Iceland: an underestimated hub for the spread of high-pathogenicity avian influenza viruses in the North Atlantic.

High-pathogenicity avian influenza viruses (HPAIVs) of the goose/Guangdong lineage are enzootically circulating in wild bird populations worldwide. This increases the risk of entry into poultry production and spill-over to mammalian species, including humans. Better understanding of the ecological and epizootiological networks of these viruses is essential to optimize mitigation measures. Based on full genome sequences of 26 HPAIV samples from Iceland, which were collected between spring and autumn 2022, as well as 1 sample from the 2023 summer period, we show that 3 different genotypes of HPAIV H5N1 clade 2.3.4.4b were circulating within the wild bird population in Iceland in 2022. Furthermore, in 2023 we observed a novel introduction of HPAIV H5N5 of the same clade to Iceland. The data support the role of Iceland as an utmost northwestern distribution area in Europe that might act also as a potential bridging point for intercontinental spread of HPAIV across the North Atlantic.

Highly pathogenic avian influenza A virus (HPAIV) H5N1 infection in two European grey seals (Halichoerus grypus) with encephalitis.

ABSTRACTRecent reports documenting sporadic infections in carnivorous mammals worldwide with highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b have raised concerns about the potential risk of adaptation to sustained transmission in mammals, including humans. We report H5N1 clade 2.3.4.4b infection of two grey seals (Halichoerus grypus) from coastal waters of The Netherlands and Germany in December 2022 and February 2023, respectively. Histological and immunohistochemical investigations showed in both animals a non-suppurative and necrotising encephalitis with viral antigen restricted to the neuroparenchyma. Whole genome sequencing showed the presence of HPAIV H5N1 clade 2.3.4.4b strains in brain tissue, which were closely related to sympatric avian influenza viruses. Viral RNA was also detected in the lung of the seal from Germany by real-time quantitative PCR. No other organs tested positive. The mammalian adaptation PB2-E627K mutation was identified in approximately 40% of the virus population present in the brain tissue of the German seal. Retrospective screening for nucleoprotein-specific antibodies, of sera collected from 251 seals sampled in this region from 2020 to 2023, did not show evidence of influenza A virus-specific antibodies. Similarly, screening by reverse transcription PCR of tissues of 101 seals that had died along the Dutch coast in the period 2020-2021, did not show evidence of influenza virus infection. Collectively, these results indicate that individual seals are sporadically infected with HPAIV-H5N1 clade 2.3.4.4b, resulting in an encephalitis in the absence of a systemic infection, and with no evidence thus far of onward spread between seals.

Investigating Environmental Matrices for Use in Avian Influenza Virus Surveillance-Surface Water, Sediments, and Avian Fecal Samples.

Surveillance of avian influenza viruses (AIV) in wild water bird populations is important for early warning to protect poultry from incursions of high-pathogenicity (HP) AIV. Access to individual water birds is difficult and restricted and limits sampling depth. Here, we focused on environmental samples such as surface water, sediments, and environmentally deposited fresh avian feces as matrices for AIV detection. Enrichment of viral particles by ultrafiltration of 10-L surface water samples using Rexeed-25-A devices was validated using a bacteriophage ϕ6 internal control system, and AIV detection was attempted using real-time RT-PCR and virus isolation. While validation runs suggested an average enrichment of about 60-fold, lower values of 10 to 15 were observed for field water samples. In total 25/36 (60%) of water samples and 18/36 (50%) of corresponding sediment samples tested AIV positive. Samples were obtained from shallow water bodies in habitats with large numbers of waterfowl during an HPAIV epizootic. Although AIV RNA was detected in a substantial percentage of samples virus isolation failed. Virus loads in samples often were too low to allow further sub- and pathotyping. Similar results were obtained with environmentally deposited avian feces. Moreover, the spectrum of viruses detected by these active surveillance methods did not fully mirror an ongoing HPAIV epizootic among waterfowl as detected by passive surveillance, which, in terms of sensitivity, remains unsurpassed. IMPORTANCE Avian influenza viruses (AIV) have a wide host range in the avian metapopulation and, occasionally, transmission to humans also occurs. Surface water plays a particularly important role in the epidemiology of AIV, as the natural virus reservoir is found in aquatic wild birds. Environmental matrices comprising surface water, sediments, and avian fecal matter deposited in the environment were examined for their usefulness in AIV surveillance. Despite virus enrichment efforts, environmental samples regularly revealed very low virus loads, which hampered further sub- and pathotyping. Passive surveillance based on oral and cloacal swabs of diseased and dead wild birds remained unsurpassed with respect to sensitivity.

Exploring surface water as a transmission medium of avian influenza viruses - systematic infection studies in mallards.

Mallards (Anas platyrhynchos) are an abundant anseriform migratory wild bird species worldwide and an important reservoir for the maintenance of low pathogenicity (LP) avian influenza viruses (AIV). They have also been implicated in the spread of high pathogenicity (HP) AIV after spill-over events from HPAIV-infected poultry. The spread of HPAIV within wild water bird populations may lead to viral contamination of natural habitats. The role of small shallow water bodies as a transmission medium of AIV among mallards is investigated here in three experimental settings. (i) Delayed onset but rapid progression of infection seeded by two mallards inoculated with either LP or HP AIV to each eight sentinel mallards was observed in groups with access to a small 100 L water pool. In contrast, groups with a bell drinker as the sole source of drinking water showed a rapid onset but lengthened course of infection. (ii) HPAIV infection also set off when virus was dispersed in the water pool; titres as low as 102 TCID50 L-1 (translating to 0.1 TCID50 mL-1) proved to be sufficient. (iii) Substantial loads of viral RNA (and infectivity) were also found on the surface of the birds' breast plumage. "Unloading" of virus infectivity from contaminated plumage into water bodies may be an efficient mechanism of virus spread by infected mallards. However, transposure of HPAIV via the plumage of an uninfected mallard failed. We conclude, surface water in small shallow water bodies may play an important role as a mediator of AIV infection of aquatic wild birds.

Improved Subtyping of Avian Influenza Viruses Using an RT-qPCR-Based Low Density Array: 'Riems Influenza a Typing Array', Version 2 (RITA-2).

Avian influenza virus (AIV) variants emerge frequently, which challenges rapid diagnosis. Appropriate diagnosis reaching the sub- and pathotype level is the basis of combatting notifiable AIV infections. Real-time RT-PCR (RT-qPCR) has become a standard diagnostic tool. Here, a total of 24 arrayed RT-qPCRs is introduced for full subtyping of 16 hemagglutinin and nine neuraminidase subtypes of AIV. This array, designated Riems Influenza A Typing Array version 2 (RITA-2), represents an updated and economized version of the RITA-1 array previously published by Hoffmann et al. RITA-2 provides improved integration of assays (24 instead of 32 parallel reactions) and reduced assay volume (12.5 µL). The technique also adds RT-qPCRs to detect Newcastle Disease (NDV) and Infectious Bronchitis viruses (IBV). In addition, it maximizes inclusivity (all sequences within one subtype) and exclusivity (no intersubtypic cross-reactions) as shown in validation runs using a panel of 428 AIV reference isolates, 15 reference samples each of NDV and IBV, and 122 clinical samples. The open format of RITA-2 is particularly tailored to subtyping influenza A virus of avian hosts and Eurasian geographic origin. Decoupling and re-arranging selected RT-qPCRs to detect specific AIV variants causing epizootic outbreaks with a temporal and/or geographic restriction is possible.