Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence.

IntroductionRepeated incursions of highly pathogenic avian influenza virus (HPAIV) H5 subtype of Gs/GD lineage pose a serious threat to poultry worldwide. We provide a detailed analysis of the spatio-temporal spread and genetic characteristics of HPAIV Gs/GD H5N8 from the 2019/20 epidemic in Poland.Material and methodsSamples from poultry and free-living birds were tested by real-time RT-PCR. Whole genome sequences from 24 (out of 35) outbreaks were generated and genetic relatedness was established. The clinical status of birds and possible pathways of spread were analysed based on the information provided by veterinary inspections combined with the results of phylogenetic studies.ResultsBetween 31 December 2019 and 31 March 2020, 35 outbreaks in commercial and backyard poultry holdings and 1 case in a wild bird were confirmed in nine provinces of Poland. Most of the outbreaks were detected in meat turkeys and ducks. All characterised viruses were closely related and belonged to a previously unrecognised genotype of HPAIV H5N8 clade 2.3.4.4b. Wild birds and human activity were identified as the major modes of HPAIV spread.ConclusionThe unprecedentedly late introduction of the HPAI virus urges for re-evaluation of current risk assessments. Continuous vigilance, strengthening biosecurity and intensifying surveillance in wild birds are needed to better manage the risk of HPAI occurrence in the future.

In April/May 2013, four outbreaks of avian influenza virus (AIV) infections caused by H9N2 subtype were diagnosed in Poland in fattening turkey flocks exhibiting a drop in feed and water intake, depression, respiratory signs and mortality. The subsequent serological survey carried out on samples collected between June 2012 and September 2013 from 92 poultry flocks detected positive sera in two additional meat turkey flocks located in the same province. The analysis of amino acids in the haemagglutinin and neuraminidase glycoproteins revealed that the detected H9N2 viruses possessed molecular profiles suggestive of low pathogenicity, avian-like SAα2,3 receptor specificity and adaptation to domestic poultry. Phylogenetic studies showed that these H9N2 AIVs grouped within the Eurasian clade of wild bird-origin AIVs and had no relationship with H9N2 AIV circulating in poultry in the Middle East and Far East Asia over the past decade. Experimentally infected SPF chickens with the index-case H9N2 virus remained healthy throughout the experiment. On the other hand, ten 3-week-old commercial turkeys infected via the oculonasal route showed respiratory signs and mortality (2/10 birds). Additional diagnostic tests demonstrated the consistent presence of DNA/RNA of Ornithobacterium rhinotracheale, Bordetella avium and, less frequently, of astro-, rota-, reo-, parvo- and adenoviruses in turkeys both from field outbreaks and laboratory experiment. Although no microbiological culture was performed, we speculate that these secondary pathogens could play a role in the pathogenicity of the current H9N2 infections.

H ighly pathogenic avian influenza (HPAI) H5 viruses belonging to clade 2.3.4.4 of the Goose/ Guangdong/96 (GS/Gd) lineage continue to pose a threat to poultry and wild birds worldwide (1-6). Reassortment events between HPAI H5 and low pathogenicity avian influenza (LPAI) viruses of wild-bird origin have led to generation of novel variants that might be periodically spread by wild birds across continents (6).After detection of the unofficially defined clade 2.3.4.4b (7) in May 2016 in Lake Uvs-Nur, Russia (8), and Qinghai Lake, China (9), the virus spread to Europe and Africa, causing one of the largest epizootics reported (1). This virus reached several countries in northern, western, eastern, central, and southern areas of Africa (10). Nigeria, Namibia, South Africa (11), and Egypt reported H5N8 cases throughout 2019, suggesting ongoing circulation of the virus in Africa.No HPAI H5N8 viruses were detected in Europe during June-November 2019 (12). We report detection of a reassortant HPAI A(H5N8) clade 2.3.4.4b virus in Europe during December 2019. The StudyIn July 2019, in the framework of active surveillance measures implemented in live bird markets in 18 of the 36 states in Nigeria, the National Veterinary Research Institute in Vom, Nigeria, identified a HPAI H5N8 virus in a guinea fowl in the southwestern state of Ogun. Months later, at the end of December 2019, a suspicion of an HPAI virus was raised in a holding of 14-week-old meat turkeys in Poland, located near water bodies (fish ponds and lakes of the Łęczna-Włodawa Lakeland). A sudden increase in deaths was observed, accompanied by neurologic signs such as trembling, inability to walk, paralysis of the wings, and pedaling movements of the legs. A total of 3,000-5,000 birds died during the first 3 days after the onset of clinical signs. Organ samples submitted to the National Reference Laboratory for Avian influenza at the National Veterinary Research Institute, Pulawy, Poland, were positive for avian influenza virus and were characterized as HPAI H5N8.We conducted antigenic characterization of the virus isolate by using the hemagglutination inhibition (HI) assay, which showed that the H5N8 virus in Poland had higher antigenic reactivity with European Union Reference Laboratory reference HPAI H5N8 A/turkey/Italy/7898/2014 (IT-7898) chicken antiserum (clade 2.3.4.4, GS/Gd lineage) compared with reactivity determined for European Union Reference Laboratory HPAI H5N1 A/chicken/Scotland/1/59 (SCOT-59) and LPAI H5N3 A/teal/England/7394-2805/06 (ENG-7394) antiserum.A comparison of HI titers obtained with the IT-7898, SCOT-59 and ENG-7394 homologous antigens and those recorded against the strain from Poland showed differences of 2 log2, 4 log2, and 5 log2, respectively. The H5N1 and H5N3 strains belong to the H5 Eurasian

Sixty-five poultry outbreaks and sixty-eight events in wild birds were reported during the highly pathogenic H5N8/H5N5 avian influenza epidemic in Poland in 2016-2017. The analysis of all gene segment sequences of selected strains revealed cocirculation of at least four different genome configurations (genotypes) generated through reassortment of clade 2.3.4.4 H5N8 viruses detected in Russia and China in mid-2016. The geographical and temporal distribution of three H5N8 genotypes indicates separate introductions. Additionally, an H5N5 virus with a different gene configuration was detected in wild birds. The compilation of the results with those from studies on the virus' diversity in Germany, Italy and the Netherlands revealed that Europe was affected by at least eight different H5N8/H5N5 reassortants. Analysis of the HA gene sequence of a larger subset of samples showed its diversification corresponding to the genotype classification. The close relationship between poultry and wild bird strains from the same locations observed in several cases points to wild birds as the primary source of the outbreaks in poultry.