Viral shedding and environmental dispersion of two clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in experimentally infected mule ducks: implications for environmental sampling.

High pathogenicity avian influenza viruses (HPAIVs) have caused major epizootics in recent years, with devastating consequences for poultry and wildlife worldwide. Domestic and wild ducks can be highly susceptible to HPAIVs, and infection leads to efficient viral replication and massive shedding (i.e., high titres for an extended time), contributing to widespread viral dissemination. Importantly, ducks are known to shed high amounts of virus in the earliest phase of infection, but the dynamics and impact of environmental contamination on the epidemiology of HPAIV outbreaks are poorly understood. In this study, we monitored mule ducks experimentally infected with two H5N8 clade 2.3.4.4b goose/Guangdong HPAIVs sampled in France in 2016-2017 and 2020-2021 epizootics. We investigated viral shedding dynamics in the oropharynx, cloaca, conjunctiva, and feathers; bird-to-bird viral transmission; and the role of the environment in viral spread and as a source of samples for early detection and surveillance. Our findings showed that viral shedding started before the onset of clinical signs, i.e., as early as 1 day post-inoculation (dpi) or post-contact exposure, peaked at 4 dpi, and lasted for up to 14 dpi. The detection of viral RNA in aerosols, dust, and water samples mirrored viral shedding dynamics, and viral isolation from these environmental samples was successful throughout the experiment. Our results confirm that mule ducks can shed high HPAIV titres through the four excretion routes tested (oropharyngeal, cloacal, conjunctival, and feather) while being asymptomatic and that environmental sampling could be a non-invasive tool for early viral RNA detection in HPAIV-infected farms.

Low Susceptibility of Pigs against Experimental Infection with HPAI Virus H5N1 Clade 2.3.4.4b.

We found that nasal and alimentary experimental exposure of pigs to highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b was associated with marginal viral replication, without inducing any clinical manifestation or pathological changes. Only 1 of 8 pigs seroconverted, pointing to high resistance of pigs to clade 2.3.4.4b infection.

Long-Term Protective Effect of Serial Infections with H5N8 Highly Pathogenic Avian Influenza Virus in Wild Ducks.

Highly pathogenic avian influenza viruses (HPAIVs) of the Goose/Guangdong (Gs/Gd) lineage are an emerging threat to wild birds. In the 2016-2017 H5N8 outbreak, unexplained variability was observed in susceptible species, with some reports of infected birds dying in high numbers and other reports of apparently subclinical infections. This experimental study was devised to test the hypothesis that previous infection with a less-virulent HPAIV (i.e., 2014 H5N8) provides long-term immunity against subsequent infection with a more-virulent HPAIV (i.e., 2016 H5N8). Therefore, two species of wild ducks-the more-susceptible tufted duck (Aythya fuligula) and the more-resistant mallard (Anas platyrhynchos)-were serially inoculated, first with 2014 H5N8 and after 9 months with 2016 H5N8. For both species, a control group of birds was first sham inoculated and after 9 months inoculated with 2016 H5N8. Subsequent infection with the more-virulent 2016 H5N8 caused no clinical signs in tufted ducks that had previously been infected with 2014 H5N8 (n = 6) but caused one death in tufted ducks that had been sham inoculated (n = 7). In mallards, 2016 H5N8 infection caused significant body weight loss in previously sham-inoculated birds (n = 8) but not in previously infected birds (n = 7). IMPORTANCE This study showed that ducks infected with a less-virulent HPAIV developed immunity that was protective against a subsequent infection with a more-virulent HPAIV 9 months later. Following 2014 H5N8 infection, the proportion of birds with detectable influenza nucleoprotein antibody declined from 100% (8/8) in tufted ducks and 78% (7/9) in mallards after 1 month to 33% (2/6) in tufted ducks and 29% (2/7) in mallards after 9 months. This finding helps predict the expected impact that an HPAIV outbreak may have on wild bird populations, depending on whether they are immunologically naive or have survived previous infection with HPAIV.

Chicken miR-148a-3p regulates immune responses against AIV by targeting the MAPK signalling pathway and IFN-γ.

MicroRNAs are involved in the immune systems of host animals and play essential roles in several immune-related pathways. In the current study, we investigated the systemic biological function of the chicken miRNA gga-miR-148a-3p on immune responses in chicken lines resistant and susceptible to HPAIV-H5N1. We found that gga-miR-148a expression in the lung tissue of H5N1-resistant chickens was significantly downregulated during HPAIV-H5N1 infection. Overexpression of gga-miR-148a and a reporter construct with wild type or mutant IFN-γ, MAPK11, and TGF-ß2 3' untranslated region (3' UTR)-luciferase in chicken fibroblasts showed that gga-miR-148a acted as a direct translational repressor of IFN-γ, MAPK11, and TGF-ß2 by targeting their 3' UTRs. Furthermore, miR-148a directly and negatively influenced the expression of signalling molecules related to the MAPK signalling pathway, including MAPK11, TGF-ß2, and Jun, and regulated antiviral responses through interferon-stimulated genes and MHC class I and class II genes by targeting IFN-γ. Downstream of the MAPK signalling pathway, several proinflammatory cytokines such as IL-1ß, IFN-γ, IL-6, TNF-α, IFN-ß, and interferon-stimulated genes were downregulated by the overexpression of gga-miR-148a. Our data suggest that gga-miR-148a-3p is an important regulator of the MAPK signalling pathway and antiviral response. These findings improve our understanding of the biological functions of gga-miR-148a-3p, the mechanisms underlying the MAPK signalling pathway, and the antiviral response to HPAIV-H5N1 infection in chickens as well as the role of gga-miR-148a-3p in improving the overall performance of chicken immune responses for breeding disease-resistant chickens.

Iceland as Stepping Stone for Spread of Highly Pathogenic Avian Influenza Virus between Europe and North America.

Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin type H5 and clade 2.3.4.4b have widely spread within the northern hemisphere since 2020 and threaten wild bird populations, as well as poultry production. We present phylogeographic evidence that Iceland has been used as a stepping stone for HPAIV translocation from northern Europe to North America by infected but mobile wild birds. At least 2 independent incursions of HPAIV H5N1 clade 2.3.4.4b assigned to 2 hemagglutinin clusters, B1 and B2, are documented for summer‒autumn 2021 and spring 2022. Spread of HPAIV H5N1 to and among colony-breeding pelagic avian species in Iceland is ongoing. Potentially devastating effects (i.e., local losses >25%) on these species caused by extended HPAIV circulation in space and time are being observed at several affected breeding sites throughout the North Atlantic.

Immunogenicity and cross-protective efficacy of recombinant H5HA1 protein of clade 2.3.2.1a highly pathogenic H5N1 avian influenza virus expressed in E.coli.

The global spread of H5N1 highly pathogenic avian influenza virus (HPAIV) in poultry has caused great economic loss to the poultry farmers and industry with significant pandemic threat. The current study involved production of recombinant HA1 protein of clade 2.3.2.1a H5N1 HPAIV (rH5HA1) in E.coli and evaluation of its protective efficacy in chickens. Purification under denaturing conditions and refolding by dialysis against buffers containing decreasing concentrations of urea was found to preserve the biological activity of the expressed recombinant protein as assessed by hemagglutination assay, Western blot and ELISA. The Montanide ISA 71 VGA adjuvanted rH5HA1 protein was used for immunization of chickens. Humoral response was maintained at a minimum of 4log2 hemagglutination inhibition (HI) titre till 154 days post 2nd booster. We evaluated the protective efficacy of rH5HA1 protein in immunized chickens by challenging them with homologous (2.3.2.1a) and heterologous (2.3.2.1c) clades of H5N1 HPAIV. In both the groups, the HI titre significantly increased (P < 0.05) after challenge and the virus shedding significantly (P < 0.05) reduced between 3rd and 14th day post challenge. The virus shedding ratio in oro-pharyngeal swabs did not differ significantly between both the groups except on 7 days post challenge and during the entire experimental period in cloacal swabs. These results indicate that rH5HA1 was able to induce homologous and cross protective immune response in chickens and could be a potential vaccine candidate used for combating the global spread of H5N1 HPAIV threat. To our knowledge, this is the first study to report immunogenicity and protective efficacy of prokaryotic recombinant H5HA1 protein in chicken.

Highly Pathogenic Avian Influenza A(H5N8) Virus Clade 2.3.4.4b, Western Siberia, Russia, 2020.

Two variants of highly pathogenic avian influenza A(H5N8) virus were detected in dead poultry in Western Siberia, Russia, during August and September 2020. One variant was represented by viruses of clade 2.3.4.4b and the other by a novel reassortant between clade 2.3.4.4b and Eurasian low pathogenicity avian influenza viruses circulating in wild birds.

Exosomal miRNA profiling from H5N1 avian influenza virus-infected chickens.

Exosomes are membrane vesicles containing proteins, lipids, DNA, mRNA, and micro RNA (miRNA). Exosomal miRNA from donor cells can regulate the gene expression of recipient cells. Here, Ri chickens were divided into resistant (Mx/A; BF2/B21) and susceptible (Mx/G; BF2/B13) trait by genotyping of Mx and BF2 genes. Then, Ri chickens were infected with H5N1, a highly pathogenic avian influenza virus (HPAIV). Exosomes were purified from blood serum of resistant chickens for small RNA sequencing. Sequencing data were analysed using FastQCv0.11.7, Cutadapt 1.16, miRBase v21, non-coding RNA database, RNAcentral 10.0, and miRDeep2. Differentially expressed miRNAs were determined using statistical methods, including fold-change, exactTest using edgeR, and hierarchical clustering. Target genes were predicted using miRDB. Gene ontology analysis was performed using gProfiler. Twenty miRNAs showed significantly different expression patterns between resistant control and infected chickens. Nine miRNAs were up-regulated and 11 miRNAs were down-regulated in the infected chickens compared with that in the control chickens. In target gene analysis, various immune-related genes, such as cytokines, chemokines, and signalling molecules, were detected. In particular, mitogen-activated protein kinase (MAPK) pathway molecules were highly controlled by differentially expressed miRNAs. The result of qRT-PCR for miRNAs was identical with sequencing data and miRNA expression level was higher in resistant than susceptible chickens. This study will help to better understand the host immune response, particularly exosomal miRNA expression against HPAIV H5N1 and could help to determine biomarkers for disease resistance.

Genetic characterization of an H5N6 avian influenza virus with multiple origins from a chicken in southern China, October 2019.

BACKGROUND: Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype pose a great threat to the poultry industry and human health. In recent years, H5N6 subtype has rapidly replaced H5N1 as the most predominate HPAIV subtype circulating in domestic poultry in China. In this study, we describe the genetic and phylogenetic characteristics of a prevalent H5N6 strain in Guangdong, China. RESULTS: Nucleotide sequencing identified a H5N6 subtype HPAIV, designated as A/chicken/Dongguan/1101/2019 (DG/19), with a multibasic cleavage site in the hemagglutinin (HA). Phylogenetic analysis revealed DG/19 was a reassortant of H5N1, H5N2, H5N8, and H6N6 subtypes of avian influenza viruses. A number of mammalian adaptive markers such as D36N in the HA were identified. CONCLUSIONS: Our results showed that HPAIV H5N6 strains still emerge in well-managed groups of chicken farms. Considering the increasing prevalence of H5N6 HPAIV, and the fact that H5N6 HPAIVs are well adapted to migratory birds, an enhanced surveillance for the East Asian-Australasian flyway should be undertaken to prevent potential threats to the poultry industry and human health.

Isolation of clade 2.3.4.4b A(H5N8), a highly pathogenic avian influenza virus, from a worker during an outbreak on a poultry farm, Russia, December 2020.

This study presents the isolation of influenza A(H5N8) virus clade 2.3.4.4b from a poultry worker during an outbreak of highly pathogenic avian influenza A(H5N8) among chickens at a poultry farm in Astrakhan, Russia in December 2020. Nasopharyngeal swabs collected from seven poultry workers were positive for influenza A(H5N8), as confirmed by RT-PCR and sequencing. The influenza A(H5N8) virus was isolated from one of the human specimens and characterised. Sporadic human influenza A(H5)2.3.4.4. infections represent a possible concern for public health.

Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses.

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 104 egg infective dose 50 (EID50) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID50 105), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.

Pathogenicity of clade 2.3.2.1 H5N1 highly pathogenic avian influenza virus in American kestrel (Falco sparverius).

Birds of prey, including endangered species, have been infected with H5 highly pathogenic avian influenza viruses (HPAIVs) in several countries. In this present study, we assessed the pathogenicity of the clade 2.3.2.1 H5N1 HPAIV in American kestrels (Falco sparverius) with a view to preventing future outbreaks in raptors. The kestrels were intranasally inoculated with the virus or fed the meat of chicks that had died from viral infection. Kestrels in both groups initially had reduced food intake, showed clinical signs such as depression and neurologic manifestations, and succumbed to the infection within 6 days. The kestrels primarily shed the virus orally from 1 day post-inoculation until death, with an average titre of 104.5-5.7 EID50/ml, which is comparable to the inoculum titre. The viruses replicated in almost all tested tissues; notably, the feather calamuses also contained infectious virions and/or viral genes. Pancreatic lesions were present in several infected birds, as shown in previous cases of HPAIV infection in raptors. These results indicate that kestrels are highly susceptible to infection by clade 2.3.2.1 H5 HPAIVs, which readily occurs through the consumption of infected bird carcasses. Early detection and removal of HPAIV infected carcasses in the field is essential for preventing outbreaks in raptors. RESEARCH HIGHLIGHTS Clade 2.3.2.1 H5 HPAIV caused lethal infection in American kestrels. Kestrels with the HPAIV showed neurologic signs and eye disorders. The HPAIV replicated in systemic tissues of kestrels, and was orally shed. The HPAIV was recovered from feather calamus of kestrels.

Genetic Characterization of Avian Influenza A(H5N6) Virus Clade 2.3.4.4, Russia, 2018.

Timely identification of pandemic influenza threats depends on monitoring for highly pathogenic avian influenza viruses. We isolated highly pathogenic avian influenza A(H5N6) virus clade 2.3.4.4, genotype G1.1, in samples from a bird in southwest Russia. The virus has high homology to human H5N6 influenza strains isolated from southeast China.

A Dual Motif in the Hemagglutinin of H5N1 Goose/Guangdong-Like Highly Pathogenic Avian Influenza Virus Strains Is Conserved from Their Early Evolution and Increases both Membrane Fusion pH and Virulence.

Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. These strains emerge from low-pathogenic precursors by the acquisition of a polybasic hemagglutinin (HA) cleavage site, the prime virulence determinant. However, required coadaptations of the HA early in HPAIV evolution remained uncertain. To address this question, we generated several HA1/HA2 chimeras and point mutants of an H5N1 clade 2.2.2 HPAIV and an H5N1 low-pathogenic strain. Initial surveys of 3,385 HPAIV H5 HA sequences revealed frequencies of 0.5% for the single amino acids 123R and 124I but a frequency of 97.5% for the dual combination. This highly conserved dual motif is still retained in contemporary H5 HPAIV, including the novel H5NX reassortants carrying neuraminidases of different subtypes, like the H5N8 and the zoonotic H5N6 strains. Remarkably, the earliest Asian H5N1 HPAIV, the Goose/Guangdong strains from 1996/1997, carried 123R only, whereas 124I appeared later in 1997. Experimental reversion in the HPAIV HA to the two residues 123S and124T, characteristic of low-pathogenic strains, prevented virus rescue, while the single substitutions attenuated the virus in both chicken and mice considerably, accompanied by a decreased HA fusion pH. This increased pH sensitivity of H5 HPAIV enables HA-mediated membrane fusion at a higher endosomal pH. Therefore, this HA adaptation may permit infection of cells with less-acidic endosomes, e.g., within the respiratory tract, resulting in an extended organ tropism. Taken together, HA coadaptation to increased acid sensitivity promoted the early evolution of H5 Goose/Guangdong-like HPAIV strains and is still required for their zoonotic potential.IMPORTANCE Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. Their prime virulence determinant is the polybasic hemagglutinin (HA) cleavage site. However, required coadaptations in the HA (and other genes) remained uncertain. Here, we identified the dual motif 123R/124I in the HA head that increases the activation pH of HA-mediated membrane fusion, essential for virus genome release into the cytoplasm. This motif is extremely predominant in H5 HPAIV and emerged already in the earliest 1997 H5N1 HPAIV. Reversion to 123S or 124T, characteristic of low-pathogenic strains, attenuated the virus in chicken and mice, accompanied by a decreased HA activation pH. This increased pH sensitivity of H5 HPAIV extends the viral tropism to cells with less-acidic endosomes, e.g., within the respiratory tract. Therefore, early HA adaptation to increased acid sensitivity promoted the emergence of H5 Goose/Guangdong-like HPAIV strains and is required for their zoonotic potential.

New Threats from H7N9 Influenza Virus: Spread and Evolution of High- and Low-Pathogenicity Variants with High Genomic Diversity in Wave Five.

H7N9 virus has caused five infection waves since it emerged in 2013. The highest number of human cases was seen in wave 5; however, the underlying reasons have not been thoroughly elucidated. In this study, the geographical distribution, phylogeny, and genetic evolution of 240 H7N9 viruses in wave 5, including 35 new isolates from patients and poultry in nine provinces, were comprehensively analyzed together with strains from first four waves. Geographical distribution analysis indicated that the newly emerging highly pathogenic (HP) and low-pathogenicity (LP) H7N9 viruses were cocirculating, causing human and poultry infections across China. Genetic analysis indicated that dynamic reassortment of the internal genes among LP-H7N9/H9N2/H6Ny and HP-H7N9, as well as of the surface genes, between the Yangtze and Pearl River Delta lineages resulted in at least 36 genotypes, with three major genotypes (G1 [A/chicken/Jiangsu/SC537/2013-like], G3 [A/Chicken/Zhongshan/ZS/2017-like], and G11 [A/Anhui/40094/2015-like]). The HP-H7N9 genotype likely evolved from G1 LP-H7N9 by the insertion of a KRTA motif at the cleavage site (CS) and then evolved into 15 genotypes with four different CS motifs, including PKGKRTAR/G, PKGKRIAR/G, PKRKRAAR/G, and PKRKRTAR/G. Approximately 46% (28/61) of HP strains belonged to G3. Importantly, neuraminidase (NA) inhibitor (NAI) resistance (R292K in NA) and mammalian adaptation (e.g., E627K and A588V in PB2) mutations were found in a few non-human-derived HP-H7N9 strains. In summary, the enhanced prevalence and diverse genetic characteristics that occurred with mammalian-adapted and NAI-resistant mutations may have contributed to increased numbers of human infections in wave 5.IMPORTANCE The highest numbers of human H7N9 infections were observed during wave 5 from October 2016 to September 2017. Our results showed that HP-H7N9 and LP-H7N9 had spread virtually throughout China and underwent dynamic reassortment with different subtypes (H7N9/H9N2 and H6Ny) and lineages (Yangtze and Pearl River Delta lineages), resulting in totals of 36 and 3 major genotypes, respectively. Notably, the NAI drug-resistant (R292K in NA) and mammalian-adapted (e.g., E627K in PB2) mutations were found in HP-H7N9 not only from human isolates but also from poultry and environmental isolates, indicating increased risks for human infections. The broad dissemination of LP- and HP-H7N9 with high levels of genetic diversity and host adaptation and drug-resistant mutations likely accounted for the sharp increases in the number of human infections during wave 5. Therefore, more strategies are needed against the further spread and damage of H7N9 in the world.

Virulence of three European highly pathogenic H7N1 and H7N7 avian influenza viruses in Pekin and Muscovy ducks.

BACKGROUND: There is paucity of data on the virulence of highly pathogenic (HP) avian influenza viruses (AIV) H7 in ducks compared to HPAIV H5. Here, the virulence of HPAIV H7N1 (designated H7N1-FPV34 and H7N1-It99) and H7N7 (designated H7N7-FPV27) was assessed in Pekin and/or Muscovy ducklings after intrachoanal (IC) or intramuscular (IM) infection. RESULTS: The morbidity rate ranged from 60 to 100% and mortality rate from 20 to 80% depending on the duck species, virus strain and/or challenge route. All Muscovy ducklings inoculated IC with H7N7-FPV27 or H7N1-FPV34 exhibited mild to severe clinical signs resulting in the death of 2/10 and 8/10 ducklings, respectively. Also, 2/10 and 6/9 of inoculated Muscovy ducklings died after IC or IM infection with H7N1-It99, respectively. Moreover, 5/10 Pekin ducklings inoculated IC or IM with H7N1-It99 died. The level of virus detected in the oropharyngeal swabs was higher than in the cloacal swabs. CONCLUSION: Taken together, HPAIV H7 cause mortality and morbidity in Muscovy and Pekin ducklings. The severity of disease in Muscovy ducklings depended on the virus strain and/or route of infection. Preferential replication of the virus in the respiratory tract compared to the gut merits further investigation.

Host Cell Mimic Polymersomes for Rapid Detection of Highly Pathogenic Influenza Virus via a Viral Fusion and Cell Entry Mechanism.

Highly pathogenic avian influenza virus (HPAIV) infections have occurred continuously and crossed the species barrier to humans, leading to fatalities. A polymerase chain reaction based molecular test is currently the most sensitive diagnostic tool for HPAIV; however, the results must be analyzed in centralized diagnosis systems by a trained individual. This requirement leads to delays in quarantine and isolation. To control the spread of HPAIV, rapid and accurate diagnostics suitable for field testing are needed, and the tests must facilitate a differential diagnosis between HPAIV and low pathogenic avian influenza virus (LPAIV), which undergo cleavage specifically by trypsin- or furin-like proteases, respectively. In this study, a differential avian influenza virus rapid test kit is developed and evaluated in vitro and using clinical specimens from HPAIV H5N1-infected animals. It is demonstrated that this rapid test kit provides highly sensitive and specific detection of HPAIV and LPAIV and is thus a useful field diagnostic tool for H5N1 HPAIV outbreaks and for rapid quarantine control of the disease.

Outbreaks among Wild Birds and Domestic Poultry Caused by Reassorted Influenza A(H5N8) Clade 2.3.4.4 Viruses, Germany, 2016.

In November 2016, an influenza A(H5N8) outbreak caused deaths of wild birds and domestic poultry in Germany. Clade 2.3.4.4 virus was closely related to viruses detected at the Russia-Mongolia border in 2016 but had new polymerase acidic and nucleoprotein segments. These new strains may be more efficiently transmitted to and shed by birds.

Reassortant Clade 2.3.4.4 Avian Influenza A(H5N6) Virus in a Wild Mandarin Duck, South Korea, 2016.

A reassortant clade 2.3.4.4 avian influenza A(H5N6) virus was isolated from a fecal sample of a Mandarin duck (Aix galericulata) in South Korea during October 2016. This virus was genetically similar to H5N6 subtype virus isolates from China, Vietnam, Laos, and Hong Kong, including human isolates.

Multiple Reassorted Viruses as Cause of Highly Pathogenic Avian Influenza A(H5N8) Virus Epidemic, the Netherlands, 2016.

In 2016, an epidemic of highly pathogenic avian influenza A virus subtype H5N8 in the Netherlands caused mass deaths among wild birds, and several commercial poultry farms and captive bird holdings were affected. We performed complete genome sequencing to study the relationship between the wild bird and poultry viruses. Phylogenetic analysis showed that the viruses are related to H5 clade 2.3.4.4 viruses detected in Russia in May 2016 but contained novel polymerase basic 2 and nucleoprotein gene segments and 2 different variants of the polymerase acidic segment. Molecular dating suggests that the reassortment events most likely occurred in wild birds in Russia or Mongolia. Furthermore, 2 genetically distinct H5N5 reassortant viruses were detected in wild birds in the Netherlands. Our study provides evidence for fast and continuing reassortment of H5 clade 2.3.4.4 viruses, which might lead to rapid changes in virus characteristics, such as pathogenicity, infectivity, transmission, and zoonotic potential.