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Highly Pathogenic Avian Influenza viruses (HPAIVs) display a tissue pantropism, which implies a possible spread in feathers. HPAIV detection from feathers had been evaluated for H5N1 or H7N1 HPAIVs. It was suggested that viral RNA loads could be equivalent or higher in samples of immature feather compared to tracheal (TS) or cloacal swabs (CS). We investigated the suitability of feathers for the detection of clade 2.3.4.4b H5N8 HPAIV in ducks and geese field samples. In the six H5N8 positive flocks that were included in this study, TS, CS and immature wing feathers were taken from at least 10 birds. Molecular loads were then estimated using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) targetting H5 and M genes. In all flocks, viral loads were at least equivalent between feather and swab samples and in most cases up to 10 3 higher in feathers. Bayesian modelling confirmed that, in infected poultry, RT-qPCR was much more likely to be positive when applied on a feather sample only (estimated sensitivity between 0.89 and 0.96 depending on the positivity threshold) than on a combination of a tracheal and a cloacal swab (estimated sensitivity between 0.45 and 0.68 depending on the positivity threshold). Viral tropism and lesions in feathers were evaluated by histopathology and immunohistochemistry. Epithelial necrosis of immature feathers and follicles was observed concurrently with positive viral antigen detection and leukocytic infiltration of pulp. Accurate detection of clade 2.3.4.4b HPAIVs in feather samples were finally confirmed with experimental H5N8 infection on 10-week-old mule ducks, as viral loads at 3, 5 and 7 days post-infection were higher in feathers than in tracheal or cloacal swabs. However, feather samples were associated with lower viral loads than tracheal swabs at day 1, suggesting better detectability of the virus in feathers in the later course of infection. These results, based on both field cases and experimental infections, suggest that feather samples should be included in the toolbox of samples for detection of clade 2.3.4.4b HPAI viruses, at least in ducks and geese.

Highly pathogenic avian influenza viruses (HPAIVs) evolve from low pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes. This evolution is characterized by the acquisition of a multi-basic cleavage site (MBCS) motif in the hemagglutinin (HA) that leads to an extended viral tropism and severe disease in poultry. One key unanswered question is whether the risk of transition to HPAIVs is similar for all LPAIVs H5 or H7 strains, or whether specific determinants in the HA sequence of some H5 or H7 LPAIV strains correlate with a higher risk of transition to HPAIVs. Here, we determined if specific features of the conserved RNA stem-loop located at the HA cleavage site-encoding region could be detected along the LPAIV to HPAIV evolutionary pathway. Analysis of the thermodynamic stability of the predicted RNA structures showed no specific patterns common to HA sequences leading to HPAIVs and distinct from those remaining LPAIVs. However, RNA structure clustering analysis revealed that most of the American lineage ancestors leading to H7 emergences via recombination shared the same viral RNA (vRNA) structure topology at the HA1/HA2 boundary region. Our study thus identified predicted secondary RNA structures present in the HA of H7 viruses, which could promote genetic recombination and acquisition of a multibasic cleavage site motif (MBCS). [ABSTRACT FROM AUTHOR]