Detection of SARS-CoV-2 Delta Variant (B.1.617.2) in Domestic Dogs and Zoo Tigers in England and Jersey during 2021.

Reverse zoonotic transmission events of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described since the start of the pandemic, and the World Organisation for Animal Health (WOAH) designated the detection of SARS-CoV-2 in animals a reportable disease. Eighteen domestic and zoo animals in Great Britain and Jersey were tested by APHA for SARS-CoV-2 during 2020-2023. One domestic cat (Felis catus), three domestic dogs (Canis lupus familiaris), and three Amur tigers (Panthera tigris altaica) from a zoo were confirmed positive during 2020-2021 and reported to the WOAH. All seven positive animals were linked with known SARS-CoV-2 positive human contacts. Characterisation of the SARS-CoV-2 variants by genome sequencing indicated that the cat was infected with an early SARS-CoV-2 lineage. The three dogs and three tigers were infected with the SARS-CoV-2 Delta variant of concern (B.1.617.2). The role of non-human species in the onward transmission and emergence of new variants of SARS-CoV-2 remain poorly defined. Continued surveillance of SARS-CoV-2 in relevant domestic and captive animal species with high levels of human contact is important to monitor transmission at the human-animal interface and to assess their role as potential animal reservoirs.

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.

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.

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.

Rapid and sensitive detection of high pathogenicity Eurasian clade 2.3.4.4b avian influenza viruses in wild birds and poultry.

Avian influenza virus (AIV) is classified as high or low pathogenicity AIV (HPAIV/LPAIV) based on intravenous pathogenicity in chickens and/or the presence or absence of multiple basic residues at the heamagglutinin (HA) cleavage site (CS). Since 2014, Europe has experienced waves of incursions of H5Nx HPAIV. Between November 2020 and March 2021, these included HPAIV H5N8, with sporadic of H5N1 and H5N5 (all clade 2.3.4.4b), detected in more than 300 "found dead" wild birds submitted through a passive surveillance programme in the United Kingdom. Currently, H5Nx HPAIV detection relies on identification of AIV RNA and H5 subtyping using real-time reverse transcription PCR (rRT-PCR) assays. The pathotype is subsequently determined by Sanger sequencing of the HA CS. Here, we report the validation and application of a rapid, more cost-effective HP H5-detection rRT-PCR assay. The HP H5 rRT-PCR assay specifically, sensitively and reproducibly detected RNA from contemporary clade 2.3.4.4b H5 HPAIVs with comparable sensitivity to the diagnostic H5-specific rRT-PCR; LPAIV H5 RNA and non-AIV RNA were not detected. On material from "found-dead" wild birds, and for statutory disease diagnosis on poultry, the HP H5 rRT-PCR results provided 100% discrimination when compared to conventional CS sequencing, significantly reducing time-to-pathotype determination and cost, enhancing the diagnostic workflow.

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.

The Emergence of H7N7 Highly Pathogenic Avian Influenza Virus from Low Pathogenicity Avian Influenza Virus Using an in ovo Embryo Culture Model.

Outbreaks of highly pathogenic avian influenza virus (HPAIV) often result in the infection of millions of poultry, causing up to 100% mortality. HPAIV has been shown to emerge from low pathogenicity avian influenza virus (LPAIV) in field outbreaks. Direct evidence for the emergence of H7N7 HPAIV from a LPAIV precursor with a rare di-basic cleavage site (DBCS) was identified in the UK in 2008. The DBCS contained an additional basic amino acid compared to commonly circulating LPAIVs that harbor a single-basic amino acid at the cleavage site (SBCS). Using reverse genetics, outbreak HPAIVs were rescued with a DBCS (H7N7DB), as seen in the LPAIV precursor or an SBCS representative of common H7 LPAIVs (H7N7SB). Passage of H7N7DB in chicken embryo tissues showed spontaneous evolution to a HPAIV. In contrast, deep sequencing of extracts from embryo tissues in which H7N7SB was serially passaged showed retention of the LPAIV genotype. Thus, in chicken embryos, an H7N7 virus containing a DBCS appears naturally unstable, enabling rapid evolution to HPAIV. Evaluation in embryo tissue presents a useful approach to study AIV evolution and allows a laboratory-based dissection of molecular mechanisms behind the emergence of HPAIV.

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.

Unexpected infection outcomes of China-origin H7N9 low pathogenicity avian influenza virus in turkeys.

The China-origin H7N9 low pathogenicity avian influenza virus (LPAIV) emerged as a zoonotic threat in 2013 where it continues to circulate in live poultry markets. Absence of overt clinical signs in poultry is a typical LPAIV infection outcome, and has contributed to its insidious maintenance in China. This study is the first description of H7N9 LPAIV (A/Anhui/1/13) infection in turkeys, with efficient transmission to two additional rounds of introduced contact turkeys which all became infected during cohousing. Surprisingly, mortality was observed in six of eight (75%) second-round contact turkeys which is unusual for LPAIV infection, with unexpected systemic dissemination to many organs beyond the respiratory and enteric tracts, but interestingly no accompanying mutation to highly pathogenic AIV. The intravenous pathogenicity index score for a turkey-derived isolate (0.39) affirmed the LPAIV phenotype. However, the amino acid change L235Q in the haemagglutinin gene occurred in directly-infected turkeys and transmitted to the contacts, including those that died and the two which resolved infection to survive to the end of the study. This polymorphism was indicative of a reversion from mammalian to avian adaptation for the H7N9 virus. This study underlined a new risk to poultry in the event of H7N9 spread beyond China.