In Ovo Models to Predict Virulence of Highly Pathogenic Avian Influenza H5-Viruses for Chickens and Ducks.

Highly pathogenic avian influenza (HPAI) H5-viruses are circulating in wild birds and are repeatedly introduced to poultry causing outbreaks in the Netherlands since 2014. The largest epizootic ever recorded in Europe was caused by HPAI H5N1 clade 2.3.4.4b viruses in the period 2021-2022. The recent H5-clade 2.3.4.4 viruses were found to differ in their virulence for chickens and ducks. Viruses causing only mild disease may remain undetected, increasing the risk of virus spread to other farms, wild birds and mammals. We developed in ovo models to determine the virulence of HPAI viruses for chickens and ducks, which are fast and have low costs. The virulence of five contemporary H5-viruses was compared studying replication rate, average time to death and virus spread in the embryo. Remarkable differences in virulence were observed between H5-viruses and between poultry species. The H5N1-2021 virus was found to have a fast replication rate in both the chicken and duck in ovo models, but a slower systemic virus dissemination compared to three other H5-clade 2.3.4.4b viruses. The results show the potential of in ovo models to quickly determine the virulence of novel HPAI viruses, and study potential virulence factors which can help to better guide the surveillance in poultry.

Zoonotic Mutation of Highly Pathogenic Avian Influenza H5N1 Virus Identified in the Brain of Multiple Wild Carnivore Species.

Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021-2022 outbreak in the Netherlands included red fox (Vulpes vulpes), polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals were submitted for testing because they showed neurological signs. In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal. Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1 virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness.

Highly Pathogenic Avian Influenza H5N1 Virus Infections in Wild Red Foxes (Vulpes vulpes) Show Neurotropism and Adaptive Virus Mutations.

During the 2020 to 2022 epizootic of highly pathogenic avian influenza virus (HPAI), several infections of mammalian species were reported in Europe. In the Netherlands, HPAI H5N1 virus infections were detected in three wild red foxes (Vulpes vulpes) that were submitted with neurological symptoms between December of 2021 and February of 2022. A histopathological analysis demonstrated that the virus was mainly present in the brain, with limited or no detection in the respiratory tract or other organs. Limited or no virus shedding was observed in throat and rectal swabs. A phylogenetic analysis showed that the three fox viruses were not closely related, but they were related to HPAI H5N1 clade 2.3.4.4b viruses that are found in wild birds. This suggests that the virus was not transmitted between the foxes. A genetic analysis demonstrated the presence of the mammalian adaptation E627K in the polymerase basic two (PB2) protein of the two fox viruses. In both foxes, the avian (PB2-627E) and the mammalian (PB2-627K) variants were present as a mixture in the virus population, which suggests that the mutation emerged in these specific animals. The two variant viruses were isolated, and virus replication and passaging experiments were performed. These experiments showed that the mutation PB2-627K increases the replication of the virus in mammalian cell lines, compared to the chicken cell line, and at the lower temperatures of the mammalian upper respiratory tract. This study showed that the HPAI H5N1 virus is capable of adaptation to mammals; however, more adaptive mutations are required to allow for efficient transmission between mammals. Therefore, surveillance in mammals should be expanded to closely monitor the emergence of zoonotic mutations for pandemic preparedness. IMPORTANCE Highly pathogenic avian influenza (HPAI) viruses caused high mortality among wild birds from 2021 to 2022 in the Netherlands. Recently, three wild foxes were found to be infected with HPAI H5N1 viruses, likely due to the foxes feeding on infected birds. Although HPAI is a respiratory virus, in these foxes, the viruses were mostly detected in the brain. Two viruses isolated from the foxes contained a mutation that is associated with adaptation to mammals. We show that the mutant virus replicates better in mammalian cells than in avian cells and at the lower body temperature of mammals. More mutations are required before viruses can transmit between mammals or can be transmitted to humans. However, infections in mammalian species should be closely monitored to swiftly detect mutations that may increase the zoonotic potential of HPAI H5N1 viruses, as these may threaten public health.

Monitoring Wind-Borne Particle Matter Entering Poultry Farms via the Air-Inlet: Highly Pathogenic Avian Influenza Virus and Other Pathogens Risk.

Wind-supported transport of particle matter (PM) contaminated with excreta from highly pathogenic avian influenza virus (HPAIv)-infected wild birds may be a HPAIv-introduction pathway, which may explain infections in indoor-housed poultry. The primary objective of our study was therefore to measure the nature and quantity of PM entering poultry houses via air-inlets. The air-inlets of two recently HPAIv-infected poultry farms (a broiler farm and a layer farm) were equipped with mosquito-net collection bags. PM was harvested every 5 days for 25 days. Video-camera monitoring registered wild bird visits. PM was tested for avian influenza viruses (AIV), Campylobacter and Salmonella with PCR. Insects, predominantly mosquitoes, were tested for AIV, West Nile, Usutu and Schmallenberg virus. A considerable number of mosquitoes and small PM amounts entered the air-inlets, mostly cobweb and plant material, but no wild bird feathers. Substantial variation in PM entering between air-inlets existed. In stormy periods, significantly larger PM amounts may enter wind-directed air-inlets. PM samples were AIV and Salmonella negative and insect samples were negative for all viruses and bacteria, but several broiler and layer farm PM samples tested Campylobacter positive. Regular wild (water) bird visits were observed near to the poultry houses. Air-borne PM and insects-potentially contaminated with HPAIv or other pathogens-can enter poultry air-inlets. Implementation of measures limiting this potential introduction route are recommended.

Multiple Introductions of Reassorted Highly Pathogenic Avian Influenza H5Nx Viruses Clade 2.3.4.4b Causing Outbreaks in Wild Birds and Poultry in The Netherlands, 2020-2021.

Highly pathogenic avian influenza (HPAI) viruses of subtype H5Nx caused outbreaks in poultry, captive birds, and wild birds in the Netherlands between October 2020 and June 2021. The full genome sequences of 143 viruses were analyzed. HPAI viruses were mainly of subtype H5N8, followed by H5N1, but also viruses of subtypes H5N3, H5N4, and H5N5 were detected. At least seven distinct genotypes were found, carrying closely related H5 segments belonging to clade 2.3.4.4b. Molecular clock analysis suggests that the reassortments of the NA gene segments likely occurred before the introduction of these viruses into the Netherlands. Genetic analysis suggested that multiple independent introductions of HPAI H5N8 viruses occurred in the Netherlands, likely followed by local spread resulting in at least two clusters of related viruses. The analysis provided evidence for independent introductions from wild birds at 10 poultry farms, whereas for two outbreaks transmission between farms could not be excluded. HPAI H5Nx viruses were detected in dead wild birds of 33 species, but mostly infected geese and swans were found. The pathogenicity of the H5N8 virus was determined for chickens and Pekin ducks, showing reduced mortality for ducks. This study provides more insight into the genetic diversity of HPAI H5Nx viruses generated by reassortment and evolution, and the spread of these viruses between wild birds and poultry. The fast and continuing evolution of H5 clade 2.3.4.4b may provide opportunities for these viruses to adapt to specific bird species, or possibly mammalian species and humans. IMPORTANCE Highly pathogenic avian influenza (HPAI) viruses are spread by migratory wild birds. Viruses causing outbreaks in wild birds and poultry in the Netherlands in 2020-2021 were genetically analyzed, which suggested that multiple virus incursions occurred. The outbreaks in poultry were likely caused by independent introductions from wild birds; only in one case virus spread between farms could not be excluded. Viruses of subtype H5N8 were mainly observed, but also other subtypes were detected that likely evolved by exchange of genetic information before these viruses were introduced into the Netherlands. Viruses were detected in many species of dead wild birds, but mostly in geese and swans. We showed that the H5N8 virus causes a higher mortality in chickens compared to ducks. This is consistent with the fact that not many wild ducks were found dead. This study provides more insight in the evolution and spread of HPAI viruses in wild birds and poultry.

Tropism of Highly Pathogenic Avian Influenza H5 Viruses from the 2020/2021 Epizootic in Wild Ducks and Geese.

Highly pathogenic avian influenza (HPAI) outbreaks have become increasingly frequent in wild bird populations and have caused mass mortality in many wild bird species. The 2020/2021 epizootic was the largest and most deadly ever reported in Europe, and many new bird species tested positive for HPAI virus for the first time. This study investigated the tropism of HPAI virus in wild birds. We tested the pattern of virus attachment of 2020 H5N8 virus to intestinal and respiratory tissues of key bird species; and characterized pathology of naturally infected Eurasian wigeons (Mareca penelope) and barnacle geese (Branta leucopsis). This study determined that 2020 H5N8 virus had a high level of attachment to the intestinal epithelium (enterotropism) of dabbling ducks and geese and retained attachment to airway epithelium (respirotropism). Natural HPAI 2020 H5 virus infection in Eurasian wigeons and barnacle geese also showed a high level of neurotropism, as both species presented with brain lesions that co-localized with virus antigen expression. We concluded that the combination of respirotropism, neurotropism, and possibly enterotropism, contributed to the successful adaptation of 2020/2021 HPAI H5 viruses to wild waterbird populations.

Incursion of Novel Highly Pathogenic Avian Influenza A(H5N8) Virus, the Netherlands, October 2020.

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.

Comparative pathogenicity and environmental transmission of recent highly pathogenic avian influenza H5 viruses.

Strategies to control spread of highly pathogenic avian influenza (HPAI) viruses by wild birds appear limited, hence timely characterization of novel viruses is important to mitigate the risk for the poultry sector and human health. In this study we characterize three recent H5-clade 2.3.4.4 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 two H5N8 viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with 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 poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, which showed 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.

Detection of Low Pathogenic Avian Influenza Virus Subtype H10N7 in Poultry and Environmental Water Samples During a Clinical Outbreak in Commercial Free-Range Layers, Netherlands 2017.

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.

Susceptibility of Chickens to Low Pathogenic Avian Influenza (LPAI) Viruses of Wild Bird- and Poultry-Associated Subtypes.

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-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.

Virus Shedding of Avian Influenza in Poultry: A Systematic Review and Meta-Analysis.

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.

The development of a multiplex serological assay for avian influenza based on Luminex technology.

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.