks. These results have important implications for highly pathogenic avian influenza virus emergence prevention, and they underscore the importance of within-host viral variant interactions in virus evolution.

Preferential Selection and Contribution of Non-Structural Protein 1 (NS1) to the Efficient Transmission of Panzootic Avian Influenza H5N8 Virus Clades 2.3.4.4A and B in Chickens and Ducks.

Highly pathogenic avian influenza virus H5N8 clade 2.3.4.4 caused outbreaks in poultry at an unprecedented global scale. The virus was spread by wild birds in Asia in two waves: clade 2.3.4.4A in 2014/2015 and clade 2.3.4.4B from 2016 up to today. Both clades were highly virulent in chickens, but only clade B viruses exhibited high virulence in ducks. Viral factors which contribute to virulence and transmission of these panzootic H5N8 2.3.4.4 viruses are largely unknown. The NS1 protein, typically composed of 230 amino acids (aa), is a multifunctional protein which is also a pathogenicity factor. Here, we studied the evolutionary trajectory of H5N8 NS1 proteins from 2013 to 2019 and their role in the fitness of H5N8 viruses in chickens and ducks. Sequence analysis and in vitro experiments indicated that clade 2.3.4.4A and clade 2.3.4.4B viruses have a preference for NS1 of 237 aa and 217 aa, respectively, over NS1 of 230 aa. NS217 was exclusively seen in domestic and wild birds in Europe. The extension of the NS1 C terminus (CTE) of clade B virus reduced virus transmission and replication in chickens and ducks and partially impaired the systemic tropism to the endothelium in ducks. Conversely, lower impact on fitness of clade A virus was observed. Remarkably, the NS1 of clade A and clade B, regardless of length, was efficient in blocking interferon (IFN) induction in infected chickens, and changes in the NS1 C terminus reduced the efficiency for interferon antagonism. Together, the NS1 C terminus contributes to the efficient transmission and high fitness of H5N8 viruses in chickens and ducks. IMPORTANCE The panzootic H5N8 highly pathogenic avian influenza viruses of clade 2.3.4.4A and 2.3.4.4B devastated the poultry industry globally. Clade 2.3.4.4A was predominant in 2014/2015 while clade 2.3.4.4B was widely spread in 2016/2017. The two clades exhibited different pathotypes in ducks. Virus factors contributing to virulence and transmission are largely unknown. The NS1 protein is typically composed of 230 amino acids (aa) and is an essential interferon (IFN) antagonist. Here, we found that the NS1 protein of clade 2.3.4.4A preferentially evolved toward long NS1 with 237 aa, while clade 2.3.4.4B evolved toward shorter NS1 with 217 aa (exclusively found in Europe) due to stop codons in the C terminus (CTE). We showed that the NS1 CTE of H5N8 is required for efficient virus replication, transmission, and endotheliotropism in ducks. In chickens, H5N8 NS1 evolved toward higher efficiency to block IFN response. These findings may explain the preferential pattern for short NS1 and high fitness of the panzootic H5N8 in birds.

Viral tropism and detection of clade 2.3.4.4b H5N8 highly pathogenic avian influenza viruses in feathers of ducks and geese.

Highly Pathogenic Avian Influenza viruses (HPAIVs) display a tissue pantropism, which implies a possible spread in feathers. HPAIV detection from feathers had been evaluated for H5N1 or H7N1 HPAIVs. It was suggested that viral RNA loads could be equivalent or higher in samples of immature feather compared to tracheal (TS) or cloacal swabs (CS). We investigated the suitability of feathers for the detection of clade 2.3.4.4b H5N8 HPAIV in ducks and geese field samples. In the six H5N8 positive flocks that were included in this study, TS, CS and immature wing feathers were taken from at least 10 birds. Molecular loads were then estimated using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) targetting H5 and M genes. In all flocks, viral loads were at least equivalent between feather and swab samples and in most cases up to 103 higher in feathers. Bayesian modelling confirmed that, in infected poultry, RT-qPCR was much more likely to be positive when applied on a feather sample only (estimated sensitivity between 0.89 and 0.96 depending on the positivity threshold) than on a combination of a tracheal and a cloacal swab (estimated sensitivity between 0.45 and 0.68 depending on the positivity threshold). Viral tropism and lesions in feathers were evaluated by histopathology and immunohistochemistry. Epithelial necrosis of immature feathers and follicles was observed concurrently with positive viral antigen detection and leukocytic infiltration of pulp. Accurate detection of clade 2.3.4.4b HPAIVs in feather samples were finally confirmed with experimental H5N8 infection on 10-week-old mule ducks, as viral loads at 3, 5 and 7 days post-infection were higher in feathers than in tracheal or cloacal swabs. However, feather samples were associated with lower viral loads than tracheal swabs at day 1, suggesting better detectability of the virus in feathers in the later course of infection. These results, based on both field cases and experimental infections, suggest that feather samples should be included in the toolbox of samples for detection of clade 2.3.4.4b HPAI viruses, at least in ducks and geese.

Respiratory disease due to mixed viral infections in poultry flocks in Egypt between 2017 and 2018: Upsurge of highly pathogenic avian influenza virus subtype H5N8 since 2018.

For several years, poultry production in Egypt has been suffering from co-circulation of multiple respiratory viruses including highly pathogenic avian influenza virus (HPAIV) H5N1 (clade 2.2.1.2) and low pathogenic H9N2 (clade G1-B). Incursion of HPAIV H5N8 (clade 2.3.4.4b) to Egypt in November 2016 via wild birds followed by spread into commercial poultry flocks further complicated the situation. Current analyses focussed on 39 poultry farms suffering from respiratory manifestation and high mortality in six Egyptian governorates during 2017-2018. Real-time RT-PCR (RT-qPCR) substantiated the co-presence of at least two respiratory virus species in more than 80% of the investigated flocks. The percentage of HPAIV H5N1-positive holdings was fairly stable in 2017 (12.8%) and 2018 (10.2%), while the percentage of HPAIV H5N8-positive holdings increased from 23% in 2017 to 66.6% during 2018. The proportion of H9N2-positive samples was constantly high (2017:100% and 2018:63%), and H9N2 co-circulated with HPAIV H5N8 in 22 out of 39 (56.8%) flocks. Analyses of 26 H5, 18 H9 and 4 N2 new sequences confirmed continuous genetic diversification. In silico analysis revealed numerous amino acid substitutions in the HA and NA proteins suggestive of increased adaptation to mammalian hosts and putative antigenic variation. For sensitive detection of H9N2 viruses by RT-qPCR, an update of primers and probe sequences was crucial. Reasons for the relative increase of HPAIV H5N8 infections versus H5N1 remained unclear, but lack of suitable vaccines against clade 2.3.4.4b cannot be excluded. A reconsideration of surveillance and control measures should include updating of diagnostic tools and vaccination strategies.

Spatiotemporal reconstruction and transmission dynamics during the 2016-17 H5N8 highly pathogenic avian influenza epidemic in Italy.

Effective control of avian diseases in domestic populations requires understanding of the transmission dynamics facilitating viral emergence and spread. In 2016-17, Italy experienced a significant avian influenza epidemic caused by a highly pathogenic A(H5N8) virus, which affected domestic premises housing around 2.7 million birds, primarily in the north-eastern regions with the highest density of poultry farms (Lombardy, Emilia-Romagna and Veneto). We perform integrated analyses of genetic, spatiotemporal and host data within a Bayesian phylogenetic framework. Using continuous and discrete phylogeography, we estimate the locations of movements responsible for the spread and persistence of the epidemic. The information derived from these analyses on rates of transmission between regions through time can be used to assess the success of control measures. Using an approach based on phylogenetic-temporal distances between domestic cases, we infer the presence of cryptic wild bird-mediated transmission, information that can be used to complement existing epidemiological methods for distinguishing transmission within the domestic population from incursions across the wildlife-domestic interface, a common challenge in veterinary epidemiology. Spatiotemporal reconstruction of the epidemic reveals a highly skewed distribution of virus movements with a high proportion of shorter distance local movements interspersed with occasional long-distance dispersal events associated with wild birds. We also show how such inference be used to identify possible instances of human-mediated movements where distances between phylogenetically linked domestic cases are unusually high.

Determinants of biosecurity practices in French duck farms after a H5N8 Highly Pathogenic Avian Influenza epidemic: The effect of farmer knowledge, attitudes and personality traits.

Biosecurity is crucial for infectious disease prevention, more importantly in the absence of vaccination. The need for improving the implementation of biosecurity practices was highlighted in French duck farms following the 2016-2017 H5N8 Highly Pathogenic Avian Influenza (HPAI) epidemic. Farmers have multiple reasons for not implementing biosecurity practices: external (time, money) and internal (socio-psychological). The purpose of this study was to determine how sets of socio-psychological factors (i.e. knowledge on biosecurity and avian influenza transmission, attitudes, personality traits, social background) affect the adoption of on-farm biosecurity practices. Biosecurity practices and socio-psychological determinants were assessed during 127 duck farm visits, in South West France, using both questionnaires and on-farm observations. Factorial analysis of mixed data (FAMD) and hierarchical clustering analysis (HCA) identified three groups of farmers with different socio-psychological profiles: the first group was characterized by minimal knowledge, negative attitudes towards biosecurity, little social pressure and a low level of conscientiousness. The second group was characterized by more extensive experience in poultry production, higher stress and social pressure. The third group was characterized by less experience in poultry production, better knowledge and positive attitudes towards biosecurity, increased self-confidence and orientation towards action. The first group had a significantly lower adoption of biosecurity measures than the two other groups. A better understanding of the factors involved in farmers' decision-making could improve the efficiency of interventions aiming at improving and maintaining the level of on-farm biosecurity in the duck industry.

Different environmental gradients associated to the spatiotemporal and genetic pattern of the H5N8 highly pathogenic avian influenza outbreaks in poultry in Italy.

Comprehensive understanding of the patterns and drivers of avian influenza outbreaks is pivotal to inform surveillance systems and heighten nations' ability to quickly detect and respond to the emergence of novel viruses. Starting in early 2017, the Italian poultry sector has been involved in the massive H5N8 highly pathogenic avian influenza epidemic that spread in the majority of the European countries in 2016/2017. Eighty-three outbreaks were recorded in north-eastern Italy, where a densely populated poultry area stretches along the Lombardy, Emilia-Romagna and Veneto regions. The confirmed cases, affecting both the rural and industrial sectors, depicted two distinct epidemic waves. We adopted a combination of multivariate statistics techniques and multi-model regression selection and inference, to investigate how environmental factors relate to the pattern of outbreaks diversity with respect to their spatiotemporal and genetic diversity. Results showed that a combination of eco-climatic and host density predictors were associated with the outbreaks pattern, and variation along gradients was noticeable among genetically and geographically distinct groups of avian influenza cases. These regional contrasts may be indicative of a different mechanism driving the introduction and spreading routes of the influenza virus in the domestic poultry population. This methodological approach may be extended to different spatiotemporal scale to foster site-specific, ecologically informed risk mitigating strategies.

Association of wild bird densities around poultry farms with the risk of highly pathogenic avian influenza virus subtype H5N8 outbreaks in the Netherlands, 2016.

Highly pathogenic (HP) avian influenza viruses (AIV) can spread globally through migratory birds and cause massive outbreaks in commercial poultry. AIV outbreaks have been associated with proximity to waterbodies, presence of waterfowl or wild bird cases near poultry farms. In this study, we compared densities of selected HPAI high-risk wild bird species around 7 locations (H farms) infected with HPAIV H5N8 in the Netherlands in 2016-2017 to densities around 21 non-infected reference farms. Nine reference farms were in low-lying water-rich areas (R-W) and 12 in higher non-water-rich areas (R-NW). Average monthly numbers/km2 of Eurasian wigeons, tufted ducks, Anatidae (ducks, geese and swans) and Laridae (gulls) were calculated between September and April in rings of 0-1, 1-3, 3-6 and 6-10 km around the farms. Linear mixed model analyses showed generally higher bird densities for H and R-W compared to R-NW farms between October and March. This was most striking for Eurasian wigeons, with in peak month December 105 (95% CI:17-642) and 40 (7-214) times higher densities around H and R-W farms, respectively, compared to R-NW farms. Increased densities around H farms for Eurasian wigeons and Anatidae were more pronounced for distances up to 10 km compared to 0-1 km that mostly consists of the farm yard, which is an unattractive habitat for waterfowl. This distance effect was not observed in gulls, nor in tufted ducks that live on large open waterbodies which are unlikely to be within 0-1 km of farms. This study provides insights into spatio-temporal density dynamics of HPAI high-risk birds around farms and their associations with poultry outbreaks. The outcomes indicate that knowledge of environmental and ecological drivers for wild bird presence and abundance may facilitate identification of priority areas for surveillance and biosecurity measures and decisions on establishments of poultry farms to reduce risk of HPAI outbreaks.

Highly pathogenic avian influenza subtype H5Nx clade 2.3.4.4 outbreaks in Dutch poultry farms, 2014-2018: Clinical signs and mortality.

In recent years, different subtypes of highly pathogenic avian influenza (HPAI) viruses caused outbreaks in several poultry types worldwide. Early detection of HPAI virus infection is crucial to reduce virus spread. Previously, the use of a mortality ratio threshold to expedite notification of suspicion in layer farms was proposed. The purpose of this study was to describe the clinical signs reported in the early stages of HPAI H5N8 and H5N6 outbreaks on chicken and Pekin duck farms between 2014 and 2018 in the Netherlands and compare them with the onset of an increased mortality ratio (MR). Data on daily mortality and clinical signs from nine egg-producing chicken farms and seven Pekin duck farms infected with HPAI H5N8 (2014 and 2016) and H5N6 (2017-2018) in the Netherlands were analysed. In 12 out of 15 outbreaks for which a MR was available, MR increase preceded or coincided with the first observation of clinical signs by the farmer. In one chicken and two Pekin duck outbreaks, clinical signs were observed prior to MR increase. On all farms, veterinarians observed clinical signs of general disease. Nervous or locomotor signs were reported in all Pekin duck outbreaks, but only in two chicken outbreaks. Other clinical signs were observed less frequently in both chickens and Pekin ducks. Compared to veterinarians, farmers observed and reported clinical signs, especially respiratory and gastrointestinal signs, less frequently. This case series suggests that a MR with a set threshold could be an objective parameter to detect HPAI infection on chicken and Pekin duck farms at an early stage. Observation of clinical signs may provide additional indication for farmers and veterinarians for notifying a clinical suspicion of HPAI infection. Further assessment and validation of a MR threshold in Pekin ducks are important as it could serve as an important tool in HPAI surveillance programs.

Enterotropism of highly pathogenic avian influenza virus H5N8 from the 2016/2017 epidemic in some wild bird species.

In 2016/2017, H5N8 highly pathogenic avian influenza (HPAI) virus of the Goose/Guangdong lineage spread from Asia to Europe, causing the biggest and most widespread HPAI epidemic on record in wild and domestic birds in Europe. We hypothesized that the wide dissemination of the 2016 H5N8 virus resulted at least partly from a change in tissue tropism from the respiratory tract, as in older HPAIV viruses, to the intestinal tract, as in low pathogenic avian influenza (LPAI) viruses, allowing more efficient faecal-oral transmission. Therefore, we determined the tissue tropism and associated lesions in wild birds found dead during the 2016 H5N8 epidemic, as well as the pattern of attachment of 2016 H5N8 virus to respiratory and intestinal tissues of four key wild duck species. We found that, out of 39 H5N8-infected wild birds of 12 species, four species expressed virus antigen in both respiratory and intestinal epithelium, one species only in respiratory epithelium, and one species only in intestinal epithelium. Virus antigen expression was association with inflammation and necrosis in multiple tissues. The level of attachment to wild duck intestinal epithelia of 2016 H5N8 virus was comparable to that of LPAI H4N5 virus, and higher than that of 2005 H5N1 virus for two of the four duck species and chicken tested. Overall, these results indicate that 2016 H5N8 may have acquired a similar enterotropism to LPAI viruses, without having lost the respirotropism of older HPAI viruses of the Goose/Guangdong lineage. The increased enterotropism of 2016 H5N8 implies that this virus had an increased chance to persist long term in the wild waterbird reservoir.

Pathologic Evaluation of Influenza A H5N8 Infection Outbreaks in Mule Ducks in Bulgaria.

This article outlines pathomorphologic findings of a study involving commercial mule ducks with confirmed influenza A H5N8 infections after a series of outbreaks in Bulgaria. Examinations were carried out after performing necropsy on dead birds from three different age groups (up to 15, 20 to 30, and 40+ days of age) fattened on different farms. Among birds of all ages, gross lesions were present as lesions affecting the heart. Histologically, the myocardium exhibited severe intermyofibrillar edema, moderate to massive hemorrhages, and degenerative changes. All lesions resulted in single or multiple and small to massive myocardial infarctions. Other affected organs included the brain, lungs, liver, spleen, and pancreas. Nonpurulent lymphocytic encephalitis was found postmortem in ducks that had shown prior clinical nervous signs. Among ducks of all ages, a viral antigen in the cardiomyocytes and the epithelium of air capillaries was found through immunohistochemical detection methods. The results of the present study allowed us to conclude that the highly pathogenic avian influenza A H5N8 viral infection may manifest itself as a systemic illness in commercial mule ducks with septicemic lesions, resulting in high morbidity and mortality rates of up to 100%. Pathomorphologic lesions were somewhat different from those previously reported in wild waterfowl.

Evaluación patológica de brotes de infección por influenza A H5N8 en patos mula en Bulgaria. Este artículo describe los hallazgos patomorfológicos de un estudio que involucró patos mula comerciales con infecciones confirmadas de influenza A H5N8 después de una serie de brotes en Bulgaria. Los exámenes se llevaron a cabo después de realizar la necropsia en aves muertas de tres grupos de edad diferentes (hasta 15 días, de 20 a 30 días y más de 40 días de edad) engordadas en diferentes granjas. Entre las aves de todas las edades, las lesiones macroscópicas estaban presentes como lesiones afectando el corazón. Histológicamente, el miocardio exhibió edema intermyofibrillar severo, hemorragias moderadas a masivas y cambios degenerativos. Todas las lesiones resultaron en infartos de miocardio simples o múltiples y de pequeños a masivos. Otros órganos afectados incluyeron el cerebro, los pulmones, el hígado, el bazo y el páncreas. Se encontró encefalitis linfocítica no supurativa se encontró post mortem en patos que habían mostrado signos nerviosos clínicos previos. Entre los patos de todas las edades, se encontró un antígeno viral en los cardiomiocitos y el epitelio de los capilares aéreos a través de métodos de detección inmunohistoquímica. Los resultados del presente estudio nos permitieron concluir que la infección viral altamente patógena de la influenza aviar H5N8 puede manifestarse como una enfermedad sistémica en patos mulos comerciales con lesiones septicémicas, lo que resulta en altas tasas de morbilidad y mortalidad de hasta el 100%. Las lesiones patomorfológicas fueron algo diferentes de las reportadas previamente en aves acuáticas silvestres.

Biosecurity risk factors for highly pathogenic avian influenza (H5N8) virus infection in duck farms, France.

Highly pathogenic avian influenza (HPAI) subtype H5N8 outbreaks occurred in poultry farms in France in 2016-2017, resulting in significant economic losses and disruption to the poultry industry. Current evidence on associations between actual on-farm biosecurity risk factors and H5N8 occurrence is limited. Therefore, a retrospective matched case-control study was undertaken to investigate the inter-relationships between on-farm biosecurity practices and H5N8 infection status to provide new insights regarding promising targets for intervention. Data were collected on 133 case and 133 control duck farms (i.e. the most affected species) located in one area of the country that was mostly affected by the disease. Data were analysed using Additive Bayesian Networks which offer a rich modelling framework by graphically illustrating the dependencies between variables. Factors indirectly and directly positively associated with farm infection were inadequate management of vehicle movements (odds ratio [OR] 9.3, 95% credible interval [CI] 4.0-22.8) and inadequate delimitation of farm and units (OR 3.0, 95% CI 1.6-5.8), respectively. Inadequate disposal of dead birds was instead negatively associated with the outcome (OR 0.1, 95% CI 0.0-0.3). The findings highlight that reinforcing farm access control systems and reducing the number of visitors are key biosecurity measures to control farm vulnerability to H5N8 infection and could help setting priorities in biosecurity practices to prevent outbreaks' re-occurrence.

Duck TRIM32 Functions in IFN-ß Signaling Against the Infection of H5N6 Highly Pathogenic Avian Influenza Virus.

In mammals, tripartite motif 32 (TRIM32) is essential for regulating host innate immune responses to viral infections. However, the antiviral effect of TRIM32 in birds has not been reported. Here, we cloned the full-length duck TRIM32 (duTRIM32) cDNA from total spleen RNA of Peking duck. DuTRIM32 consists of 682 amino acids and has 95.5% similarity in amino acid sequences with chicken TRIM32 and 84.9% similarity with human TRIM32, respectively. DuTRIM32 mRNA was found to be ubiquitously expressed in all tested tissues from healthy ducks. Overexpression of duTRIM32 significantly activated the IFN-ß promoter and upregulated the mRNA levels of IFN-ß, IRF7, and Mx, which indicates that duTRIM32 is involved in the type I IFN pathway. Furthermore, duTRIM32 was found to directly interact with duck STING (duSTING) and to contribute to the expression of IFN-ß mediated by duSTING. The mRNA level of duTRIM32 was significantly upregulated in the lungs and spleens of H5N6 highly pathogenic avian influenza virus (HPAIV) infected ducks 3 days post-infection (DPI). Furthermore, overexpression of duTRIM32 could inhibit the replication of H5N6 HPAIV in duck embryo fibroblasts (DEFs). Therefore, these results indicate that duTRIM32 is involved in the type I IFN pathway and exhibit an antiviral effect against H5N6 HPAIV infection.

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

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

Modulation of lethal HPAIV H5N8 clade 2.3.4.4B infection in AIV pre-exposed mallards.

In 2016/2017, a severe epidemic of HPAIV H5N8 clade 2.3.4.4 group B (H5N8B) affected Europe. To analyse the role of mallards in the spatiotemporal dynamics of global HPAIV H5N8B dispersal, mallards (Anas platyrhynchos), naturally exposed to various AIV and therefore seropositive, were challenged with H5N8B. All experiments were controlled by infection and co-housing of seronegative juvenile Pekin ducklings. All ducks that survived the first infection were re-challenged 21 dpi with the homologous H5N8B strain. After the first H5N8B infection, seropositive mallards showed only mild clinical symptoms. Moderate to low viral shedding, occurring particularly from the oropharynx and lasting for 7 days maximum, led to severe clinical disease of all contact ducklings. All challenged seronegative Pekin ducks and contact ducklings died or had to be euthanized. H5-specific antibodies were detected in surviving birds within 2 weeks. Virus and viral RNA could be isolated from several water samples until 6 and 9 dpi, respectively. Conversely, upon re-infection with homologous H5N8B neither inoculated nor contact ducklings showed any clinical symptoms, nor was an antibody titer increase of seropositive mallards or any seroconversion of contact ducklings observed. Mallard ducks naturally pre-exposed to LPAIV can play a role as a clinically unsuspicious virus reservoir for H5N8B effective in virus transmission. Mallards with homologous immunity did not contribute to virus transmission.

Domestic ducks play a major role in the maintenance and spread of H5N8 highly pathogenic avian influenza viruses in South Korea.

The H5N8 highly pathogenic avian influenza viruses (HPAIVs) belonging to clade 2.3.4.4 spread from Eastern China to Korea in 2014 and caused outbreaks in domestic poultry until 2016. To understand how H5N8 HPAIVs spread at host species level in Korea during 2014-2016, a Bayesian phylogenetic analysis was used for ancestral state reconstruction and estimation of the host transition dynamics between wild waterfowl, domestic ducks and chickens. Our data support that H5N8 HPAIV most likely transmitted from wild waterfowl to domestic ducks, and then maintained in domestic ducks followed by dispersal of HPAIV from domestic ducks to chickens, suggesting domestic duck population plays a central role in the maintenance, amplification and spread of wild HPAIV to terrestrial poultry in Korea.