High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

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.

Effect of 2020-21 and 2021-22 Highly Pathogenic Avian Influenza H5 Epidemics on Wild Birds, the Netherlands.

The number of highly pathogenic avian influenza (HPAI) H5-related infections and deaths of wild birds in Europe was high during October 1, 2020-September 30, 2022. To quantify deaths among wild species groups with known susceptibility for HPAI H5 during those epidemics, we collected and recorded mortality data of wild birds in the Netherlands. HPAI virus infection was reported in 51 bird species. The species with the highest numbers of reported dead and infected birds varied per epidemic year; in 2020-21, they were within the Anatidae family, in particular barnacle geese (Branta leucopsis) and in 2021-22, they were within the sea bird group, particularly Sandwich terns (Thalasseus sandvicensis) and northern gannet (Morus bassanus). Because of the difficulty of anticipating and modeling the future trends of HPAI among wild birds, we recommend monitoring live and dead wild birds as a tool for surveillance of the changing dynamics of HPAI.

Immunoadjuvant efficacy of CpG plasmids for H9N2 avian influenza inactivated vaccine in chickens with maternal antibodies.

Maternal-derived antibodies (MDAs) are one of reasons why vaccination with the H9N2 inactivated whole virus (IWV) vaccine failed in poultry. Unmethylated CpG motif-containing oligodeoxynucleotides (CpG ODN) shows great potential to overcome MDAs interference in mammals, but whether it has similar characteristics in poultry is still unknown. In the present study, different classes and various copies of CpG ODN motifs were cloned into two different plasmids (pCDNA3.1 or T vector). Immunomodulatory activities and immunoadjuvant efficacy of these CpG ODN plasmids were tested in vitro and in vivo in the presence of passively transferred antibodies (PTAs) that were used to mimic MDAs. Results showed that the T vector enriched with 30 copies of CpG-A ODN and 20 copies of CpG-B ODN (T-CpG-AB) significantly up-regulated mRNA expression of chicken-interferon-α (ch-IFN-α), chicken-interferon-ß (ch-IFN-ß) and chicken-interleukin-12 protein 40 (ch-IL-12p40). When administered as adjuvant of the H9N2 IWV vaccine, the minimal dose of T-CpG-AB plasmid was 30 µg per one-day-old chicken, which could induce strong humoral immune responses in the presence of PTAs. Furthermore, T-CpG-AB plasmid-based vaccine triggered both strong humoral immune responses and cytokines expression in the presence of PTAs in chickens. Overall, our findings suggest that T-CpG-AB plasmid can be an excellent adjuvant candidate for the H9N2 IWV vaccine to overcome MDAs interference in chickens.

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.

Mass Mortality Caused by Highly Pathogenic Influenza A(H5N1) Virus in Sandwich Terns, the Netherlands, 2022.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research.

Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021.

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. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade 2.3.4.4b, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm shift in the epidemiology of HPAI in Europe.

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.

Tissue tropism and pathology of highly pathogenic avian influenza H5N6 virus in chickens and Pekin ducks.

The highly pathogenic avian influenza (HPAI) H5N6 virus caused outbreaks on commercial poultry farms in the Netherlands in 2017-2018, holding chickens and Pekin ducks. Intravenous pathogenicity index (IVPI) tests confirmed the high pathogenicity of the virus. Tissues derived from birds from infected farms (natural infection) and IVPI tests (experimental infection) were used to compare histopathology and virus distribution in both poultry species. After natural infection in chickens, histopathologic changes were present in the respiratory tract and several internal organs in both chickens and Pekin ducks. Viral antigen expression in the tissues of chickens varied from that in ducks. Virus expression was found in epithelial, mononuclear and endothelial cells in chickens. In contrast to the major role infected endothelial cells seem to play in systemic infections of chickens, in ducks the number of infected endothelial cells was very limited. Therefore, endothelial cell infection likely does not play a major role in systemic infection and disease progression in HPAI H5N6 virus infected Pekin ducks.

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.

Molecular and Antigenic Characterization of Avian H9N2 Viruses in Southern China.

The H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in BJ94 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 × 10-3 substitutions/site/year, which was 1.5-fold faster than that of the average H9N2 HA rate (3.95 × 10-3 substitutions/site/year). Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses. IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry (especially in chicken in South Korea and China). Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 AIVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.

The PB1 gene from H9N2 avian influenza virus showed high compatibility and increased mutation rate after reassorting with a human H1N1 influenza virus.

BACKGROUND: Reassortment between human and avian influenza viruses (AIV) may result in novel viruses with new characteristics that may threaten human health when causing the next flu pandemic. A particular risk may be posed by avian influenza viruses of subtype H9N2 that are currently massively circulating in domestic poultry in Asia and have been shown to infect humans. In this study, we investigate the characteristics and compatibility of a human H1N1 virus with avian H9N2 derived genes. METHODS: The polymerase activity of the viral ribonucleoprotein (RNP) complex as combinations of polymerase-related gene segments derived from different reassortment events was tested in luciferase reporter assays. Reassortant viruses were generated by reverse genetics. Gene segments of the human WSN-H1N1 virus (A/WSN/1933) were replaced by gene segments of the avian A2093-H9N2 virus (A/chicken/Jiangsu/A2093/2011), which were both the Hemagglutinin (HA) and Neuraminidase (NA) gene segments in combination with one of the genes involved in the RNP complex (either PB2, PB1, PA or NP). The growth kinetics and virulence of reassortant viruses were tested on cell lines and mice. The reassortant viruses were then passaged for five generations in MDCK cells and mice lungs. The HA gene of progeny viruses from different passaging paths was analyzed using Next-Generation Sequencing (NGS). RESULTS: We discovered that the avian PB1 gene of H9N2 increased the polymerase activity of the RNP complex in backbone of H1N1. Reassortant viruses were able to replicate in MDCK and DF1 cells and mice. Analysis of the NGS data showed a higher substitution rate for the PB1-reassortant virus. In particular, for the PB1-reassortant virus, increased virulence for mice was measured by increased body weight loss after infection in mice. CONCLUSIONS: The higher polymerase activity and increased mutation frequency measured for the PB1-reassortant virus suggests that the avian PB1 gene of H9N2 may drive the evolution and adaptation of reassortant viruses to the human host. This study provides novel insights in the characteristics of viruses that may arise by reassortment of human and avian influenza viruses. Surveillance for infections with H9N2 viruses and the emergence of the reassortant viruses in humans is important for pandemic preparedness.

Efficacy of a recombinant turkey herpesvirus (H9) vaccine against H9N2 avian influenza virus in chickens with maternal-derived antibodies.

Although vaccines have been widely used for many years, they have failed to control H9N2 avian influenza virus (AIV) in the field in China. The high level of maternal-derived antibodies (MDAs) against H9N2 virus contributes to the H9N2 influenza vaccine failure in poultry. The study aimed to generate a new vaccine to overcome MDAs interference in H9N2 vaccination in chickens. We used turkey herpesvirus (HVT) as a vaccine vector to express H9 hemagglutinin (HA) proteins. The recombinant HVT expressing H9 HA proteins (rHVT-H9) was successfully generated and characterized in primary chicken embryonic fibroblasts (CEFs). Western blot and indirect immunofluorescence assay (IFA) showed that the rHVT-H9 consistently expressed HA proteins. In addition, the rHVT-H9 had similar growth kinetics to the parent HVT. Preliminary animal experiments showed that compared to the conventional inactivated whole virus (IWV) vaccine, the rHVT-H9 stimulated robust humoral immunity in chickens with passively transferred antibodies (PTAs) that were used to mimic MDAs. Transmission experiments showed that the rHVT-H9 induced both humoral and cellular immunity in chickens with PTAs. Furthermore, we used mathematical models to quantify the vaccine's efficacy in preventing the transmission of H9N2 AIV. The results showed that the rHVT-H9 reduced the virus shedding period and decreased the reproduction ratio (R) value in chickens with PTAs after homologous challenge. However, the vaccination in this trial did not yet bring R < 1. In summary, we generated a new rHVT-H9 vaccine, which stimulated strong humoral and cellular immunity, reducing virus shedding and transmission of H9N2 AIV even in the presence of PTAs in chickens.

Vaccination with inactivated virus against low pathogenic avian influenza subtype H9N2 does not prevent virus transmission in chickens.

H9N2 subtype avian influenza has spread dramatically in China ever since first reported in the 1990s. A national vaccination program for poultry was initiated in 1998. Field isolation data show that the widely used inactivated H9N2 vaccine does not provide effective control of the transmission of this low pathogenic avian influenza (LPAI) virus in poultry. Current research has focused on two reasons: (i) insufficient immune response triggered by the vaccination with the inactivated virus, (ii) the occurrence of escape mutants selected by vaccine-induced immune pressure. However, the lack of effectivity of the inactivated virus vaccine to sufficiently reduce transmission has been noticed. We mimicked the natural infection and transmission process of the H9N2 virus in vaccinated and non-vaccinated chickens. A statistical model was used to estimate the transmission rate parameters among vaccinated chickens, varying in serum hemagglutinin inhibition titers (HIT) and non-vaccinated chickens. We demonstrate, for the first time, that the transmission is not sufficiently reduced by the H9N2 vaccine, even when vaccinated chickens have an IgG serum titer (HIT>23), which is considered protective for vaccination against homologous highly pathogenic avian influenza (HPAI) virus. Our study does, on the other hand, cast new light on virus transmission and immune escape of LPAI H9N2 virus in vaccinated chickens populations, and shows that new mitigation strategies against LPAI viruses in poultry are needed.

Highly Pathogenic Avian Influenza A(H5N1) Virus in Wild Red Foxes, the Netherlands, 2021.

We detected infection with highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b in 2 red fox (Vulpes vulpes) cubs found in the wild with neurologic signs in the Netherlands. The virus is related to avian influenza viruses found in wild birds in the same area.

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.

Seasonal risk of low pathogenic avian influenza virus introductions into free-range layer farms in the Netherlands.

Poultry can become infected with avian influenza viruses (AIV) via (in) direct contact with infected wild birds. Free-range chicken farms in the Netherlands were shown to have a higher risk for introduction of low pathogenic avian influenza (LPAI) virus than indoor chicken farms. Therefore, during outbreaks of highly pathogenic avian influenza (HPAI), free-range layers are confined indoors as a risk mitigation measure. In this study, we characterized the seasonal patterns of AIV introductions into free-range layer farms, to determine the high-risk period. Data from the LPAI serological surveillance programme for the period 2013-2016 were used to first estimate the time of virus introduction into affected farms and then assess seasonal patterns in the risk of introduction. Time of introduction was estimated by fitting a mathematical model to seroprevalence data collected longitudinally from infected farms. For the period 2015-2016, longitudinal follow-up included monthly collections of eggs for serological testing from a cohort of 261 farms. Information on the time of introduction was then used to estimate the monthly incidence and seasonality by fitting harmonic and Poisson regression models. A significant yearly seasonal risk of introduction that lasted around 4 months (November to February) was identified with the highest risk observed in January. The risk for introduction of LPAI viruses in this period was on average four times significantly higher than the period of low risk around the summer months. Although the data for HPAI infections were limited in the period 2014-2018, a similar risk period for introduction of HPAI viruses was observed. The results of this study can be used to optimize risk-based surveillance and inform decisions on timing and duration of indoor confinement when HPAI viruses are known to circulate in the wild bird population.

Molecular and virological characterization of the first poultry outbreaks of Genotype VII.2 velogenic avian orthoavulavirus type 1 (NDV) in North-West Europe, BeNeLux, 2018.

After two decades free of Newcastle disease, Belgium encountered a velogenic avian orthoavulavirus type 1 epizootic in 2018. In Belgium, 20 cases were diagnosed, of which 15 occurred in hobby flocks, 2 in professional poultry flocks and 3 in poultry retailers. The disease also disseminated from Belgium towards the Grand Duchy of Luxembourg by trade. Independently, the virus was detected once in the Netherlands, almost simultaneously to the first Belgian detection. As such Newcastle disease emerged in the entire BeNeLux region. Both the polybasic sequence of the fusion gene cleavage site and the intracerebral pathotyping assay demonstrated the high pathogenicity of the strain. This paper represents the first notification of this specific VII.2 subgenotype in the North-West of Europe. Time-calibrated full genome phylogenetic analysis indicated the silent or unreported circulation of the virus prior to the emergence of three genetic clusters in the BeNeLux region without clear geographical or other epidemiological correlation. The Dutch strain appeared as an outgroup to the Belgian and Luxembourgian strains in the time-correlated genetic analysis and no epidemiological link could be identified between the Belgian and Dutch outbreaks. In contrast, both genetic and epidemiological outbreak investigation data linked the G.D. Luxembourg case to the Belgian outbreak. The genetic links between Belgian viruses from retailers and hobby flocks only partially correlated with epidemiological data. Two independent introductions into the professional poultry sector were identified, although their origin could not be determined. Animal experiments using 6-week- old specific pathogen-free chickens indicated a systemic infection and efficient transmission of the virus. The implementation of re-vaccination in the professional sector, affected hobby and retailers, as well as the restriction on assembly and increased biosecurity measures, possibly limited the epizootic and resulted in the disappearance of the virus. These findings emphasize the constant need for awareness and monitoring of notifiable viruses in the field.

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.