Transmission dynamics and pathogenesis differ between pheasants and partridges infected with clade 2.3.4.4b H5N8 and H5N1 high-pathogenicity avian influenza viruses.

During the UK 2020-2021 epizootic of H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs), high mortality occurred during incursions in commercially farmed common pheasants (Phasianus colchicus). Two pheasant farms, affected separately by H5N8 and H5N1 subtypes, included adjacently housed red-legged partridges (Alectoris rufa), which appeared to be unaffected. Despite extensive ongoing epizootics, H5Nx HPAIV partridge outbreaks were not reported during 2020-2021 and 2021-2022 in the UK, so it is postulated that partridges are more resistant to HPAIV infection than other gamebirds. To assess this, pathogenesis and both intra- and inter-species transmission of UK pheasant-origin H5N8-2021 and H5N1-2021 HPAIVs were investigated. Onward transmission to chickens was also assessed to better understand the risk of spread from gamebirds to other commercial poultry sectors. A lower infectious dose was required to infect pheasants with H5N8-2021 compared to H5N1-2021. However, HPAIV systemic dissemination to multiple organs within pheasants was more rapid following infection with H5N1-2021 than H5N8-2021, with the former attaining generally higher viral RNA levels in tissues. Intraspecies transmission to contact pheasants was successful for both viruses and associated with viral environmental contamination, while interspecies transmission to a first chicken-contact group was also efficient. However, further onward transmission to additional chicken contacts was only achieved with H5N1-2021. Intra-partridge transmission was only successful when high-dose H5N1-2021 was administered, while partridges inoculated with H5N8-2021 failed to shed and transmit, although extensive tissue tropism was observed for both viruses. Mortalities among infected partridges featured a longer incubation period compared to that in pheasants, for both viruses. Therefore, the susceptibility of different gamebird species and pathogenicity outcomes to the ongoing H5Nx clade 2.3.4.4b HPAIVs varies, but pheasants represent a greater likelihood of H5Nx HPAIV introduction into galliforme poultry settings. Consequently, viral maintenance within gamebird populations and risks to poultry species warrant enhanced investigation.

Different Outcomes of Chicken Infection with UK-Origin H5N1-2020 and H5N8-2020 High-Pathogenicity Avian Influenza Viruses (Clade 2.3.4.4b).

Clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses (HPAIVs) of the "goose/Guangdong" lineage have caused a series of European epizootics since 2014. During autumn/winter 2020-2021, several H5Nx subtypes were detected in the UK, with H5N8 being the dominant subtype in wild birds and poultry. Despite the greater subtype diversity (due to viral neuraminidase gene reassortment) reported in wild birds, only H5N8 and H5N1 subtypes caused clade 2.3.4.4 UK HPAIV poultry outbreaks during this period. The direct inoculation of layer chickens showed that H5N8-2020 was more infectious than H5N1-2020, which supported the European H5N8 dominance during that season. However, the mean death time was longer for H5N8-2020 (3.42 days) than for H5N1-2020 (2.17 days). Transmission from directly infected to naive in-contact chickens was inefficient for both subtypes. Histological lesions, the tissue dissemination of viral antigen, and nucleic acid were more extensive and abundant and accumulated more rapidly for H5N1-2020 compared with H5N8-2020. Although inefficient, H5N1-2020 transmission was faster, with its greater virulence indicating that this subtype posed a major concern, as subsequently shown during H5N1 dominance of the clade 2.3.4.4 epizootic since autumn 2021. An evaluation of these in vivo viral characteristics is key to understanding the continuing poultry threats posed by clade 2.3.4.4 H5Nx HPAIVs.

Understanding the disease and economic impact of avirulent avian paramyxovirus type 1 (APMV-1) infection in Great Britain.

Newcastle disease (ND) is a notifiable disease affecting chickens and other avian species caused by virulent strains of Avian paramyxovirus type 1 (APMV-1). While outbreaks of ND can have devastating consequences, avirulent strains of APMV-1 generally cause subclinical infections or mild disease. However, viruses can cause different levels of disease in different species and virulence can evolve following cross-species transmission events. This report describes the detection of three cases of avirulent APMV-1 infection in Great Britain (GB). Case 1 emerged from the 'testing to exclude' scheme in chickens in Shropshire while cases 2 and 3 were made directly from notifiable avian disease investigations in chicken broilers in Herefordshire and on premises in Wiltshire containing ducks and mixed species, respectively). Class II/genotype I.1.1 APMV-1 from case 1 shared 99.94% identity to the Queensland V4 strain of APMV-1. Class II/genotype II APMV-1 was detected from case 2 while the class II/genotype I.2 virus from case 3 aligned closely with strains isolated from Anseriformes. Exclusion of ND through rapid detection of avirulent APMV-1 is important where clinical signs caused by avirulent or virulent APMV-1s could be ambiguous. Understanding the diversity of APMV-1s circulating in GB is critical to understanding disease threat from these adaptable viruses.

Investigating the Genetic Diversity of H5 Avian Influenza Viruses in the United Kingdom from 2020-2022.

Since 2020, the United Kingdom and Europe have experienced annual epizootics of high-pathogenicity avian influenza virus (HPAIV). The first epizootic, during the autumn/winter of 2020-2021, involved six H5Nx subtypes, although H5N8 HPAIV dominated in the United Kingdom. While genetic assessments of the H5N8 HPAIVs within the United Kingdom 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 detections of H5N1 in wild birds over the summer of 2021, the autumn/winter of 2021-2022 saw another European H5 HPAIV epizootic that 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 suggest that the H5N1 viruses circulating in Europe during late 2020 continued to circulate in wild birds throughout 2021, with minimal adaptation, but then went on to reassort with AIVs in the wild bird population. We have undertaken an in-depth genetic assessment of H5 HPAIVs detected in the United Kingdom over 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 incursions of infections from wild birds. This provides key supporting data for mitigation activities. IMPORTANCE High-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 United Kingdom has experienced two successive outbreaks of H5 HPAIV. While H5N8 HPAIV was predominant during the 2020-2021 outbreak, other H5 subtypes were also detected. The following year, there was a shift in the subtype dominance to H5N1 HPAIV, but multiple H5N1 genotypes were detected. Through the thorough utilization of whole-genome sequencing, it was possible to track and characterize the genetic evolution of these H5 HPAIVs in United Kingdom poultry and wild birds. This enabled us to assess the risk posed by these viruses at the poultry-wild bird and the avian-human interfaces and to investigate the potential lateral spread between infected premises, a key factor in understanding the threat to the commercial sector.

Clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) from the 2021/22 epizootic is highly duck adapted and poorly adapted to chickens.

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.

Pathogenesis and infection dynamics of high pathogenicity avian influenza virus (HPAIV) H5N6 (clade 2.3.4.4b) in pheasants and onward transmission to chickens.

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.

JMM Profile: Avian influenza: a veterinary pathogen with zoonotic potential.

Avian influenza viruses (AIVs) are classified as either low pathogenicity (LP; generally causing sub-clinical to mild infections) or high pathogenicity (HP; capable of causing significant mortality events in birds). To date, HPAIVs appear o be restricted to the haemagglutinin (HA) glycoprotein H5 and H7 AIV subtypes. Both LPAIV and HPAIV H5 and H7 AIV subtypes are classified as the causative agents of notifiable disease in poultry. A broad range of non-H5/non-H7 LPAIVs also exist that have been associated with more severe disease outcomes in avian species. As a result, the constant threat from AIVs causes significant economic damage in poultry production systems worldwide. The close proximity between mammalian and susceptible avian species in some environments provides the opportunity for both inter-host transmission and mammalian adaptation, potentially resulting in novel AIV strains capable of infecting humans.

A universal RT-qPCR assay for "One Health" detection of influenza A viruses.

The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

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.

Ducks Are Susceptible to Infection with a Range of Doses of H5N8 Highly Pathogenic Avian Influenza Virus (2016, Clade 2.3.4.4b) and Are Largely Resistant to Virus-Specific Mortality, but Efficiently Transmit Infection to Contact Turkeys.

Widespread H5N8 highly pathogenic avian influenza virus (HPAIV; clade 2.3.4.4b) infections occurred in wild birds and poultry across Europe during winter 2016-17. Four different doses of H5N8 HPAIV (A/wigeon/Wales/052833/2016 [wg-Wal-16]) were used to infect 23 Pekin ducks divided into four separate pens, with three contact turkeys introduced for cohousing per pen at 1 day postinfection (dpi). All doses resulted in successful duck infection, with four sporadic mortalities recorded among the 23 (17%) infected ducks, which appeared unrelated to the dose. The ducks transmitted wg-Wal-16 efficiently to the contact turkeys; all 12 (100%) turkeys died. Systemic viral dissemination was detected in multiple organs in two duck mortalities, with limited viral dissemination in another duck, which died after resolution of shedding. Systemic viral tropism was observed in two of the turkeys. The study demonstrated the utility of Pekin ducks as surrogates of infected waterfowl to model the wild bird/gallinaceous poultry interface for introduction of H5N8 HPAIV into terrestrial poultry, where contact turkeys served as a susceptible host. Detection of H5N8-specific antibody up to 58 dpi assured the value of serologic surveillance in farmed ducks by hemagglutination inhibition and anti-nucleoprotein ELISAs.

Los patos son susceptibles a la infección con un rango de dosis del virus de la influenza aviar altamente patógena subtipo H5N8 (2016, clado 2.3.4.4b) son muy resistentes a la mortalidad específica por el virus, pero transmiten la infección de manera eficiente a pavos por contacto. La diseminación de la infección por el virus de la influenza aviar altamente patógeno H5N8 (con las siglas en inglés HPAIV); clado 2.3.4.4b ocurrió en aves silvestres y avicultura comercial en toda Europa durante el invierno 2016­17. Se usaron cuatro dosis diferentes del virus de alta patogenicidad H5N8 (A/wigeon/Gales/052833/2016 [wg-Wal-16]) para infectar a 23 patos Pekin divididos en cuatro corrales, cohabitando con tres pavos en el corral para determinar transmisión por contacto al primer día después de la infección (dpi). Todas las dosis dieron como resultado una infección exitosa de los patos, con mortalidad esporádica en cuatro aves (17%) registradas entre los 23 patos infectados, que no parecieron estar relacionadas con la dosis. Los patos transmitieron el virus wg-Wal-16 de manera eficiente a los pavos por contacto; los 12 pavos (100%) murieron. La diseminación viral sistémica se detectó en múltiples órganos en dos patos muertos, con diseminación viral limitada en otro pato que murió después de la resolución de la eliminación viral. Se observó tropismo viral sistémico en dos de los pavos. El estudio demostró la utilidad de los patos Pekin como sustitutos de las aves acuáticas infectadas en un modelo de la interface entre aves silvestres y aves comerciales gallináceas para la introducción del subtipo H5N8 del virus de influenza aviar de alta patogenicidad en las aves comerciales terrestres. Los pavos de contacto sirvieron como hospedadores susceptibles. La detección de anticuerpos específicos contra el subtipo H5N8 hasta 58 días después de la inoculación justificó el valor de la vigilancia serológica en las granjas de patos mediante la inhibición de la hemaglutinación y por estuches de ELISA dirigidos contra la nucleoproteína.

Two Single Incursions of H7N7 and H5N1 Low Pathogenicity Avian Influenza in U.K. Broiler Breeders During 2015 and 2016.

Low pathogenicity (LP) avian influenza viruses (AIVs) have a natural reservoir in wild birds. These cause few (if any) overt clinical signs, but include H5 and H7 LPAIVs, which are notifiable in poultry. In the European Union, notifiable avian disease (NAD) demands laboratory confirmation with prompt statutory interventions to prevent dissemination of infection to multiple farms. Crucially, for H5 and H7 LPAIVs, movement restrictions and culling limit the further risk of mutation to the corresponding highly pathogenic (HP) H5 and H7 AIVs in gallinaceous poultry. An H7N7 LPAIV outbreak occurred during February 2015 at a broiler breeder chicken premise in England. Full genome sequencing suggested an avian origin closely related to contemporary European H7 LPAIV wild bird strains with no correlates for human adaptation. However, a high similarity of PB2, PB1, and NA genes with H10N7 viruses from European seals during 2014 was observed. An H5N1 LPAIV outbreak during January 2016 affecting broiler breeder chickens in Scotland resulted in rapid within-farm spread. An interesting feature from this case was that although viral tropism occurred in heart and kidney endothelial cells, suggesting HPAIV infection, the H5N1 virus had the molecular cleavage site signature of an LPAIV belonging to an indigenous European H5 lineage. There was no genetic evidence for human adaptation or antiviral drug resistance. The source of the infection was also likely to be via indirect contact with wild birds mediated via fomite spread from the nearby environment. Both LPAIV outbreaks were preceded by local flooding events that attracted wild waterfowl to the premises. Prompt detection of both outbreaks highlighted the value of the "testing to exclude" scheme launched in the United Kingdom for commercial gallinaceous poultry in 2014 as an early warning surveillance mechanism for NAD.

Dos incursiones únicas de influenza aviar de baja patogenicidad H7N7 y H5N1 en criadores de pollos de engorde en el Reino Unido durante 2015 y 2016. Los virus de influenza aviar de baja patogenicidad tienen un reservorio natural en aves silvestres. Estos causan pocos (si es que se presentan) signos clínicos evidentes, pero se incluyen los virus de influenza de baja patogenicidad H5 y H7, que son notificables en avicultura. En la Unión Europea, las enfermedades aviares notificables (NAD, por sus siglas en inglés) requieren de confirmación de laboratorio con intervenciones reglamentarias rápidas para prevenir la diseminación de la infección a múltiples granjas. De manera crucial, para las los virus de baja patogenicidad H5 y H7, las restricciones de movimiento y el sacrificio limitan el riesgo adicional de mutación hacia los correspondientes virus H5 y H7 altamente patógenos en aves comerciales. Un brote de influenza aviar de baja patogenicidad H7N7 ocurrió en febrero del 2015 en una granja de pollos reproductores de pollos de engorde en Inglaterra. La secuenciación completa del genoma sugirió un origen aviar estrechamente relacionado con las cepas de aves silvestres contemporáneas europeas de baja patogenicidad H7 sin indicios para la adaptación humana. Sin embargo, se observó una alta similitud de los genes PB2, PB1 y NA con los virus H10N7 de focas europeas durante el 2014. Un brote de influenza aviar de baja patogenicidad por H5N1 en enero del 2016 que afectó a los pollos reproductores de pollos de engorde en Escocia resultó en una rápida propagación dentro de la granja. Una característica interesante de este caso fue que, aunque el tropismo viral ocurrió en las células endoteliales del corazón y el riñón, lo que sugería una infección por un virus de alta patogenicidad, el virus H5N1 tenía el sitio de disociación molecular característico de un virus de baja patogenicidad perteneciente a un linaje indígena H5 europeo. No se observó evidencia genética para la adaptación humana o la resistencia a los medicamentos antivirales. También es probable que la fuente de la infección fue a través del contacto indirecto con las aves silvestres mediadas a través de la propagación de fómites desde el entorno cercano. Ambos brotes de influenza aviar de baja patogenicidad fueron precedidos por inundaciones locales que atrajeron aves acuáticas silvestres a las instalaciones. La rápida detección de ambos brotes resaltó el valor del esquema de "Diagnóstico para Excluir" establecido en el Reino Unido para la avicultura comercial en el 2014 como un mecanismo de vigilancia de alerta temprana para las enfermedades aviares notificables.

Proceedings Paper-Avian Diseases 10th AI Symposium Issue Development and Application of Real-Time PCR Assays for Specific Detection of Contemporary Avian Influenza Virus Subtypes N5, N6, N7, N8, and N9.

Previously published NA subtype-specific real-time reverse-transcriptase PCRs (RRT-PCRs) were further validated for the detection of five avian influenza virus (AIV) NA subtypes, namely N5, N6, N7, N8, and N9. Testing of 30 AIV isolates of all nine NA subtypes informed the assay assessments, with the N5 and N9 RRT-PCRs retained as the original published assays while the N7 and N8 assays were modified in the primer-probe sequences to optimize detection of current threats. The preferred N6 RRT-PCR was either the original or the modified variant, depending on the specific H5N6 lineage. Clinical specimen (n = 137) testing revealed the ability of selected N5, N6, and N8 RRT-PCRs to sensitively detect clade 2.3.4.4b highly pathogenic AIV (HPAIV) infections due to H5N5, H5N6, and H5N8 subtypes, respectively, all originating from European poultry and wild bird cases during 2016-2018. Similar testing (n = 32 clinical specimens) also showed the ability of N7 and N9 RRT-PCRs to sensitively detect European H7N7 HPAIV and China-origin H7N9 low pathogenicity AIV infections, respectively.

Desarrollo y aplicación de ensayos de PCR en tiempo real para la detección específica de subtipos contemporáneos de influenza aviar Virus N5, N6, N7, N8 y N9. Métodos de transcripción reversa y PCR en tiempo real (RRT) específicos para subtipo específico de NA que fueron publicados anteriormente se validaron completamente para la detección de cinco subtipos de NA del virus de la influenza aviar (AIV), incluyendo N5, N6, N7, N8 y N9. El análisis de 30 aislamientos del virus de la influenza aviar de los nueve subtipos de NA proporcionaron información acerca de las evaluaciones de los ensayos, con los métodos de RRT-PCR N5 y N9 evaluados de acuerdo a los ensayos originales, mientras que los métodos N7 y N8 se modificaron en las secuencias del iniciador y de la sonda para optimizar la detección de los virus que constituyen amenazas actuales. Los métodos de RRT-PCR para el subtipo N6 preferido fueron tanto el dirigido al virus original o el dirigido a la variante modificada, dependiendo del linaje específico de H5N6. Las pruebas de muestras clínicas (n=137) revelaron que los métodos RRT-PCR seleccionados para N5, N6 y N8 detectaron con sensibilidad los subtipos del clado 2.3.4.4b del virus de la influenza aviar altamente patógenos H5N5, H5N6 y H5N8, respectivamente, todos originados en Europa de casos en avicultura comercial y de aves silvestres durante el año 2016 al 2018. Estudios similares (muestras clínicas n = 32) también mostraron que los métodos de RRT-PCR para los subtipos N7 y N9 detectaron con sensibilidad las infecciones por el virus H7N7 de alta patogenicidad europeo y por el subtipo H7N9 de origen chino de baja patogenicidad, respectivamente.

Detection of non-notifiable H4N6 avian influenza virus in poultry in Great Britain.

A 12-month pilot project for notifiable avian disease (NAD) exclusion testing in chicken and turkey flocks in Great Britain (GB) offered, in partnership with industry, opportunities to carry out differential diagnosis in flocks where NAD was not suspected, and to identify undetected or undiagnosed infections. In May 2014, clinical samples received from a broiler breeder chicken premises that had been experiencing health and production problems for approximately one week tested positive by avian influenza (AI) real-time reverse transcription polymerase chain reaction (RRT-PCR). Following immediate escalation to an official, statutory investigation to rule out the presence of notifiable AI virus (AIV; H5 or H7 subtypes), a non-notifiable H4N6 low pathogenicity (LP) AIV was detected through virus isolation in embryonated specific pathogen free (SPF) fowls' eggs, neuraminidase inhibition test, cleavage site sequencing and AIV subtype H4-specific serology. Premises movement restrictions were lifted, and no further disease control measures were implemented as per the United Kingdom (UK) legislation. Phylogenetic analysis of the haemagglutinin and neuraminidase genes of the virus revealed closest relationships to viruses from Mallard ducks in Sweden during 2007 and 2009. In June 2014, clinical suspicion of NAD was reported in a flock of free-range laying chickens elsewhere in GB, due to increasing daily mortality and reduced egg production over a five-day period. An H4N6 LPAIV with an intravenous pathogenicity index of 0.50 was isolated. This virus was genetically highly similar, but not identical, to the virus detected during May 2014. Full viral genome analyses showed characteristics of a strain that had not recently transferred from wild birds, implying spread within the poultry sector had occurred. A stalk deletion in the neuraminidase gene sequence indicated an adaptation of the virus to poultry. Furthermore, there was unexpected evidence of systemic spread of the virus on post-mortem. No other cases were reported. Infection with LPAIVs often result in variable clinical presentation in poultry, making detection of disease more difficult.