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Highly pathogenic H5N1 avian influenza viruses cause devastating outbreaks in farmed poultry with serious consequences for animal welfare and economic losses. Zoonotic infection of humans through close contact with H5N1 infected birds is often severe and fatal. England experienced an outbreak of H5N1 in turkeys in 1991 that led to thousands of farmed bird mortalities. Isolation of clonal populations of one such virus from this outbreak uncovered amino acid differences in the virus haemagglutinin (HA) gene whereby the different genotypes could be associated with distinct pathogenic outcomes in chickens; both low pathogenic (LP) and high pathogenic (HP) phenotypes could be observed despite all containing a multi-basic cleavage site (MBCS) in the HA gene. Using reverse genetics, three amino acid substitutions in HA were examined for their ability to affect pathogenesis in the chicken. Restoration of amino acid polymorphisms close to the receptor binding site that are commonly found in H5 viruses only partially improved viral fitness in vitro and in vivo . A third novel substitution in the fusion peptide, HA 2 G4R, enabled the HP phenotype. HA 2 G4R decreased the pH stability of HA and increased the pH of HA fusion. The substitutions close to the receptor binding site optimised receptor binding while modulating the pH of HA fusion. Importantly, this study revealed pathogenic determinants beyond the MBCS.

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 (H7N7 DB ), as seen in the LPAIV precursor or an SBCS representative of common H7 LPAIVs (H7N7 SB ). Passage of H7N7 DB in chicken embryo tissues showed spontaneous evolution to a HPAIV. In contrast, deep sequencing of extracts from embryo tissues in which H7N7 SB 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.

Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness - infectivity, spread and pathogenesis - is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field.

In April 2013, an H9N2 low pathogenicity avian influenza (LPAI) virus was isolated in a turkey breeder farm in Eastern England comprising 4966 birds. Point-of-lay turkey breeding birds had been moved from a rearing site and within 5 days had shown rapid onset of clinical signs of dullness, coughing, and anorexia. Three houses were involved, two contained a total of 4727 turkey hens, and the third housed 239 male turkeys. Around 50% of the hens were affected, whereas the male turkeys demonstrated milder clinical signs. Bird morbidity rose from 10% to 90%, with an increase in mortality in both houses of turkey hens to 17 dead birds in one house and 27 birds in the second house by day 6. The birds were treated with an antibiotic but were not responsive. Postmortem investigation revealed air sacculitis but no infraorbital sinus swellings or sinusitis. Standard samples were collected, and influenza A was detected. H9 virus infection was confirmed in all three houses by detection and subtyping of hemagglutinating agents in embryonated specific-pathogen-free fowls' eggs, which were shown to be viruses of H9N2 subtype using neuraminidase inhibition tests and a suite of real-time reverse transcription PCR assays. LPAI virus pathotype was suggested by cleavage site sequencing, and an intravenous pathogenicity index of 0.00 confirmed that the virus was of low pathogenicity. Therefore, no official disease control measures were required, and despite the high morbidity, birds recovered and were kept in production. Neuraminidase sequence analysis revealed a deletion of 78 nucleotides in the stalk region, suggesting an adaptation of the virus to poultry. Hemagglutinin gene sequences of two of the isolates clustered with a group of H9 viruses containing other contemporary European H9 strains in the Y439/Korean-like group. The closest matches to the two isolates were A/turkey/Netherlands/11015452/11 (H9N2; 97.9-98% nucleotide identity) and A/mallard/Finland/Li13384/10 (H9N2; 97% nucleotide identity). Both PB2 partial sequences were a 100% nucleotide identity with A/mallard/France/090360/09, indicating a European origin of the causative virus. Furthermore, partial sequencing analysis of the remaining genes revealed the virus to be genotypically of European avian origin and therefore of lower risk to public health compared with contemporary viruses in Central and Eastern Asia. Occupational health risks were assessed, and preventative measures were taken.

We report the first occurrence of pandemic (H1N1) 2009 virus [A(H1N1)pdm09] infection on two epidemiologically linked turkey breeder premises in the United Kingdom during December 2010 and January 2011. Clinically, the birds showed only mild signs of disease, with the major presenting sign being an acute and marked reduction in egg production, leading to the prompt reporting of suspected avian notifiable disease for official investigation. Presence of A(H1N1)pdm09 infection in the United Kingdom turkey breeder flocks was confirmed by detailed laboratory investigations including virus isolation in embryonated specific pathogen-free fowls' eggs, two validated real-time reverse transcription-PCR tests, and nucleotide sequencing of the hemagglutinin and neuraminidase genes. These investigations revealed high nucleotide identity with currently circulating human A(H1N1)pdm09 strains, suggesting that human-to-poultry transmission (reverse zoonosis) was the most likely route of infection. Peak levels of human influenza-like illness community transmission also coincided with the onset of clinical signs in both affected turkey breeder flocks. This case demonstrated the value of the existing passive surveillance framework and associated veterinary and laboratory infrastructure that enables the detection and management of both exotic and new and emerging disease hazards and risks. The case also presents further evidence of the susceptibility of turkeys to infection with influenza A viruses of nonavian origin.