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Highly pathogenic avian influenza A(H5N8) virus was detected in mute swans in the Netherlands during October 2020. The virus shares a common ancestor with clade 2.3.4.4b viruses detected in Egypt during 2018–2019 and has similar genetic composition. The virus is not directly related to H5N8 viruses from Europe detected in the first half of 2020.

Highly pathogenic avian influenza (HPAI) viruses of H5 clade 2.3.4.4 have spread to many countries in Asia, Europe and North America by migratory wild birds, causing outbreaks on hundreds of poultry farms. Strategies to control spread by wild birds appear limited, hence timely characterization of novel viruses is important to limit the risk for the poultry sector and human health. In this study we characterize three recent viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the H5N8 group A and B viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent virus, the H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with the group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds, and between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, showing that chickens are efficiently infected by (fecal)contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector.

The HPAI H5N8 virus detected in mute swans in the Netherlands in October 2020 shares a common ancestor with clade 2.3.4.4b viruses last detected in Egypt in 2018-2019 and has a similar genetic composition. The virus is not directly related to European H5N8 viruses detected in first half of 2020.

Wild birds are the natural reservoir of the avian influenza virus (AIV) and may transmit AIV to poultry via direct contact or indirectly through the environment. In the Netherlands, a clinically suspected free-range layer flock was reported to the veterinary authorities by the farmer. Increased mortality, a decreased feed intake, and a drop in egg production were observed. Subsequently, an infection with low pathogenic avian influenza virus was detected. This study describes the diagnostic procedures used for detection and subtyping of the virus. In addition to routine diagnostics, the potential of two different environmental diagnostic methods was investigated for detecting AIV in surface water. AIV was first detected using rRT-PCR and isolated from tracheal and cloacal swabs collected from the hens. The virus was subtyped as H10N7. Antibodies against the virus were detected in 28 of the 31 sera tested. An intravenous pathogenicity index (IVPI) experiment was performed, but no clinical signs (IVPI = 0) were observed. Post-mortem examination and histology confirmed the AIV infection. Multiple water samples were collected longitudinally from the free-range area and waterway near the farm. Both environmental diagnostic methods allowed the detection of the H10N7 virus, demonstrating the potential of these methods in detection of AIV. The described methods could be a useful additional procedure for AIV surveillance in water-rich areas with large concentrations of wild birds or in areas around poultry farms. In addition, these methods could be used as a tool to test if the environment or free-range area is virus-free again, at the end of an AIV epidemic.

Avian influenza (AI) is an infectious disease in birds with enormous impact on the poultry sector. AI viruses are divided into different subtypes based on the antigenicity of their surface proteins haemagglutinin (HA) and neuraminidases (NA). In birds, 16 HA subtypes and 9 NA subtypes are detected in different combinations. Traditional serological methods for the subtyping of AI antibodies are labour-intensive and have to be performed for each HA and NA subtype separately. This study describes the development of a multiplex serological assay for subtyping AI antibodies in poultry sera using Luminex xMAP technology. This multiplex assay allows the detection of all AI serotypes in one single assay. For all HA and NA subtypes, recombinant proteins were purified and coupled to colour-coded magnetic bead sets. Using the Luminex MAGPIX device, binding of serum antibodies to the antigens on the bead sets is detected by fluorescent secondary antibodies, and the different bead sets are identified. The results of the multiplex assay were compared with that of the traditional singleplex assays. We show that serotyping using the novel multiplex serological assay is consistent with the results of the traditional assays in 97.8% of the reference sera and in 90.8% of the field sera. The assay has a higher sensitivity than the traditional assays, and requires a smaller sample volume. Therefore, the assay will allow complete AI-serotyping in small volumes of field sera, which will improve the monitoring of AI subtypes circulating in poultry significantly.

Understanding virus shedding patterns of avian influenza virus (AIV) in poultry is important for understanding host-pathogen interactions and developing effective control strategies. Many AIV strains were studied in challenge experiments in poultry, but no study has combined data from those studies to identify general AIV shedding patterns. These systematic review and meta-analysis were performed to summarize qualitative and quantitative information on virus shedding levels and duration for different AIV strains in experimentally infected poultry species. Methods were designed based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Four electronic databases were used to collect literature. A total of 1155 abstract were screened, with 117 studies selected for the qualitative analysis and 71 studies for the meta-analysis. A large heterogeneity in experimental methods was observed and the quantitative analysis showed that experimental variables such as species, virus origin, age, inoculation route and dose, affect virus shedding (mean, peak and duration) for highly pathogenic AIV (HPAIV), low pathogenic AIV (LPAIV) or both. In conclusion, this study highlights the need to standardize experimental procedures, it provides a comprehensive summary of the shedding patterns of AIV strains by infected poultry and identifies the variables that influence the level and duration of AIV shedding.

Analysis of low pathogenic avian influenza (LPAI) viruses circulating in the Netherlands in a previous study revealed associations of specific hemagglutinin (HA) and neuraminidase (NA) subtypes with wild bird or poultry hosts. In this study, we identified putative host associations in LPAI virus internal proteins. We show that LPAI viruses isolated from poultry more frequently carried the allele A variant of the nonstructural protein (NS) gene, compared to wild bird viruses. We determined the susceptibility of chickens to wild bird&ndash, associated subtypes H3N8 and H4N6 and poultry-associated subtypes H8N4 and H9N2, carrying either NS allele A or B, in an infection experiment. We observed variations in virus shedding and replication patterns, however, these did not correlate with the predicted wild bird- or poultry-associations of the viruses. The experiment demonstrated that LPAI viruses of wild bird-associated subtypes can replicate in chickens after experimental infection, despite their infrequent detection in poultry. Although the NS1 protein is known to play a role in immune modulation, no differences were detected in the limited innate immune response to LPAI virus infection. This study contributes to a better understanding of the infection dynamics of LPAI viruses in chickens.