Pathological and phylogenetic characteristics of fowl AOAV-1 and H5 isolated from naturally infected Meleagris Gallopavo.

BACKGROUND: In this study, we investigated the prevalence of respiratory viruses in four Hybrid Converter Turkey (Meleagris gallopavo) farms in Egypt. The infected birds displayed severe respiratory signs, accompanied by high mortality rates, suggesting viral infections. Five representative samples from each farm were pooled and tested for H5 & H9 subtypes of avian influenza viruses (AIVs), Avian Orthoavulavirus-1 (AOAV-1), and turkey rhinotracheitis (TRT) using real-time RT-PCR and conventional RT-PCR. Representative tissue samples from positive cases were subjected to histopathology and immunohistochemistry (IHC). RESULTS: The PCR techniques confirmed the presence of AOAV-1 and H5 AIV genes, while none of the tested samples were positive for H9 or TRT. Microscopic examination of tissue samples revealed congestion and hemorrhage in the lungs, liver, and intestines with leukocytic infiltration. IHC revealed viral antigens in the lungs, liver, and intestines. Phylogenetic analysis revealed that H5 HA belonged to 2.3.4.4b H5 sublineage and AOAV-1 belonged to VII 1.1 genotype. CONCLUSIONS: The study highlights the need for proper monitoring of hybrid converter breeds for viral diseases, and the importance of vaccination programs to prevent unnecessary losses. To our knowledge, this is the first study that reports the isolation of AOAV-1 and H5Nx viruses from Hybrid Converter Turkeys in Egypt.

Natural Infection with H5N1 Highly Pathogenic Influenza (HPAI) Virus in 5- and 10-Day-Old Commercial Pekin Ducklings (Anas platyrhynchos domesticus).

Highly pathogenic avian influenza (HPAI) has resulted in catastrophic economic losses globally in poultry. This case report describes the diagnostic detection and pathology of HPAI H5N1 in 5-day-old commercial ducklings, which is an atypical age for detection of natural infection of HPAI in poultry. The pathology observed at 5 days of age was also compared to lesions observed in ducklings from the same flock evaluated at 10 days of age before depopulation. The California Animal Health and Food Safety (CAHFS) Laboratory, Tulare, received ten 5-day-old Pekin duckling (Anas platyrhynchos domesticus) carcasses for diagnostic evaluation due to mortality that started increasing at 3 days of age. The most common gross findings included bilateral pulmonary edema with congestion and enlarged, mottled livers and spleens. Microscopically, cerebral neuronophagia, pancreatic necrosis, and interstitial pneumonia with pulmonary edema were observed in the 5-day-old ducklings. Oropharyngeal and cloacal swabs were positive for avian influenza virus (AIV) by real-time reverse transcriptase PCR. The AIV was typed as HPAI, EA/AM 2.3.4.4b H5N1 goose/Guangdong clade lineage by the National Veterinary Services Laboratory. Ducks at the affected premises were depopulated 4 days after the 5-day-old ducklings were submitted to the CAHFS lab, at which time additional tissue samples were collected for comparison to 10-day-old ducklings on the same premises. Differences in microscopic lesions and AIV tissue distribution were observed between the 5-day and 10-day tissues collected. Notably, microscopic lesions were more severe in the brain and pancreas at 10 days of age. Findings in 10-day-old ducklings included cerebral lymphoplasmacytic perivascular cuffing, gliosis, neuronal degeneration, and pancreatic necrosis. AIV antigen distribution and intensity was greatest in the cerebral tissue of the brains at 10 days and in the lungs at 5 days of age. To the authors' knowledge, published studies are limited on AIV natural infection in domestic ducks less than 9 days of age.

Infección natural con el virus de la influenza altamente patógena (HPAI) H5N1 en patitos Pekín comerciales (Anas platyrhynchos domesticus) de 5 y 10 días de edad. La influenza aviar altamente patógena (HPAI) ha provocado pérdidas económicas catastróficas en todo el mundo entre las aves de corral. Este reporte de caso describe la detección diagnóstica y la patología de la infección por un virus de influenza aviar de alta patogenicidad H5N1 en patitos comerciales de 5 días de edad, que es una edad atípica para la detección de la infección natural del virus de la influenza aviar de alta patogenicidad en avicultura. La patología observada a los 5 días de edad también se comparó con las lesiones observadas en patitos de la misma parvada evaluados a los 10 días de edad, antes de la despoblación. El Laboratorio de Salud Animal y Seguridad Alimentaria de California (CAHFS), con sede Tulare, recibió 10 cadáveres de patito Pekín (Anas platyrhynchos domesticus) de 5 días de edad para su evaluación diagnóstica debido a que la mortalidad comenzó a aumentar a los 3 días de edad. Los hallazgos macroscópicos más comunes incluyeron edema pulmonar bilateral con congestión en hígado y bazos agrandados y moteados. Microscópicamente se observó neuronofagia cerebral, necrosis pancreática y neumonía intersticial con edema pulmonar en los patitos de 5 días de edad. Los hisopos orofaríngeos y cloacales fueron positivos para el virus de la influenza aviar (AIV) mediante transcripción reversa y PCR en tiempo real. El Laboratorio Nacional de Servicios Veterinarios clasificó al virus como de alta patogenicidad EA/AM 2.3.4.4b H5N1 clado de linaje de ganso/clado Guangdong. Los patos en las instalaciones afectadas fueron despoblados 4 días después de que los patitos de 5 días fueran enviados al laboratorio de CAHFS, momento en el cual se recolectaron muestras de tejido adicionales para compararlas con patitos de 10 días de las mismas instalaciones. Se observaron diferencias en las lesiones microscópicas y la distribución del tejido del AIV entre los tejidos recolectados de 5 y 10 días. En particular, las lesiones microscópicas fueron más severas en el cerebro y en el páncreas a los 10 días de edad. Los hallazgos en patitos de 10 días incluyeron infiltraciones linfoplasmocitarias perivasculares en el cerebro, gliosis, degeneración neuronal y necrosis pancreática. La distribución e intensidad del antígeno de influenza aviar fue mayor en el tejido cerebral de los cerebros a los 10 días y en los pulmones a los 5 días de edad. De acuerdo al conocimiento de los autores, los estudios publicados sobre la infección natural por el virus de la influenza aviar en patos domésticos de menos de 9 días de edad son limitados.

Neurological examination of clinically healthy pigeons (Columba livia domestica), mute swans (Cygnus olor), common buzzards (Buteo buteo), common kestrels (Falco tinnunculus) and northern goshawks (Accipiter gentilis).

BACKGROUND: A neurological examination is essential for determining the localisation of neurological lesions. However, in avian species, quantitative data regarding the practicability and feasibility of neurological tests are very limited. Therefore, the aim of this study was to establish normative data for the neurological examination of clinically healthy birds of different species. METHODS: Forty-two domestic and feral pigeons (Columba livia domestica), 42 mute swans (Cygnus olor), 12 common buzzards (Buteo buteo), 24 common kestrels (Falco tinnunculus) and six northern goshawks (Accipiter gentilis) were examined. All birds underwent a predefined neurological examination. Interobserver variations between three examiners were investigated in 11 pigeons and 11 mute swans. RESULTS: All postural reaction tests, except for the drop and flap reaction in mute swans, provoked a consistent response in pigeons and mute swans, whereas postural reaction tests of the legs in raptors were often not performable. Cranial nerve tests and most of the spinal reflexes revealed variable responses in all birds. The gastrocnemius reflex was not provokable in any bird. Interobserver agreement was almost perfect (Gwet's AC1 coefficient ≥0.81) for 16 of 21 parameters in the examination in pigeons and for 14 of 21 in mute swans. LIMITATIONS: The inclusion of free-ranging birds, which were not used to handling and for which limited information regarding age, history of previous diseases, etc. was available, may have influenced the results. CONCLUSION: The normative neurological examination data provided in this study will help improve clinicians' interpretation of neurological examination results in the respective bird species.

Lesions and viral antigen distribution in bald eagles, red-tailed hawks, and great horned owls naturally infected with H5N1 clade 2.3.4.4b highly pathogenic avian influenza virus.

An epidemic of highly pathogenic avian influenza (HPAI) began in North America in the winter of 2021. The introduced Eurasian H5N1 clade 2.3.4.4b virus subsequently reassorted with North American avian influenza strains. This postmortem study describes the lesions and influenza A virus antigen distribution in 3 species of raptors, including bald eagles (Haliaeetus leucocephalus, n = 6), red-tailed hawks (Buteo jamaicensis, n = 9), and great horned owls (Bubo virginianus, n = 8), naturally infected with this virus strain based on positive reverse transcriptase polymerase chain reaction and sequencing results from oropharyngeal swabs. The birds presented with severe neurologic signs and either died or were euthanized because of the severity of their clinical signs and suspected influenza virus infection. Gross lesions were uncommon and included forebrain hemorrhages in 2 eagles, myocarditis in 1 hawk, and multifocal pancreatic necrosis in 3 owls. Histological lesions were common and included encephalitis, myocarditis, multifocal pancreas necrosis, multifocal adrenal necrosis, histiocytic splenitis, and anterior uveitis in decreasing frequency. Influenza A viral antigen was detected in brain, heart, pancreas, adrenal gland, kidney, spleen, liver, and eye. In conclusion, bald eagles, red-tailed hawks, and great horned owls infected with the HPAI clade 2.3.4.4b virus strain and showing neurological signs of illness may develop severe or fatal disease with histologically detectable lesions in the brain that are frequently positive for viral antigen.

Highly pathogenic avian influenza virus H5N1 infection in skua and gulls in the United Kingdom, 2022.

The reemergence of the highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in the United Kingdom in 2021-2022 has caused unprecedented epizootic events in wild birds and poultry. During the summer of 2022, there was a shift in virus transmission dynamics resulting in increased HPAIV infection in seabirds, and consequently, a profound impact on seabird populations. To understand the pathological impact of HPAIV in seabirds, we evaluated the virus antigen distribution and associated pathological changes in the tissues of great skua (Stercorarius skua, n = 8), long-tailed skua (Stercorarius longicaudus, n = 1), European herring gull (Larus argentatus, n = 5), and black-headed gull (Chroicocephalus ridibundus, n = 4), which succumbed to natural infection of HPAIV during the summer of 2022. Cases were collected from Shetland, including Scatness (mainland), No Ness (mainland), Clumlie (mainland), Hermaness (island), Fair Isle (island), Noss (island), and the West Midlands, South East, and South West of England. Grossly, gizzard ulceration was observed in one great skua and pancreatic necrosis was observed in 4 herring gulls, with intralesional viral antigen detected subsequently. Microscopical analysis revealed neuro-, pneumo-, lymphoid-, and cardiomyotropism of HPAIV H5N1, with the most common virus-associated pathological changes being pancreatic and splenic necrosis. Examination of the reproductive tract of the great skua revealed HPAIV-associated oophoritis and salpingitis, and virus replication within the oviductal epithelium. The emergence of HPAIV in seabirds Stercorariidae and Laridae, particularly during summer 2022, has challenged the dogma of HPAIV dynamics, posing a significant threat to wild bird life with potential implications for the reproductive performance of seabirds of conservation importance.

Naturally occurring highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b infection in three domestic cats in North America during 2023.

The Eurasian strain of highly pathogenic avian influenza (HPAI) H5N1 is a devastating pathogen for birds that also has the capacity to infect mammals. This report describes the presentation, clinical case findings (including haemogram and serum biochemistry), gross and microscopic lesions and virus detection in three HPAI H5N1-infected domestic cats from the USA in 2023. All three cats presented with neurological abnormalities and were euthanized due to a poor prognosis within 2 days (two cats) or 10 days (one cat) of known clinical disease onset. Necropsy consistently revealed pulmonary congestion and oedema, and cerebrocortical malacia with haemorrhage was also seen in the cat that survived for 10 days. On histology, all cats had necrotizing encephalitis and interstitial pneumonia with pulmonary congestion, oedema, vasculitis and vascular thrombosis. One cat also had microscopic multifocal necrosis in the liver, pancreas and an adrenal gland. To our knowledge, this report is the first to detail pathological findings in HPAI H5N1 naturally-infected cats during the widespread outbreak in North America beginning in 2021, and that describes a cat surviving for 10 days after onset of HPAI H5N1 encephalitis.

Controlling the emergence of highly pathogenic avian influenza viruses: one of the challenges of the 21st century? / La lutte contre l'émergence des virus influenza aviaires hautement pathogènes : un des défis du XXIe siècle ?

Millions of farmed birds culled, hundreds of gannets dead on the coast, dying marine mammals suffering from neurological disorders: these events regularly make the headlines. What do they have in common? Highly pathogenic avian influenza viruses (HPAIV). HPAIVs are viruses capable of replicating systemically, causing both asymptomatic infections and devastating mortality, depending on the susceptibility of the host species. Known for several decades now, these viruses have seen their circulation particularly increased in recent years, and have been responsible for massive epizootics on several continents. In addition to the devastating effects they can cause in poultry and wildlife, HPAIVs are also capable of crossing the species barrier. Improving knowledge about these viruses and better control of their spread therefore has several objectives: to protect public health, to guarantee food safety, to preserve biodiversity and the economy of the poultry industry. This article reviews the current state of knowledge on HPAIVs: from their epidemiology to the mechanisms of emergence and control measures.

Des millions d'oiseaux d'élevage abattus, des centaines de fous de Bassan morts sur le littoral, des mammifères marins agonisants atteints de troubles neurologiques : ces événements font régulièrement la une de l'actualité. Leur point commun ? Les virus influenza aviaires hautement pathogènes (VIAHP). Les VIAHP sont des virus capables de se répliquer de manière systémique, engendrant des infections asymptomatiques comme une mortalité foudroyante, en fonction de la sensibilité des espèces hôtes. Connus depuis maintenant plusieurs décennies, ces virus ont vu leur circulation particulièrement augmenter ces dernières années et ont été responsables d'épizooties massives sur plusieurs continents. Aux effets dévastateurs qu'ils peuvent causer dans les filières avicoles et dans la faune sauvage, les VIAHP sont également capables de franchir la barrière d'espèce. Mieux les connaître et mieux maîtriser leur diffusion revêt donc plusieurs objectifs : protéger la santé publique, garantir la sécurité alimentaire, préserver la biodiversité ou encore l'économie des filières avicoles. Cet article de synthèse fait un état de l'art des connaissances actuelles sur les VIAHP : de leur épidémiologie aux mécanismes d'émergence en passant par les mesures de lutte.

First Report of Sarconema eurycerca Heart Infection in Mute Swans in Serbia.

BACKGROUND: The Sarconema eurycerca heart infection may be one of the main causes of death in swans and geese due to severe heart lesions. To date, there have been no reports of Sarconema eurycerca in mute swans (Cygnus olor) in Serbia and this parasite has never been morphologically characterized in wild birds from this region. METHODS: In spring 2021, eight dead mute swans (Cygnus olor) were collected during avian influenza surveillance on the territory of the Province of Vojvodina and submitted to Scientific Veterinary Institute "Novi Sad" to determine the cause of death. The presence of highly pathogenic avian influenza subtype H5N1 was determined in all swans using molecular methods. RESULTS: Necropsy of the swans revealed changes characteristic for highly pathogenic avian influenza infection, such as severe haemorrhages and necrosis in different organs. Three of the eight swans showed massive subepicardial haemorrhages and myocarditis with intralesional 2-4 cm long whitish roundworms. Histopathologically, haemorrhages and many adult heart worms were observed in the myocardium. Based on morphological features of parasites, infection by Sarconema eurycerca was concluded. CONCLUSION: According to the available scientific literature, the presence of heart roundworms in swans has not yet been confirmed in our country. This report is the first published case of cardiac filariasis associated with S. eurycerca infection in mute swans in the Republic of Serbia.

Gross pathology associated with highly pathogenic avian influenza H5N8 and H5N1 in naturally infected birds in the UK (2020-2021).

BACKGROUND: Multiple outbreaks with highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b viruses, including H5N8 and H5N1, have occurred in the United Kingdom, as well as in other European countries, since late 2020. METHODS: This report describes the pathology among poultry species (chickens, turkeys, ducks, and pheasants) and captive birds (Black Swans, a whistling duck and peregrine falcons) naturally infected with HPAIV from 22 cases of HPAIV H5N8 and two cases of HPAIV H5N1 outbreaks investigated between October 2020 and April 2021. RESULTS: On gross examination, pancreatic necrosis was easily identified and most commonly observed in galliformes infected with both subtypes of HPAIV but rarely in anseriformes. In addition, splenic necrosis was also frequently observed in chickens and turkeys infected with HPAIV H5N8. Other less common lesions included cardiac petechiae, serosal haemorrhages and ascites in a variety of species. CONCLUSION: Given the widespread dissemination of HPAIV infection in susceptible avian species during autumn/winter 2020-2021, these data, when evaluated along with clinical information, is a valuable first step for both veterinarians and field services to evaluate gross pathology at post-mortem to support the diagnosis of HPAIV infection.

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.

Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses.

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 104 egg infective dose 50 (EID50) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID50 105), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.

Single dose of multi-clade virus-like particle vaccine protects chickens against clade 2.3.2.1 and clade 2.3.4.4 highly pathogenic avian influenza viruses.

Virus-like particles (VLPs) are recognized as an alternative vaccine platform that provide effective protection against various highly pathogenic avian influenza viruses (HPAIVs). Here, we developed multi-clade VLPs expressing two HAs (a chimera of clade 2.3.2.1c and clade 2.3.4.4c HA) within a single vector. We then compared its protective efficacy with that of a monovalent VLP and evaluated its potency against each homologous strain. Chickens vaccinated with the multi-clade VLP shed less virus and were better protected against challenge than birds receiving monovalent vaccines. Single vaccination with a multi-clade VLP resulted in 100% survival, with no clinical symptoms and high levels of pre-challenge protective immunity (7.6-8.5 log2). Moreover, the multi-clade VLP showed high productivity (128-256 HAU) both in the laboratory and on a large scale, making it cheaper than whole inactivated vaccines produced in eggs. However, the PD50 (protective dose 50%) of the multi-clade VLP against clades 2.3.2.1c and 2.3.4.4c was < 50 PD50 (28 and 42 PD50, respectively), and effective antibody response was maintained for 2-3 months. This multi-clade VLP protects against both clades of HPAI viruses and can be produced in high amounts at low cost. Thus, the vaccine has potential as a pandemic preparedness vaccine.

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.

H7N7 Avian Influenza Virus Mutation from Low to High Pathogenicity on a Layer Chicken Farm in the UK.

Avian influenza virus (AIV) subtypes H5 and H7 are capable of mutating from low to high pathogenicity strains, causing high mortality in poultry with significant economic losses globally. During 2015, two outbreaks of H7N7 low pathogenicity AIV (LPAIV) in Germany, and one each in the United Kingdom (UK) and The Netherlands occurred, as well as single outbreaks of H7N7 high pathogenicity AIV (HPAIV) in Germany and the UK. Both HPAIV outbreaks were linked to precursor H7N7 LPAIV outbreaks on the same or adjacent premises. Herein, we describe the clinical, epidemiological, and virological investigations for the H7N7 UK HPAIV outbreak on a farm with layer chickens in mixed free-range and caged units. H7N7 HPAIV was identified and isolated from clinical samples, as well as H7N7 LPAIV, which could not be isolated. Using serological and molecular evidence, we postulate how the viruses spread throughout the premises, indicating potential points of incursion and possible locations for the mutation event. Serological and mortality data suggested that the LPAIV infection preceded the HPAIV infection and afforded some clinical protection against the HPAIV. These results document the identification of a LPAIV to HPAIV mutation in nature, providing insights into factors that drive its manifestation during outbreaks.

Estimating epidemiological parameters using diagnostic testing data from low pathogenicity avian influenza infected turkey houses.

Limiting spread of low pathogenicity avian influenza (LPAI) during an outbreak is critical to reduce the negative impact on poultry producers and local economies. Mathematical models of disease transmission can support outbreak control efforts by estimating relevant epidemiological parameters. In this article, diagnostic testing data from each house on a premises infected during a LPAI H5N2 outbreak in the state of Minnesota in the United States in 2018 was used to estimate the time of virus introduction and adequate contact rate, which determines the rate of disease spread. A well-defined most likely time of virus introduction, and upper and lower 95% credibility intervals were estimated for each house. The length of the 95% credibility intervals ranged from 11 to 22 with a mean of 17 days. In some houses the contact rate estimates were also well-defined; however, the estimated upper 95% credibility interval bound for the contact rate was occasionally dependent on the upper bound of the prior distribution. The estimated modes ranged from 0.5 to 6.0 with a mean of 2.8 contacts per day. These estimates can be improved with early detection, increased testing of monitored premises, and combining the results of multiple barns that possess similar production systems.

Pathology of an outbreak of highly pathogenic avian influenza A(H5N1) virus of clade 2.3.2.1a in turkeys in Bangladesh.

A mixed-aged flock of 130 turkeys in Bangladesh reported the sudden death of 1 bird in September 2017. Highly pathogenic avian influenza A(H5N1) virus was detected in 3 turkeys, and phylogenetic analysis placed the viruses in the reassortant clade 2.3.2.1a. The birds had clinical signs of depression, diarrhea, weakness, closed eyes, and finally death. The mortality rate of the flock was 13% over the 6 d prior to the flock being euthanized. At autopsy, we observed congestion in lungs and brain, hemorrhages in the trachea, pancreas, breast muscle, coronary fat, intestine, bursa of Fabricius, and kidneys. Histopathology revealed hemorrhagic pneumonia, hemorrhages in the liver and kidneys, and hemorrhages and necrosis in the spleen and pancreas. Significant changes in the brain included gliosis, focal encephalomalacia and encephalitis, and neuronophagia.

Emergence of Novel Reassortant H1N1 Avian Influenza Viruses in Korean Wild Ducks in 2018 and 2019.

Influenza A virus subtype H1N1 has caused global pandemics like the "Spanish flu" in 1918 and the 2009 H1N1 pandemic several times. H1N1 remains in circulation and survives in multiple animal sources, including wild birds. Surveillance during the winter of 2018-2019 in Korea revealed two H1N1 isolates in samples collected from wild bird feces: KNU18-64 (A/Greater white-fronted goose/South Korea/KNU18-64/2018(H1N1) and WKU19-4 (A/wild bird/South Korea/WKU19-4/2019(H1N1). Phylogenetic analysis indicated that M gene of KNU18-64(H1N1) isolate resembles that of the Alaskan avian influenza virus, whereas WKU19-4(H1N1) appears to be closer to the Mongolian virus. Molecular characterization revealed that they harbor the amino acid sequence PSIQRSGLF and are low-pathogenicity influenza viruses. In particular, the two isolates harbored three different mutation sites, indicating that they have different virulence characteristics. The mutations in the PB1-F2 and PA protein of WKU19-4(H1N1) indicate increasing polymerase activity. These results corroborate the kinetic growth data for WKU19-4 in MDCK cells: a dramatic increase in the viral titer after 12 h post-inoculation compared with that in the control group H1N1 (CA/04/09(pdm09)). The KNU18-64(H1N1) isolate carries mutations indicating an increase in mammal adaptation; this characterization was confirmed by the animal study in mice. The KNU18-64(H1N1) group showed the presence of viruses in the lungs at days 3 and 6 post-infection, with titers of 2.71 ± 0.16 and 3.71 ± 0.25 log10(TCID50/mL), respectively, whereas the virus was only detected in the WKU19-4(H1N1) group at day 6 post-infection, with a lower titer of 2.75 ± 0.51 log10(TCID50/mL). The present study supports the theory that there is a relationship between Korea and America with regard to reassortment to produce novel viral strains. Therefore, there is a need for increased surveillance of influenza virus circulation in free-flying and wild land-based birds in Korea, particularly with regard to Alaskan and Asian strains.

Continued Evolution of H5Nx Avian Influenza Viruses in Bangladeshi Live Poultry Markets: Pathogenic Potential in Poultry and Mammalian Models.

The genesis of novel influenza viruses through reassortment poses a continuing risk to public health. This is of particular concern in Bangladesh, where highly pathogenic avian influenza viruses of the A(H5N1) subtype are endemic and cocirculate with other influenza viruses. Active surveillance of avian influenza viruses in Bangladeshi live poultry markets detected three A(H5) genotypes, designated H5N1-R1, H5N1-R2, and H5N2-R3, that arose from reassortment of A(H5N1) clade 2.3.2.1a viruses. The H5N1-R1 and H5N1-R2 viruses contained HA, NA, and M genes from the A(H5N1) clade 2.3.2.1a viruses and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. H5N2-R3 viruses contained the HA gene from circulating A(H5N1) clade 2.3.2.1a viruses, NA and M genes from concurrently circulating A(H9N2) influenza viruses, and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. Representative viruses of all three genotypes and a parental clade 2.3.2.1a strain (H5N1-R0) infected and replicated in mice without prior adaptation; the H5N2-R3 virus replicated to the highest titers in the lung. All viruses efficiently infected and killed chickens. All viruses replicated in inoculated ferrets, but no airborne transmission was detected, and only H5N2-R3 showed limited direct-contact transmission. Our findings demonstrate that although the A(H5N1) viruses circulating in Bangladesh have the capacity to infect and replicate in mammals, they show very limited capacity for transmission. However, reassortment does generate viruses of distinct phenotypes.IMPORTANCE Highly pathogenic avian influenza A(H5N1) viruses have circulated continuously in Bangladesh since 2007, and active surveillance has detected viral evolution driven by mutation and reassortment. Recently, three genetically distinct A(H5N1) reassortant viruses were detected in live poultry markets in Bangladesh. Currently, we cannot assign pandemic risk by only sequencing viruses; it must be conducted empirically. We found that the H5Nx highly pathogenic avian influenza viruses exhibited high virulence in mice and chickens, and one virus had limited capacity to transmit between ferrets, a property considered consistent with a higher zoonotic risk.

Experimental infection and pathology of two highly pathogenic avian influenza H5N1 viruses isolated from crow and chicken in house crows (Corvus splendens).

We investigated the experimental infection of two highly pathogenic avian influenza H5N1 viruses isolated from crow (A/crow/Assam/142119/2008) and chicken (A/chicken/Sikkim/151466/2009) in house crows (Corvus splendens). Both viruses caused infection in crows, where four out of six and three out of six crows succumbed to H5N1 infection within 11 days post challenge by crow and chicken viruses, respectively. The major clinical signs in crows were wing paralysis, circling and torticollis. The virus shedding detected from swabs was not persistent in both crow nor chicken viruses. Both viruses were isolated more frequently from oral swabs than from cloacal swabs. Both virus strains were isolated from brain, lungs, heart, liver, pancreas, spleen, large intestines of crows that succumbed to H5N1 infection. The surviving birds seroconverted in response to H5N1 virus infection. Microscopically, both viruses caused coagulative necrosis in pancreas and kidneys. Brain showed gliosis and neuronal degeneration. This experimental study highlights that crows could be infected with H5N1 viruses from different hosts with minor differences in pathogenicity. Therefore, it is imperative to carry out surveillance of highly pathogenic avian influenza H5N1 virus in synanthropic birds along with biosecurity measures to mitigate the H5N1 spread in poultry population. Keywords: chicken virus; crow virus; highly pathogenic avian influenza; house crows.

The pathogenesis of a North American H5N2 clade 2.3.4.4 group A highly pathogenic avian influenza virus in surf scoters (Melanitta perspicillata).

BACKGROUND: Aquatic waterfowl, particularly those in the order Anseriformes and Charadriiformes, are the ecological reservoir of avian influenza viruses (AIVs). Dabbling ducks play a recognized role in the maintenance and transmission of AIVs. Furthermore, the pathogenesis of highly pathogenic AIV (HPAIV) in dabbling ducks is well characterized. In contrast, the role of diving ducks in HPAIV maintenance and transmission remains unclear. In this study, the pathogenesis of a North American A/Goose/1/Guangdong/96-lineage clade 2.3.4.4 group A H5N2 HPAIV, A/Northern pintail/Washington/40964/2014, in diving sea ducks (surf scoters, Melanitta perspicillata) was characterized. RESULTS: Intrachoanal inoculation of surf scoters with A/Northern pintail/Washington/40964/2014 (H5N2) HPAIV induced mild transient clinical disease whilst concomitantly shedding high virus titers for up to 10 days post-inoculation (dpi), particularly from the oropharyngeal route. Virus shedding, albeit at low levels, continued to be detected up to 14 dpi. Two aged ducks that succumbed to HPAIV infection had pathological evidence for co-infection with duck enteritis virus, which was confirmed by molecular approaches. Abundant HPAIV antigen was observed in visceral and central nervous system organs and was associated with histopathological lesions. CONCLUSIONS: Collectively, surf scoters, are susceptible to HPAIV infection and excrete high titers of HPAIV from the respiratory and cloacal tracts whilst being asymptomatic. The susceptibility of diving sea ducks to H5 HPAIV highlights the need for additional research and surveillance to further understand the contribution of diving ducks to HPAIV ecology.