Efficient and Informative Laboratory Testing for Rapid Confirmation of H5N1 (Clade 2.3.4.4) High-Pathogenicity Avian Influenza Outbreaks in the United Kingdom.

During the early stages of the UK 2021-2022 H5N1 high-pathogenicity avian influenza virus (HPAIV) epizootic in commercial poultry, 12 infected premises (IPs) were confirmed by four real-time reverse-transcription-polymerase chain reaction (RRT)-PCRs, which identified the viral subtype and pathotype. An assessment was undertaken to evaluate whether a large sample throughput would challenge laboratory capacity during an exceptionally large epizootic; hence, assay performance across our test portfolio was investigated. Statistical analysis of RRT-PCR swab testing supported it to be focused on a three-test approach, featuring the matrix (M)-gene, H5 HPAIV-specific (H5-HP) and N1 RRT-PCRs, which was successfully assessed at 29 subsequent commercial IPs. The absence of nucleotide mismatches in the primer/probe binding regions for the M-gene and limited mismatches for the H5-HP RRT-PCR underlined their high sensitivity. Although less sensitive, the N1 RRT-PCR remained effective at flock level. The analyses also guided successful surveillance testing of apparently healthy commercial ducks from at-risk premises, with pools of five oropharyngeal swabs tested by the H5-HP RRT-PCR to exclude evidence of infection. Serological testing at anseriform H5N1 HPAIV outbreaks, together with quantitative comparisons of oropharyngeal and cloacal shedding, provided epidemiological information concerning the chronology of initial H5N1 HPAIV incursion and onward spread within an IP.

Detection of Highly Pathogenic Avian Influenza Virus H5N1 Clade 2.3.4.4b in Great Skuas: A Species of Conservation Concern in Great Britain.

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.

Incursion of H5N8 high pathogenicity avian influenza virus (HPAIV) into gamebirds in England.

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.

Regional Transmission and Reassortment of 2.3.4.4b Highly Pathogenic Avian Influenza (HPAI) Viruses in Bulgarian Poultry 2017/18.

Between 2017 and 2018, several farms across Bulgaria reported outbreaks of H5 highly-pathogenic avian influenza (HPAI) viruses. In this study we used genomic and traditional epidemiological analyses to trace the origin and subsequent spread of these outbreaks within Bulgaria. Both methods indicate two separate incursions, one restricted to the northeastern region of Dobrich, and another largely restricted to Central and Eastern Bulgaria including places such as Plovdiv, Sliven and Stara Zagora, as well as one virus from the Western region of Vidin. Both outbreaks likely originate from different European 2.3.4.4b virus ancestors circulating in 2017. The viruses were likely introduced by wild birds or poultry trade links in 2017 and have continued to circulate, but due to lack of contemporaneous sampling and sequences from wild bird viruses in Bulgaria, the precise route and timing of introduction cannot be determined. Analysis of whole genomes indicates a complete lack of reassortment in all segments but the matrix protein gene (MP), which presents as multiple smaller clusters associated with different European 2.3.4.4b viruses. Ancestral reconstruction of host states of the hemagglutinin (HA) gene of viruses involved in the outbreaks suggests that transmission is driven by domestic ducks into galliform poultry. Thus, according to present evidence, we suggest the surveillance of domestic ducks as they are an epidemiologically relevant species for subclinical infection. Monitoring the spread due to movement between farms within regions and links to poultry production systems in European countries can help to predict and prevent future outbreaks. The 2.3.4.4b lineage which caused the largest recorded poultry epidemic in Europe continues to circulate, and the risk of further transmission by wild birds during migration remains.

Detection of H3N8 influenza A virus with multiple mammalian-adaptive mutations in a rescued Grey seal (Halichoerus grypus) pup.

Avian influenza A viruses (IAVs) in different species of seals display a spectrum of pathogenicity, from sub-clinical infection to mass mortality events. Here we present an investigation of avian IAV infection in a 3- to 4-month-old Grey seal (Halichoerus grypus) pup, rescued from St Michael's Mount, Cornwall in 2017. The pup underwent medical treatment but died after two weeks; post-mortem examination and histology indicated sepsis as the cause of death. IAV NP antigen was detected by immunohistochemistry in the nasal mucosa, and sensitive real-time reverse transcription polymerase chain reaction assays detected trace amounts of viral RNA within the lower respiratory tract, suggesting that the infection may have been cleared naturally. IAV prevalence among Grey seals may therefore be underestimated. Moreover, contact with humans during the rescue raised concerns about potential zoonotic risk. Nucleotide sequencing revealed the virus to be of subtype H3N8. Combining a GISAID database BLAST search and time-scaled phylogenetic analyses, we inferred that the seal virus originated from an unsampled, locally circulating (in Northern Europe) viruses, likely from wild Anseriformes. From examining the protein alignments, we found several residue changes in the seal virus that did not occur in the bird viruses, including D701N in the PB2 segment, a rare mutation, and a hallmark of mammalian adaptation of bird viruses. IAVs of H3N8 subtype have been noted for their particular ability to cross the species barrier and cause productive infections, including historical records suggesting that they may have caused the 1889 pandemic. Therefore, infections such as the one we report here may be of interest to pandemic surveillance and risk and help us better understand the determinants and drivers of mammalian adaptation in influenza.

Surveillance and investigative diagnosis of a poultry flock in Great Britain co-infected with an influenza A virus and an avirulent avian avulavirus type 1.

A detailed veterinary and laboratory investigation revealed an unusual case of concurrent avian avulavirus type 1 (AAvV-1, formerly called avian paramyxovirus type 1) and low pathogenicity avian influenza (LPAI) virus infections of chickens during March 2010 in a mixed poultry and livestock farm in Great Britain. Respiratory signs and daily mortality of 5-6 birds in a broiler flock 8-weeks of age prompted submission of two carcasses to an Animal and Plant Health Agency (APHA) regional laboratory. Infectious bronchitis virus infection was suspected initially and virus isolation in SPF embryonated fowls' eggs was attempted at APHA-Weybridge. Avirulent AAvV-1 was detected in the first sampling. Both in vitro nucleotide sequencing of the fusion gene and in vivo pathotyping by intracerebral pathogenicity index revealed an avirulent AAvV-1 not definitively ascribed to licensed vaccine. Upon initial detection of the AAvV-1 virus, statutory restrictions were placed on the farm, an official veterinary visit was performed and further samples were submitted to APHA-Weybridge for official statutory disease investigation. An H2N3 LPAI virus was subsequently isolated from tissue samples and swabs submitted from the follow-up statutory investigation. The subtype was confirmed by haemagglutination inhibition test (HAIT) and neuraminidase inhibition (NI) tests on egg-amplified virus. As neither virus was notifiable according to the internationally recognized EU and OIE standards, and/or definitions of disease, statutory farm restrictions were lifted. Veterinary investigations identified the broiler flock to be free-range, next to a river and duck pen, reinforcing the suspicion of wild bird origin for both viruses which may have been co-circulating in ducks. It could not, however, be established as to whether there were separate introductions of the two viruses or whether there had been a single co-introduction of the viruses. The described case highlights the value of integrated surveillance and laboratory approaches, including veterinary field investigations, international standards and definitions of notifiable avian disease, validated RRT-PCR assays, and virus isolation in achieving rapid and accurate diagnostic results.