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Influenza A viruses (FLUAV) of the H9N2 subtype are zoonotic pathogens that cause significant economic damage to the poultry industry. Vaccination to prevent and control H9N2 infections in poultry is widely employed in the Middle East and Asia. We used phylogenetics and antigenic analysis to study the antigenic properties of the H9 hemagglutinin (HA) using sera produced in Japanese quail (Coturnix c. japonica). Consensus HA1 sequences were generated to capture antigenic diversity among isolates. We constructed chimeric H9N2 viruses containing the HA1 of each consensus sequence on a constant isogenic backbone. The resulting viruses were used to generate antisera from quail, a common and significant minor poultry species whose anti-HA response profiles remain poorly defined. Antigenic maps were generated by plotting the cross-hemagglutination inhibition (HI) data from the panel of quail sera against the chimeric constructs and 51 H9 field isolates. The chimeric antigens were divided into four different antigenic profiles (cyan, blue, orange, and red). Site-directed mutagenesis analysis showed 9 amino acid positions of antigenic relevance. Substitutions at amino acid positions 149, 150, and 180 (H9 HA numbering) had relatively significant impact on HI activity using quail sera. Substitutions E180A and R131K/E180A led to the most significant antigenic change transitions. This study provides insights into the antigenic profile of H9 FLUAVs, with important implications for understanding antigenic drift and improving vaccine development for use in minor poultry species.IMPORTANCEDetermining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 HA using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species.

The 2021/2022 epizootic of high pathogenicity avian influenza (HPAIV) remains one of the largest ever in the UK, being caused by a clade 2.3.4.4b H5N1 HPAIV. This epizootic affected more than 145 poultry premises, most likely through independent incursion from infected wild birds, supported by more than 1700 individual detections of H5N1 from wild bird mortalities. Here an H5N1 HPAIV, representative of this epizootic (H5N1-21), was used to investigate its virulence, pathogenesis and transmission in layer chickens and Pekin ducks, two species of epidemiological importance. We inoculated both avian species with decreasing H5N1-21 doses. The virus was highly infectious in ducks, with high infection levels and accompanying shedding of viral RNA, even in ducks inoculated with the lowest dose, reflecting the strong waterfowl adaptation of the clade 2.3.4.4 HPAIVs. Duck-to-duck transmission was very efficient, coupled with high environmental contamination. H5N1-21 was frequently detected in water sources, serving as likely sources of infection for ducks, but inhalable dust and aerosols represented low transmission risks. In contrast, chickens inoculated with the highest dose exhibited lower rates of infection compared to ducks. There was no evidence for experimental H5N1-21 transmission to any naive chickens, in two stocking density scenarios, coupled with minimal and infrequent contamination being detected in the chicken environment. Systemic viral dissemination to multiple organs reflected the pathogenesis and high mortalities in both species. In summary, the H5N1-21 virus is highly infectious and transmissible in anseriformes, yet comparatively poorly adapted to galliformes, supporting strong host preferences for wild waterfowl. Key environmental matrices were also identified as being important in the epidemiological spread of this virus during the continuing epizootic.

During 2021-22 High Pathogenicity Avian Influenza (HPAI) killed thousands of wild birds across Europe and North America, suggesting a change in infection dynamics and a shift to new hosts, including seabirds. Northern Gannets (Morus bassanus) appeared especially severely impacted, but limited understanding of how the virus spread across the metapopulation, or the demographic consequences of mass mortality limit our understanding of its severity. Accordingly, we collate information on HPAIV outbreaks across most North Atlantic gannet colonies and for the largest colony (Bass Rock, UK), provide impacts on population size, breeding success, adult survival, and preliminary results on serology. Unusually high numbers of dead gannets were first noted in Iceland during April 2022. Outbreaks in May occurred in many Scottish colonies, followed by colonies in Canada, Germany and Norway. By the end of June, outbreaks had occurred in five Canadian colonies and in the Channel Islands. Outbreaks in 12 UK and Ireland colonies appeared to follow a clockwise pattern with the last infected colonies recorded in late August/September. Unusually high mortality was recorded at 40 colonies (75% of global total colonies). Dead birds testing positive for HPAIV H5N1 were associated with 58% of these colonies. At Bass Rock, the number of occupied sites decreased by at least 71%, breeding success declined by ∼66% compared to the long-term UK mean and adult survival between 2021 and 2022 was 42% lower than the preceding 10-year average. Serological investigation detected antibodies specific to H5 in apparently healthy birds indicating that some gannets recover from HPAIV infection. Further, most of these recovered birds had black irises, suggestive of a phenotypic indicator of previous infection. Untangling the impacts of HPAIV infection from other key pressures faced by seabirds is key to establishing effective conservation strategies for threatened seabird populations, HPAIV being a novel and pandemic threat.

Since October 2021, Europe has experienced the largest avian influenza virus (AIV) epizootic, caused by clade 2.3.4.4b H5N1 high pathogenicity AIV (HPAIV), with over 320 poultry and captive bird infected premises (IPs) and 2480 dead H5N1 positive wild birds detected in Great Britain alone. Many IPs have been detected as geographical clusters, raising questions around potential lateral spread between premises by airborne particles. Airborne transmission over short distances has been reported for some AIVs strains. However, the risk of airborne spread of this strain remains to be elucidated. We conducted extensive sampling from IPs where clade 2.3.4.4b H5N1 HPAIVs was confirmed during the 2022/23 epizootic, each representing a major poultry species (ducks, turkeys, and chickens). A range of environmental samples were collected inside and outside houses, including deposited dust, feathers, and other potential fomites. Viral RNA (vRNA) and infectious virus were detected in air samples collected from inside and outside, but in close proximity, of infected houses, with vRNA alone being detected greater distances (>10m) outside. Some dust samples collected outside of the affected houses contained infectious virus, while feathers from the affected houses, located up to 60m away, only contained vRNA. Together, these data suggest that airborne particles harbouring infectious HPAIV can be translocated short distances (IMPORTANCEUnderstanding the routes of avian influenza virus incursion into poultry premises is critical to ensure the implementation of effective preventative measures, reducing the risk of spread of the virus. Extensive environmental sampling across three infected premises, representing the major global commercial poultry species, detected viral RNA and infectious virus in air samples, dust, and other fomites inside of the houses containing infected poultry, as well as within the immediate proximity of infected houses. This suggests that particles harbouring infectious virus may travel short distances outside of affected houses, which may potentially contribute to house-to-house transmission on the same premises, but not at distances likely to contribute to the transmission to another poultry premises. Consequently, control efforts should be focused on traditional, robust biosecurity measures at key barriers on poultry houses. Human behaviour in and around poultry houses as factors contributing to biosecurity risks should be considered.

This focus article has been prepared by Marco Falchieri, Scott M. Reid, Craig S. Ross, Joe James, Alexander M. P. Byrne, Madalina Zamfir, Ian H. Brown and Ashley C. Banyard of the APHA; Glen Tyler and Emma Philip of NatureScot; and Will Miles of Scottish Oceans Institute, School of Biology, University of St Andrews.

Angiotensin converting enzyme 2 (ACE2) is a host cell membrane protein (receptor) that mediates the binding of coronavirus, most notably SARS coronaviruses in the respiratory and gastrointestinal tracts. Although SARS-CoV-2 infection is mainly confined to humans, there have been numerous incidents of spillback (reverse zoonoses) to domestic and captive animals. An absence of information on the spatial distribution of ACE2 in animal tissues limits our understanding of host species susceptibility. Here, we describe the distribution of ACE2 using immunohistochemistry (IHC) on histological sections derived from carnivores, ungulates, primates and chiroptera. Comparison of mink (Neovison vison) and ferret (Mustela putorius furo) respiratory tracts showed substantial differences, demonstrating that ACE2 is present in the lower respiratory tract of mink but not ferrets. The presence of ACE2 in the respiratory tract in some species was much more restricted as indicated by limited immunolabelling in the nasal turbinate, trachea and lungs of cats (Felis catus) and only the nasal turbinate in the golden Syrian hamster (Mesocricetus auratus). In the lungs of other species, ACE2 could be detected on the bronchiolar epithelium of the sheep (Ovis aries), cattle (Bos taurus), European badger (Meles meles), cheetah (Acinonyx jubatus), tiger and lion (Panthera spp.). In addition, ACE2 was present in the nasal mucosa epithelium of the serotine bat (Eptesicus serotinus) but not in pig (Sus scrofa domestica), cattle or sheep. In the intestine, ACE2 immunolabelling was seen on the microvillus of enterocytes (surface of intestine) across various taxa. These results provide anatomical evidence of ACE2expression in a number of species which will enable further understanding of host susceptibility and tissue tropism of ACE2 receptor-mediated viral infection

The re-emergence of the high pathogenicity 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 have evaluated the virus 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). Grossly there was gizzard ulceration in one great skua and pancreatic necrosis in four herring gulls, which were confirmed for virus infectionin situby immunohistochemistry. Microscopical analysis revealed neuro-, pneumo-, lymphoidand cardiotropism 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. Across the birds, epitheliotropism was evident in the intestine, nasal turbinate, and trachea. This was, in contrast, not observed in the 2021 summer mortality event in great skuas and may be significant for the disease epidemiology observed in 2022. The emergence of HPAIV in seabirds, particularly during summer 2022, has challenged the dogma of HPAIV dynamics, posing a significant threat to wild bird life with potential implications to the reproductive performance of seabirds of conservation importance.

Newcastle Disease (ND), caused by virulent forms of Avian orthoavulavirus serotype-1 (AOAV-1) is an economically important avian disease worldwide. The past two incursions of ND into the United Kingdom occurred in game bird populations during 2005 and 2006. The nature of the game bird semi-feral rearing system, which can bring these birds into close contact with both wild birds and commercial or backyard poultry, has been hypothesized to act as a bridge between these two environments. As such, the risk that AOAV-1-infected game birds may pose to the UK poultry industry was investigated. Pheasants, partridges and chickens were experimentally infected with the virulent strain APMV-1/Chicken/Bulgaria/112/13, a genotype VII.2 virus associated with ND outbreaks in Eastern Europe. The study demonstrated that both chickens and pheasants are susceptible to infection with APMV-1/Chicken/Bulgaria/112/13, which results in high mortality and onward transmission. Partridges by contrast are susceptible to infection, but mortality was reduced, as was onward transmission. However, the data indicated that both pheasants and partridges may serve as intermediate hosts of AOAV-1 and may bridge the wild bird–domestic poultry interface enabling transmission into an economically damaging environment where morbidity and mortality may be high.

Swine influenza is an acute respiratory disease of swine caused by swine influenza A virus (SwIAV). The ability of SwIAV to spread bidirectionally from animals to humans (zoonotic), and from humans to animals (reverse zoonotic), drives coinfection that can result in gene segment exchange and elevates the risk of generating viruses with pandemic potential. Compared to human-origin influenza A viruses, current data indicate a greater diversity amongst circulating SwIAVs, with three major subtypes (classified by haemagglutinin and neuraminidase) circulating globally in swine (H1N1, H1N2 and H3N2). The lack of protection afforded by human seasonal influenza vaccines against SwIAVs exacerbates the risk associated with reassortment of human, swine and potentially avian viruses. As such, global monitoring of SwIAVs is important for both human and animal health as they represent a true ‘One Health’ challenge with pandemic potential.

Since 2020, the UK and Europe, have experienced annual epizootics of high pathogenicity avian influenza virus (HPAIV). The first during autumn/winter 2020/21 involved the detected with six H5Nx subtypes although H5N8 HPAIV dominated in the UK. Whilst genetic assessment of the H5N8 HPAIVs within the UK demonstrated relative homogeneity, there was a background of other genotypes circulating at a lower degree with different neuraminidase and internal genes. Following a small number of summer detections of H5N1 in wild birds over the summer of 2021, autumn/winter 2021/22 saw another European H5 HPAIV epizootic, that has dwarfed the prior epizootic. This second epizootic was dominated almost exclusively by H5N1 HPAIV, although six distinct genotypes were defined. We have used genetic analysis to evaluate the emergence of different genotypes and proposed reassortment events that have been observed. The existing data suggests that the H5N1 circulating in Europe during late 2020, continued to circulate in wild birds throughout 2021, with minimal adaptation, but has then gone on to reassort with AIVs in the wild bird population. We have undertaken an in-depth genetic assessment of H5 HPAIVs detected in the UK, over the last two winter seasons and demonstrate the utility of in-depth genetic analyses in defining the diversity of H5 HPAIVs circulating in avian species, the potential for zoonotic risk and whether incidents of lateral spread can be defined over independent incursion of infection from wild birds. Key supporting data for mitigation activities.ImportanceHigh pathogenicity avian influenza virus (HPAIV) outbreaks devastate avian species across all sectors having both economic and ecological impacts through mortalities in poultry and wild birds, respectively. These viruses can also represent a significant zoonotic risk. Since 2020, the UK has experienced two successive outbreaks of H5 HPAIV. Whilst H5N8 HPAIV was predominant during the 2020/21 outbreak, other H5 subtypes were also detected. The following year there was a shift in subtype dominance to H5N1 HPAIV, but multiple H5N1 genotypes were detected. Through thorough utilisation of whole-genome sequencing, it was possible to track and characterise the genetic evolution of these H5 HPAIVs in UK poultry and wild birds. This has enabled us to assess the risk posed by these viruses at the poultry:wild bird and the avian:human interface and to investigate potential lateral spread between infected premises, a key factor in understanding threat to the commercial sector.

Since 2003, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been maintained in poultry, periodically spilling back into wild migratory birds and spread to other geographic regions, with re-introduction to domestic birds causing severe impacts for poultry health, production and food sustainability. Successive waves of infection have also resulted in substantial genetic evolution and reassortment, enabling the emergence of multiple clades and subtypes within the H5 2.3.4.4 HPAI viruses. Control of AI is principally through either culling or through vaccination using conventional vaccines. Here, we antigenically and genetically characterise the emerging 2020/21 H5NX clade 2.3.4.4 strains and assess cross-reactivity to putative vaccine strains using chicken antisera. We demonstrate significant antigenic differences between commercially available poultry vaccines and currently circulating viruses suggesting that vaccination options might be suboptimal in the current outbreaks.

From 2016 to 2020, high pathogenicity avian influenza (HPAI) H5 viruses circulated in Asia, Europe, and Africa, causing waves of infections and the deaths of millions of wild and domestic birds and presenting a zoonotic risk. In late 2021, H5N1 HPAI viruses were isolated from poultry in Canada and also retrospectively from a great black-backed gull (Larus marinus), raising concerns that the spread of these viruses to North America was mediated by migratory wild bird populations. In February and April 2022, H5N1 HPAI viruses were isolated from a bald eagle (Haliaeetus leucocephalus) and broiler chickens in British Columbia, Canada. Phylogenetic analysis showed that the virus from bald eagle was genetically related to H5N1 HPAI virus isolated in Hokkaido, Japan, in January 2022. The virus identified from broiler chickens was a reassortant H5N1 HPAI virus with unique constellation genome segments containing PB2 and NP from North American lineage LPAI viruses, and the remaining gene segments were genetically related to the original Newfoundland-like H5N1 HPAI viruses detected in November and December 2021 in Canada. This is the first report of H5 HPAI viruses’ introduction to North America from the Pacific and the North Atlantic-linked flyways and highlights the expanding risk of genetically distinct virus introductions from different geographical locations and the potential for local reassortment with both the American lineage LPAI viruses in wild birds and with both Asian-like and European-like H5 HPAI viruses. We also report the presence of some amino acid substitutions across each segment that might contribute to the replicative efficiency of these viruses in mammalian host, evade adaptive immunity, and pose a potential zoonotic risk.

Avian influenza virus (AIV) subtypes H5N1 and H9N2 co-circulate in poultry in Bangladesh, causing significant bird morbidity and mortality. Despite their importance to the poultry value chain, the role of farms in spreading and maintaining AIV infections remains poorly understood in most disease-endemic settings. To address this crucial gap, we conducted a cross-sectional study between 2017 and 2019 in the Chattogram Division of Bangladesh in clinically affected and dead chickens in farms with suspected AIV infection. Viral prevalence of each subtype was approximately 10% among farms for which veterinary advice was sought, indicating high levels of virus circulation in chicken farms despite the low number of reported outbreaks. Co-circulation of both subtypes was common in farms, with our findings suggest that in the field, the co-circulation of H5N1 and H9N2 can modulate disease severity, which could facilitate an underestimated level of AIV transmission in the poultry value chain. Finally, using newly generated whole-genome sequences, we investigate the evolutionary history of a small subset of H5N1 and H9N2 viruses. Our analyses revealed that for both subtypes, the sampled viruses were genetically most closely related to other viruses isolated in Bangladesh and represented multiple independent incursions. However, due to lack of longitudinal surveillance in this region, it is difficult to ascertain whether these viruses emerged from endemic strains circulating in Bangladesh or from neighbouring countries. We also show that amino acids at putative antigenic residues underwent a distinct replacement during 2012 which coincides with the use of H5N1 vaccines.

Analyses of HPAI H5 viruses from poultry outbreaks across a wide Eurasian region since July 2020 including the Russian Federation, Republics of Iraq and Kazakhstan, and recent detections in migratory waterfowl in the Netherlands, revealed undetected maintenance of H5N8, likely in galliform poultry since 2017/18 and both H5N5 and H5N1. All viruses belong to A/H5 clade 2.3.4.4b with closely related HA genes. Heterogeneity in Eurasian H5Nx HPAI emerging variants threatens poultry production, food security and veterinary public health.

BackgroundThe 2009 pandemic H1N1 (A(H1N1)pdm09) influenza A virus (IAV) has replaced the previous seasonal H1N1 strain in humans and continues to circulate worldwide. The comparative performance of inactivated A(H1N1)pdm09 influenza vaccines remains of considerable interest. The objective of this study was to evaluate the efficacy of two licensed A(H1N1)pdm09 inactivated vaccines (AS03B adjuvanted split virion Pandemrix from GlaxoSmithKline and referred here as (V1) and non‐adjuvanted whole virion Celvapan from Baxter and referred here as (V2)) in ferrets as a pre‐clinical model for human disease intervention.MethodsNaïve ferrets were divided into two groups (V1 and V2) and immunised intramuscularly with two different A/California/07/2009‐derived inactivated vaccines, V1 administered in a single dose and V2 administered in 2 doses separated by 21 days. Six weeks after the first immunisation, vaccinated animals and a non‐vaccinated control (NVC) group were intra‐nasally challenged with 106.5 TCID50 of the isolate A/England/195/2009 A(H1N1)pdm09 with 99.1% amino acid identity to the vaccine strain. Clinical signs, lung histopathology, viral quantification and antibody responses were evaluated.Results and ConclusionsResults revealed important qualitative differences in the performance of both inactivated vaccines in relation to protection against challenge with a comparable virus in a naive animal (ferret) model of human disease. Vaccine V1 limited and controlled viral shedding and reduced lower respiratory tract infection. In contrast, vaccine V2 did not control infection and animals showed sustained viral shedding and delayed lower respiratory infection, resulting in pulmonary lesions, suggesting lower efficacy of V2 vaccine.

Influenza A(H1N1)pdm09 (pH1N1) virus has become established in swine in the United Kingdom and currently co-circulates with previously enzootic swine influenza A virus (IAV) strains, including avian-like H1N1 and human-like H1N2 viruses. During 2010, a swine influenza A reassortant virus, H1N2r, which caused mild clinical disease in pigs in the United Kingdom, was isolated. This reassortant virus has a novel gene constellation, incorporating the internal gene cassette of pH1N1-origin viruses and hemagglutinin and neuraminidase genes of swine IAV H1N2 origin. We investigated the pathogenesis and infection dynamics of the H1N2r isolate in pigs (the natural host) and in ferrets, which represent a human model of infection. Clinical and virologic parameters were mild in both species and both intraspecies and interspecies transmission was observed when initiated from either infected pigs or infected ferrets. This novel reassortant virus has zoonotic and reverse zoonotic potential, but no apparent increased virulence or transmissibility, in comparison to pH1N1 viruses.

The 2016–17 European outbreak of H5N8 HPAIV (Clade 2.3.4.4b) affected a wider range of avian species than the previous H5N8 outbreak (2014–15), including an incursion of H5N8 HPAIV into gamebirds in England. Natural infection of captive-reared pheasants (Phasianus colchicus) led to variable disease presentation; clinical signs included ruffled feathers, reluctance to move, bright green faeces, and/or sudden mortality. Several birds exhibited neurological signs (nystagmus, torticollis, ataxia). Birds exhibiting even mild clinical signs maintained substantial levels of virus replication and shedding, with preferential shedding via the oropharyngeal route. Gross pathology was consistent with HPAIV, in gallinaceous species but diphtheroid plaques in oropharyngeal mucosa associated with necrotising stomatitis were novel but consistent findings. However, minimal or modest microscopic pathological lesions were detected despite the systemic dissemination of the virus. Serology results indicated differences in the timeframe of exposure for each case (n = 3). This supported epidemiological conclusions confirming that the movement of birds between sites and other standard husbandry practices with limited hygiene involved in pheasant rearing (including several fomite pathways) contributed to virus spread between premises.

On 5 January 2022, high pathogenicity avian influenza A(H5N1) was confirmed in an individual who kept a large flock of ducks at their home in England. The individual remained asymptomatic. H5N1 was confirmed in 19/20 sampled live birds on 22 December 2021. Comprehensive contact tracing (n = 11) revealed no additional primary cases or secondary transmissions. Active surveillance of exposed individuals is essential for case identification. Asymptomatic swabbing helped refine public health risk assessment and facilitated case management given changes in avian influenza epidemiology.

The UK and Europe have seen successive outbreaks of highly pathogenic avian influenza across the 2020/21 and 2021/22 autumn/winter seasons. Understanding both the epidemiology and transmission of these viruses in different species is critical to aid mitigating measures where outbreaks cause extensive mortalities in both land- and waterfowl. Infection of different species can result in mild or asymptomatic outcomes, or acute infections that result in high morbidity and mortality levels. Definition of disease outcome in different species is of great importance to understanding the role different species play in the maintenance and transmission of these pathogens. Further, the infection of species that have conservation value is also important to recognise and characterise to understand the impact on what might be limited wild populations. Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b has been detected in great skuas (Stercorarius skua) across different colonies on islands off the shore of Scotland, Great Britain during summer 2021. A large number of great skuas were observed as developing severe clinical disease and dying during the epizootic and mortalities were estimated to be high where monitored. Of eight skuas submitted for post-mortem examination, seven were confirmed as being infected with this virus using a range of diagnostic assays. Here we overview the outbreak event that occurred in this species, listed as species of conservation concern in Great Britain and outline the importance of this finding with respect to virus transmission and maintenance.

High pathogenicity avian influenza viruses clade 2.3.4.4 H5 have spread among wild birds worldwide during recent years causing annual die-offs among wild birds and outbreaks in poultry in multiple European countries. The outbreaks significantly impact the poultry and game bird sectors. Infected game birds may act as a bridging species potentially enabling spread of virus into commercial and backyard premises. In this study, the pathogenesis and transmission of a HPAIV clade 2.3.4.4b H5N6 virus was investigated in pheasants and chickens. Efficient virus transmission was detected between pheasants over multiple rounds of naïve pheasant introductions and onwards to chickens. Mortality of up to 100% was observed for both infected pheasants and chickens. Intra-species transmission from chicken to chicken was less efficient. The study confirmed that clade 2.3.4.4b H5N6 HPAIV is highly virulent in pheasants and emphasises the role of pheasants as a bridging host for the infection of commercial poultry.

This focus article has been prepared by Paul Duff, Paul Holmes, James Aegerter, Cat Man, Ed Fullick, Scott Reid, Fabian Lean, Alex Nunez, Rowena Hansen, Joanna Tye, Levon Stephan and Ian Brown of the APHA and Caroline Robinson of SRUC.

We report a disease and mortality event involving swans, seals, and a fox at a wildlife rehabilitation center in the United Kingdom during late 2020. Five swans had onset of highly pathogenic avian influenza virus infection while in captivity. Subsequently, 5 seals and a fox died (or were euthanized) after onset of clinical disease. Avian-origin influenza A virus subtype H5N8 was retrospectively determined as the cause of disease. Infection in the seals manifested as seizures, and immunohistochemical and molecular testing on postmortem samples detected a neurologic distribution of viral products. The fox died overnight after sudden onset of inappetence, and postmortem tissues revealed neurologic and respiratory distribution of viral products. Live virus was isolated from the swans, seals, and the fox, and a single genetic change was detected as a potential adaptive mutation in the mammalian-derived viral sequences. No human influenza-like illness was reported in the weeks after the event

Numerous outbreaks of high-pathogenicity avian influenza (HPAI) were reported during 2020–2021. In Africa, H5Nx has been detected in Benin, Burkina Faso, Nigeria, Senegal, Lesotho, Namibia and South Africa in both wild birds and poultry. Botswana reported its first outbreak of HPAI to the World Organisation for Animal Health (WOAH) in 2021. An H5N1 virus was detected in a fish eagle, doves, and chickens. Full genome sequence analysis revealed that the virus belonged to clade 2.3.4.4b and showed high identity within haemagglutinin (HA) and neuraminidase proteins (NA) for viruses identified across a geographically broad range of locations. The detection of H5N1 in Botswana has important implications for disease management, wild bird conservation, tourism, public health, economic empowerment of vulnerable communities and food security in the region.

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

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.

Previous studies have described RT-LAMP methodology for the rapid detection of SARS-CoV-2 in nasopharyngeal (NP) and oropharyngeal (OP) swab and saliva samples. This study describes the validation of an improved sample preparation method for extraction free RT-LAMP and defines the clinical performance of four different RT-LAMP assay formats for detection of SARS-CoV-2 within a multisite clinical evaluation. Direct RT-LAMP was performed on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva from asymptomatic and symptomatic individuals across healthcare and community settings. For Direct RT-LAMP, overall diagnostic sensitivity (DSe) of 70.35% (95% CI 63.48-76.60%) on swabs and 84.62% (79.50-88.88%) on saliva was observed, with diagnostic specificity (DSp) of 100% (98.98-100.00%) on swabs and 100% (99.72-100.00%) on saliva when compared to RT-qPCR; analysing samples with RT-qPCR ORF1ab CT values of ≤25 and ≤33, DSe of 100% (96.34-100%) and 77.78% (70.99-83.62%) for swabs were observed, and 99.01% (94.61-99.97%) and 87.61% (82.69-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and DSp were 96.06% (92.88-98.12%) and 99.99% (99.95-100%) for swabs, and 80.65% (73.54-86.54%) and 99.99% (99.95-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use-cases, including frequent, interval-based testing of saliva with Direct RT-LAMP from asymptomatic individuals that may otherwise be missed using symptomatic testing alone.

Wild bird migration and illegal trade of infected poultry, eggs, and poultry products have been associated with the spread of avian influenza viruses (AIV). During 1992-1996, H7N1 and H7N8 low pathogenic AIV (LPAIV) were identified from captive wild birds; such as Pekin robin (Leiothrix lutea), magpie robin (Copsychus saularis), flycatcher sp. (genus Empidonax), a species of softbill and parakeet, sun conure (Aratinga solstitialis), painted conure (Pyrrhura picta), fairy bluebird (Irena puella), and common iora (Aegithina tiphia), kept in aviaries or quarantine stations in England, The Netherlands, Singapore and the United States (U.S.). In this study, we sequenced these H7 viruses isolated from quarantine facilities and aviaries using next-generation sequencing and conducted a comparative phylogenetic analysis of complete genome sequences to elucidate spread patterns. The complete genome sequencing and phylogenetic analysis suggested that H7 viruses originated from a common source, even though they were obtained from birds in distant geographical regions. All H7N1 and H7N8 viruses were LPAIV, except a H7N1 highly pathogenic AIV (HPAIV), A/Pekin robin/California/30412/1994(H7N1) virus. Our results support the continued need for regulation of the captive wild bird trade to reduce the risk of introduction and dissemination of both LPAIV and HPAIV throughout the world.

The progress of colonization of ash stems from ascospore inocula of Hymenoscyphus fraxineus was examined by light and electron microscopy. The main aim of the study was to characterize the cytology of the biotroph to necrotroph transition during lesion formation. Following direct penetration into epidermal cells, the fungus produced intracellular hyphae that invaded up to five cells before plant cells died. A lack of close attachment between the hyphal cell wall and plant cell membrane was revealed by plasmolysis of epidermal cells. Plant cells died at the centre of the infection but hyphae at the edge were typically found in living plant cells even around large lesions. During biotrophic invasion, the cytoplasm of penetrated plant cells showed very little response despite the plant cell membrane being in direct contact with the fungal cell wall. Before plant cell death, dark staining of the cytoplasm and proliferation of small vesicles was noted, but organelles retained normal ultrastructure. Dead plant cells contained dark brown, osmiophilic droplets. Penetration between epidermal or collenchyma cells was usually targeted to shared pits and involved constriction of hyphae. The transition to necrotrophy was not associated with a clear change in hyphal morphology. Biotrophic intracellular hyphae contained dense cytoplasm but hyphae in dead plant cells were more vacuolated. Remarkably little plant cell wall degradation was observed despite the fungus penetrating up to 18 cells deep into stem tissue. Features of the development of the ash dieback fungus are compared with other hemibiotrophic pathogens.

Swineinfluenza A virus(SwIV) infection has considerable economic and animal welfare consequences and, because of the zoonotic potential, can also have public health implications. The 2009 pandemicH1N1‘swine-origin’ infection is now endemic in both pigs and humans. In Europe, avian-like H1avN1, human-like H1huN2, human-like swineH3N2and, since 2009, pandemic H1N1 (pH1N1) lineage viruses andreassortants, constitute the dominant subtypes. In this study, we used a swine pH1N1 challenge virus to investigate the efficacy ofwhole inactivated virus vaccineshomologous or heterologous to the challenge virus as well as a commercial vaccine. We found that vaccine-mediated protection was most effective when vaccine antigen and challenge virus were homologous and correlated with the specific production ofneutralising antibodiesand a cellular response to the challenge virus. We conclude that a conventional whole inactivated SwIV vaccine must be antigenically matched to the challenge strain to be an effective control measure.

High pathogenicity avian influenza virus (HPAIV) clade 2.3.4.4b has re-emerged in the United Kingdom in 2021-2022 winter season, with over 90 cases of HPAIV detected among poultry and captive birds in England, Scotland, Wales, and Northern Ireland. Globally, HPAIV H5N1 has also had a wide geographical dispersion, causing outbreaks in Europe, North America, Asia, and Africa, impacting on socioeconomic and wildlife conservation. It is important to raise awareness of the gross pathological features of HPAIV and subsequently aid disease investigation through definition of pathological indicators following natural infection. In this study, we report on the gross pathology of HPAI H5N1 in poultry species (chicken, turkey, pheasant, guineafowl, duck, goose), and captive or wild birds (mute swan, tufted duck, jackdaw, peahen, white-tailed eagle) that tested positive between October 2021 and February 2022. Pancreatic and splenic necrosis were the common pathological findings in both Galliformes and Anseriformes. In addition to the more severe lesions documented in Galliformes, we also noted increased detection of pathological changes in a broader range of Anseriformes particularly in domestic ducks, in contrast to those reported in previous seasons with other H5Nx HPAIV subtypes. A continual effort to characterise the pathological impact of the disease is necessary to update on the presentation of HPAIV for both domestic/captive and wild birds whilst guiding early presumptive diagnosis.

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016-17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016-17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005-06 and 2016-17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016-17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control.

We conducted a cross-sectional study in live bird markets (LBMs) in Dhaka and Chittagong, Bangladesh, to estimate the prevalence of avian influenza A(H5) and A(H9) viruses in different types of poultry and environmental areas by using Bayesian hierarchical logistic regression models. We detected these viruses in nearly all LBMs. Prevalence of A(H5) virus was higher in waterfowl than in chickens, whereas prevalence of A(H9) virus was higher in chickens than in waterfowl and, among chicken types, in industrial broilers than in cross-breeds and indigenous breeds. LBMs with >1 wholesaler were more frequently contaminated by A(H5) virus than retail-only LBMs. Prevalence of A(H9) virus in poultry and level of environmental contamination were also higher in LBMs with >1 wholesaler. We found a high level of circulation of both avian influenza viruses in surveyed LBMs. Prevalence was influenced by type of poultry, environmental site, and trading.

SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced ‘COVID-19 like-illness’, and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. A pVNT correlated strongly with SARS-CoV-2 VNT and was able to effectively discriminate SARS-CoV-2 antibody positive and negative serum with the same efficiency as the VNT. Moreover, the pVNT was performed with the same level of discrimination across multiple separate institutions. Therefore, the pVNT is a sensitive, specific, and reproducible lower biosafety level alternative to VNT for detecting SARS-CoV-2 antibodies for diagnostic and research applications. Our data illustrate the potential utility of applying VNT or pVNT and ELISA antibody tests in parallel to enhance the sensitivity of exposure to infection.

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.