Strong and consistent effects of waterbird composition on HPAI H5 occurrences across Europe.

Since 2014, highly pathogenic avian influenza (HPAI) H5 viruses of clade 2.3.4.4 have been dominating the outbreaks across Europe, causing massive deaths among poultry and wild birds. However, the factors shaping these broad-scale outbreak patterns, especially those related to waterbird community composition, remain unclear. In particular, we do not know whether these risk factors differ from those of other H5 clades. Addressing this knowledge gap is important for predicting and preventing future HPAI outbreaks. Using extensive waterbird survey datasets from about 6883 sites, we here explored the effect of waterbird community composition on HPAI H5Nx (clade 2.3.4.4) spatial patterns in the 2016/2017 and 2020/2021 epidemics in Europe, and compared it with the 2005/2006 HPAI H5N1 (clade 2.2) epidemic. We showed that HPAI H5 occurrences in wild birds in the three epidemics were strongly associated with very similar waterbird community attributes, which suggested that, in nature, similar interspecific transmission processes operate between the HPAI H5 subtypes or clades. Importantly, community phylogenetic diversity consistently showed a negative association with H5 occurrence in all three epidemics, suggesting a dilution effect of phylogenetic diversity. In contrast, waterbird community variables showed much weaker associations with HPAI H5Nx occurrence in poultry. Our results demonstrate that models based on previous epidemics can predict future HPAI H5 patterns in wild birds, implying that it is important to include waterbird community factors in future HPAI studies to predict outbreaks and improve surveillance activities.

Vaccination and Antiviral Treatment against Avian Influenza H5Nx Viruses: A Harbinger of Virus Control or Evolution.

Despite the panzootic nature of emergent highly pathogenic avian influenza H5Nx viruses in wild migratory birds and domestic poultry, only a limited number of human infections with H5Nx viruses have been identified since its emergence in 1996. Few countries with endemic avian influenza viruses (AIVs) have implemented vaccination as a control strategy, while most of the countries have adopted a culling strategy for the infected flocks. To date, China and Egypt are the two major sites where vaccination has been adopted to control avian influenza H5Nx infections, especially with the widespread circulation of clade 2.3.4.4b H5N1 viruses. This virus is currently circulating among birds and poultry, with occasional spillovers to mammals, including humans. Herein, we will discuss the history of AIVs in Egypt as one of the hotspots for infections and the improper implementation of prophylactic and therapeutic control strategies, leading to continuous flock outbreaks with remarkable virus evolution scenarios. Along with current pre-pandemic preparedness efforts, comprehensive surveillance of H5Nx viruses in wild birds, domestic poultry, and mammals, including humans, in endemic areas is critical to explore the public health risk of the newly emerging immune-evasive or drug-resistant H5Nx variants.

Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines induced cross-reactive protection in mice and ferrets.

IMPORTANCE: Clade 2.3.4.4 H5Nx avian influenza viruses (AIVs) have circulated globally and caused substantial economic loss. Increasing numbers of humans have been infected with Clade 2.3.4.4 H5N6 AIVs in recent years. Only a few human influenza vaccines have been licensed to date. However, the licensed live attenuated influenza virus vaccine exhibited the potential of being recombinant with the wild-type influenza A virus (IAV). Therefore, we developed a chimeric cold-adapted attenuated influenza vaccine based on the Clade 2.3.4.4 H5 AIVs. These H5 vaccines demonstrate the advantage of being non-recombinant with circulated IAVs in the future influenza vaccine study. The findings of our current study reveal that these H5 vaccines can induce cross-reactive protective efficacy in mice and ferrets. Our H5 vaccines may provide a novel option for developing human-infected Clade 2.3.4.4 H5 AIV vaccines.

Functional traits explain waterbirds' host status, subtype richness, and community-level infection risk for avian influenza.

Species functional traits can influence pathogen transmission processes, and consequently affect species' host status, pathogen diversity, and community-level infection risk. We here investigated, for 143 European waterbird species, effects of functional traits on host status and pathogen diversity (subtype richness) for avian influenza virus at species level. We then explored the association between functional diversity and HPAI H5Nx occurrence at the community level for 2016/17 and 2021/22 epidemics in Europe. We found that both host status and subtype richness were shaped by several traits, such as diet guild and dispersal ability, and that the community-weighted means of these traits were also correlated with community-level risk of H5Nx occurrence. Moreover, functional divergence was negatively associated with H5Nx occurrence, indicating that functional diversity can reduce infection risk. Our findings highlight the value of integrating trait-based ecology into the framework of diversity-disease relationship, and provide new insights for HPAI prediction and prevention.

Genetic evolution of hemagglutinin and neuraminidase genes of H5N1 highly pathogenic avian influenza viruses in Thailand.

Background: Ongoing outbreaks of H5N1 highly pathogenic avian influenza (HPAI) viruses and the emergence of the genetic-related hemagglutinin (HA) gene of reassortant H5Nx viruses currently circulating in wild birds and poultries pose a great global public health concern. In this study, we comprehensively analyzed the genetic evolution of Thai H5N1 HA and neuraminidase (NA) genes between 2003 and 2010. The H5N1 Thailand virus clade 2.3.4 was also genetically compared to the currently circulating clade 2.3.4.4 of H5Nx viruses. Methods: Full-length nucleotide sequences of 178 HA and 143 NA genes of H5N1 viruses circulating between 2003 and 2010 were phylogenetically analyzed using maximum likelihood (ML) phylogenetic construction. Bayesian phylogenetic trees were reconstructed using BEAST analysis with a Bayesian Markov chain Monte Carlo (MCMC) approach. The maximum clade credibility (MCC) tree was determined, and the time of the most recent common ancestor (tMRCA) was estimated. The H5N1 HA nucleotide sequences of clade 2.3.4 Thailand viruses were phylogenetically analyzed using ML phylogenetic tree construction and analyzed for nucleotide similarities with various subtypes of reassortant H5Nx HA clade 2.3.4.4. Results: ML phylogenetic analysis revealed two distinct HA clades, clade 1 and clade 2.3.4, and two distinct NA groups within the corresponding H5 clade 1 viruses. Bayesian phylogenetic reconstruction for molecular clock suggested that the Thai H5N1 HA and NA emerged in 2001.87 (95% HPD: 2001.34-2002.49) and 2002.38 (95% HPD: 2001.99-2002.82), respectively, suggesting that the virus existed before it was first reported in 2004. The Thai H5N1 HA clade 2.3.4 was grouped into corresponding clades 2.3.4, 2.3.4.1, 2.3.4.2, and 2.3.4.3, and shared nucleotide similarities to reassortant H5Nx clade 2.3.4.4 ranged from 92.4-96.8%. Phylogenetic analysis revealed monophyletic H5Nx clade 2.3.4.4 evolved from H5N1 clade 2.3.4. Conclusion: H5N1 viruses existed, and were presumably introduced and circulated in avian species in Thailand, before they were officially reported in 2004. HA and NA genes continuously evolved during circulation between 2004 and 2010. This study provides a better understanding of genetic evolution with respect to molecular epidemiology. Monitoring and surveillance of emerging variants/reassortants should be continued.

Multiple HA substitutions in highly pathogenic avian influenza H5Nx viruses contributed to the change in the NA subtype preference.

Novel highly pathogenic avian influenza (HPAI) H5Nx viruses are predominantly circulating worldwide, with an increasing potential threat of an outbreak in humans. It remains largely unknown how the stably maintained HPAI H5N1 suddenly altered its neuraminidase (NA) to other NA subtypes, which resulted in the emergence and evolution of H5Nx viruses. Here, we found that a combination of four specific amino acid (AA) substitutions (S123P-T156A-D183N- S223 R) in the hemagglutinin (HA) protein consistently observed in the H5Nx markedly altered the NA preference of H5N1 viruses. These molecular changes in H5N1 impaired its fitness, particularly viral growth and the functional activities of the HA and NA proteins. Among the AA substitutions identified, the T156A substitution, which contributed to the NA shift, also dramatically altered the antigenicity of H5N1 viruses, suggesting an occurrence of antigenic drift triggered by selective pressure. Our study shows the importance of how HA and NA complement each other and that antigenic drift in HA can potentially cause a shift in the NA protein in influenza A virus evolution.

Development of a Rapid Fluorescent Diagnostic System for Early Detection of the Highly Pathogenic Avian Influenza H5 Clade 2.3.4.4 Viruses in Chicken Stool.

Rapid diagnosis is essential for the control and prevention of H5 highly pathogenic avian influenza viruses (HPAIVs). However, highly sensitive and rapid diagnostic systems have shown limited performance due to specific antibody scarcity. In this study, two novel specific monoclonal antibodies (mAbs) for clade 2.3.4.4 H5Nx viruses were developed by using an immunogen from a reversed genetic influenza virus (RGV). These mAbs were combined with fluorescence europium nanoparticles and an optimized lysis buffer, which were further used for developing a fluorescent immunochromatographic rapid strip test (FICT) for early detection of H5Nx influenza viruses on chicken stool samples. The result indicates that the limit of detection (LoD) of the developed FICT was 40 HAU/mL for detection of HPAIV H5 clade 2.3.4.4b in spiked chicken stool samples, which corresponded to 4.78 × 104 RNA copies as obtained from real-time polymerase chain reaction (RT-PCR). An experimental challenge of chicken with H5N6 HPAIV is lethal for chicken three days post-infection (DPI). Interestingly, our FICT could detect H5N6 in stool samples at 2 DPI earlier, with 100% relative sensitivity in comparison with RT-PCR, and it showed 50% higher sensitivity than the traditional colloidal gold-based rapid diagnostic test using the same mAbs pair. In conclusion, our rapid diagnostic method can be utilized for the early detection of H5Nx 2.3.4.4 HPAIVs in avian fecal samples from poultry farms or for influenza surveillance in wild migratory birds.

Highly Pathogenic Avian Influenza H5Nx in Poland in 2020/2021: a Descriptive Epidemiological Study of a Large-scale Epidemic.

Introduction: Highly pathogenic avian influenza (HPAI) outbreaks caused by the Gs/Gd lineage of H5Nx viruses occur in Poland with increased frequency. The article provides an update on the HPAI situation in the 2020/2021 season and studies the possible factors that caused the exceptionally fast spread of the virus. Material and Methods: Samples from poultry and wild birds delivered for HPAI diagnosis were tested by real-time RT-PCR and a representative number of detected viruses were submitted for partial or full-genome characterisation. Information yielded by veterinary inspection was used for descriptive analysis of the epidemiological situation. Results: The scale of the epidemic in the 2020/2021 season was unprecedented in terms of duration (November 2020-August 2021), number of outbreaks in poultry (n = 357), wild bird events (n = 92) and total number of affected domestic birds (approximately ~14 million). The major drivers of the virus spread were the harsh winter conditions in February 2020 followed by the introduction of the virus to high-density poultry areas in March 2021. All tested viruses belonged to H5 clade 2.3.4.4b with significant intra-clade diversity and in some cases clearly distinguished clusters. Conclusion: The HPAI epidemic in 2020/2021 in Poland struck with unprecedented force. The conventional control measures may have limited effectiveness to break the transmission chain in areas with high concentrations of poultry.

Protective immunity induced by oral vaccination with a recombinant Lactococcus lactis vaccine against H5Nx in chickens.

BACKGROUND: The development of an influenza vaccine for poultry that provides broadly protective immunity against influenza H5Nx viruses is a challenging goal. RESULTS: Lactococcus lactis (L. lactis)/pNZ8149-HA1-M2 expressing hemagglutinin-1 (HA1) of A/chicken/Vietnam/NCVD-15A59/2015 (H5N6) and the conserved M2 gene of A/Vietnam/1203/2004 (H5N1) was generated. L. lactis/pNZ8149-HA1-M2 could induce significant humoral, mucosal and cell-mediated immune responses, as well as neutralization antibodies. Importantly, L. lactis/pNZ8149-HA1-M2 could prevent disease symptoms without significant weight loss and confer protective immunity in a chicken model against lethal challenge with divergent influenza H5Nx viruses, including H5N6 and H5N1. CONCLUSIONS: L. lactis/pNZ8149-HA1-M2 can serve as a promising vaccine candidate in poultry industry for providing protection against H5Nx virus infection in the field application.

Next-Generation Computationally Designed Influenza Hemagglutinin Vaccines Protect against H5Nx Virus Infections.

H5N1 COBRA hemagglutinin (HA) sequences, termed human COBRA-2 HA, were constructed through layering of HA sequences from viruses isolated from humans collected between 2004-2007 using only clade 2 strains. These COBRA HA proteins, when expressed on the surface of virus-like particles (VLP), elicited protective immune responses in mice, ferrets, and non-human primates. However, these vaccines were not as effective at inducing neutralizing antibodies against newly circulating viruses. Therefore, COBRA HA-based vaccines were updated in order to elicit protective antibodies against the current circulating clades of H5Nx viruses. Next-generation COBRA HA vaccines were designed to encompass the newly emerging viruses circulating in wild avian populations. HA amino acid sequences from avian and human H5 influenza viruses isolated between 2011-2017 were downloaded from the GISAID (Global Initiative on Sharing All Influenza Data). Mice were vaccinated with H5 COBRA rHA that elicited antibodies with hemagglutinin inhibition (HAI) activity against H5Nx viruses from five clades. The H5 COBRA rHA vaccine, termed IAN8, elicited protective immune responses against mice challenged with A/Sichuan/26621/2014 and A/Vietnam/1203/2004. This vaccine elicited antibodies with HAI activity against viruses from clades 2.2, 2.3.2.1, 2.3.4.2, 2.2.1 and 2.2.2. Lungs from vaccinated mice had decreased viral titers and the levels of cellular infiltration in mice vaccinated with IAN-8 rHA were similar to mice vaccinated with wild-type HA comparator vaccines or mock vaccinated controls. Overall, these next-generation H5 COBRA HA vaccines elicited protective antibodies against both historical H5Nx influenza viruses, as well as currently circulating clades of H5N1, H5N6, and H5N8 influenza viruses.

Risk Assessment for Highly Pathogenic Avian Influenza A(H5N6/H5N8) Clade 2.3.4.4 Viruses.

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease.

Multiple Gene Segments Are Associated with Enhanced Virulence of Clade 2.3.4.4 H5N8 Highly Pathogenic Avian Influenza Virus in Mallards.

Highly pathogenic avian influenza (HPAI) viruses from the H5Nx Goose/Guangdong/96 lineage continue to cause outbreaks in domestic and wild bird populations. Two distinct genetic groups of H5N8 HPAI viruses, hemagglutinin (HA) clades 2.3.4.4A and 2.3.4.4B, caused intercontinental outbreaks in 2014 to 2015 and 2016 to 2017, respectively. Experimental infections using viruses from these outbreaks demonstrated a marked difference in virulence in mallards, with the H5N8 virus from 2014 causing mild clinical disease and the 2016 H5N8 virus causing high mortality. To assess which gene segments are associated with enhanced virulence of H5N8 HPAI viruses in mallards, we generated reassortant viruses with 2014 and 2016 viruses. For single-segment reassortants in the genetic backbone of the 2016 virus, pathogenesis experiments in mallards revealed that morbidity and mortality were reduced for all eight single-segment reassortants compared to the parental 2016 virus, with significant reductions in mortality observed with the polymerase basic protein 2 (PB2), nucleoprotein (NP), and matrix (M) reassortants. No differences in morbidity and mortality were observed with reassortants that either have the polymerase complex segments or the HA and neuraminidase (NA) segments of the 2016 virus in the genetic backbone of the 2014 virus. In vitro assays showed that the NP and polymerase acidic (PA) segments of the 2014 virus lowered polymerase activity when combined with the polymerase complex segments of the 2016 virus. Furthermore, the M segment of the 2016 H5N8 virus was linked to filamentous virion morphology. Phylogenetic analyses demonstrated that gene segments related to the more virulent 2016 H5N8 virus have persisted in the contemporary H5Nx HPAI gene pool until 2020. IMPORTANCE Outbreaks of H5Nx HPAI viruses from the goose/Guangdong/96 lineage continue to occur in many countries and have resulted in substantial impact on wild birds and poultry. Epidemiological evidence has shown that wild waterfowl play a major role in the spread of these viruses. While HPAI virus infection in gallinaceous species causes high mortality, a wide range of disease outcomes has been observed in waterfowl species. In this study, we examined which gene segments contribute to severe disease in mallards infected with H5N8 HPAI viruses. No virus gene was solely responsible for attenuating the high virulence of a 2016 H5N8 virus, but the PB2, NP, and M segments significantly reduced mortality. The findings herein advance our knowledge on the pathobiology of avian influenza viruses in waterfowl and have potential implications on the ecology and epidemiology of H5Nx HPAI in wild bird populations.

The pathogenesis of a North American H5N2 clade 2.3.4.4 group A highly pathogenic avian influenza virus in surf scoters (Melanitta perspicillata).

BACKGROUND: Aquatic waterfowl, particularly those in the order Anseriformes and Charadriiformes, are the ecological reservoir of avian influenza viruses (AIVs). Dabbling ducks play a recognized role in the maintenance and transmission of AIVs. Furthermore, the pathogenesis of highly pathogenic AIV (HPAIV) in dabbling ducks is well characterized. In contrast, the role of diving ducks in HPAIV maintenance and transmission remains unclear. In this study, the pathogenesis of a North American A/Goose/1/Guangdong/96-lineage clade 2.3.4.4 group A H5N2 HPAIV, A/Northern pintail/Washington/40964/2014, in diving sea ducks (surf scoters, Melanitta perspicillata) was characterized. RESULTS: Intrachoanal inoculation of surf scoters with A/Northern pintail/Washington/40964/2014 (H5N2) HPAIV induced mild transient clinical disease whilst concomitantly shedding high virus titers for up to 10 days post-inoculation (dpi), particularly from the oropharyngeal route. Virus shedding, albeit at low levels, continued to be detected up to 14 dpi. Two aged ducks that succumbed to HPAIV infection had pathological evidence for co-infection with duck enteritis virus, which was confirmed by molecular approaches. Abundant HPAIV antigen was observed in visceral and central nervous system organs and was associated with histopathological lesions. CONCLUSIONS: Collectively, surf scoters, are susceptible to HPAIV infection and excrete high titers of HPAIV from the respiratory and cloacal tracts whilst being asymptomatic. The susceptibility of diving sea ducks to H5 HPAIV highlights the need for additional research and surveillance to further understand the contribution of diving ducks to HPAIV ecology.

Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry.

Since the initial detection of H5N1, a highly pathogenic avian influenza (HPAI) virus, in 1996 in China, numerous HPAI H5 lineages have been classified, and they continue to pose a threat to animal and human health. In this study, we developed a novel primer/probe set that can be employed to simultaneously detect pan-H5 HPAI and two clades, 2.3.2.1 and 2.3.4.4, of H5Nx viruses using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The sensitivity and specificity of these primer sets and probes were confirmed with a number of different subtypes of influenza virus and the H5-HA gene plasmid DNA. In particular, the multiplex RT-qPCR assay was successfully applied to the simultaneous detection of H5 HPAI and different virus clades in clinical field samples from a poultry farm. Therefore, this multiplex assay and a novel detection primer set and probes will be useful for the laboratory diagnosis and epidemiological field studies of different circulating H5 HPAI virus clades in poultry and migratory wild birds.

The PB2 and M genes are critical for the superiority of genotype S H9N2 virus to genotype H in optimizing viral fitness of H5Nx and H7N9 avian influenza viruses in mice.

Genotype S H9N2 avian influenza virus, which has been predominant in China since 2010, contributed its entire internal gene cassette to the genesis of novel reassortant influenza viruses, including H5Nx, H7N9 and H10N8 viruses that pose great threat to poultry and humans. A key feature of the genotype S H9N2 virus is the substitution of G1-like M and PB2 genes for the earlier F/98-like M and PB2 of genotype H virus. However, how this gene substitution has influenced viral adaptability of emerging influenza viruses in mammals remains unclear. We report here that reassortant H5Nx and H7N9 viruses with the genotype S internal gene cassette displayed enhanced replication and virulence over those with genotype H internal gene cassette in cell cultures as well as in the mouse models. We showed that the G1-like PB2 gene was associated with increased polymerase activity and improved nuclear accumulation compared with the F/98-like counterpart, while the G1-like M gene facilitated effective translocation of RNP to cytoplasm. Our findings suggest that the genotype S H9N2 internal gene cassette, which possesses G1-like M and PB2 genes, is superior to that of genotype H, in optimizing viral fitness, and thus have implications for assessing the potential risk of these gene introductions to generate emerging influenza viruses.

Genetic Characterization and Zoonotic Potential of Highly Pathogenic Avian Influenza Virus A(H5N6/H5N5), Germany, 2017-2018.

We genetically characterized highly pathogenic avian influenza virus A(H5N6) clade 2.3.4.4b isolates found in Germany in 2017-2018 and assessed pathogenicity of representative H5N5 and H5N6 viruses in ferrets. These viruses had low pathogenicity; however, continued characterization of related isolates is warranted because of their high potential for reassortment.

The PB2 and M genes of genotype S H9N2 virus contribute to the enhanced fitness of H5Nx and H7N9 avian influenza viruses in chickens.

Genotype S H9N2 viruses frequently donate their internal genes to facilitate the generation of novel influenza viruses, e.g., H5N6, H7N9, and H10N8, which have caused human infection. Genotype S was originated from the replacement of F/98-like M and PB2 genes of the genotype H with those from G1-like lineage. However, whether this gene substitution will influence the viral fitness of emerging influenza viruses remains unclear. We found that H5Nx and H7N9 viruses with G1-like PB2 or M gene exhibited higher virulence and replication than those with F/98-like PB2 or M in chickens. We also determined the functional significance of G1-like PB2 in conferring increased polymerase activity and improved nucleus transportation efficiency, and facilitated RNP nuclear export by G1-like M. Our results suggest that G1-like PB2 and M genes optimize viral fitness, and thus play a crucial role in the genesis of emerging influenza viruses that cause rising prevalence in chickens.

Adaptive Evolution of Human-Isolated H5Nx Avian Influenza A Viruses.

Avian influenza A viruses (AIVs) H5N1, first identified in 1996, are highly pathogenic in domestic poultry and continue to occasionally infect humans. In this study, we sought to identify genetic changes that occurred during their multiple invasions to humans. We evaluated all available H5Nx AIV genomes. Significant signals of positive selection were detected in 29 host-shift branches. 126 parallel evolution sites were detected on these branches, including 17 well-known sites (such as T271A, A274T, T339M, Q591K, E627K, and D701N in PB2; A134V, D154N, S223N, and R497K in HA) that play roles in allowing AIVs to cross species barriers. Our study suggests that during human infections, H5Nx viruses have experienced adaptive evolution (positive selection and convergent evolution) that allowed them to adapt to their new host environments. Analyses of adaptive evolution should be useful in identifying candidate sites that play roles in human infections, which can be tested by functional experiments.

The Emergence and Decennary Distribution of Clade 2.3.4.4 HPAI H5Nx.

Reassortment events among influenza viruses occur naturally and may lead to the development of new and different subtypes which often ignite the possibility of an influenza outbreak. Between 2008 and 2010, highly pathogenic avian influenza (HPAI) H5 of the N1 subtype from the A/goose/Guangdong/1/96-like (Gs/GD) lineage generated novel reassortants by introducing other neuraminidase (NA) subtypes reported to cause most outbreaks in poultry. With the extensive divergence of the H5 hemagglutinin (HA) sequences of documented viruses, the WHO/FAO/OIE H5 Evolutionary Working Group clustered these viruses into a systematic and unified nomenclature of clade 2.3.4.4 currently known as "H5Nx" viruses. The rapid emergence and circulation of these viruses, namely, H5N2, H5N3, H5N5, H5N6, H5N8, and the regenerated H5N1, are of great concern based on their pandemic potential. Knowing the evolution and emergence of these novel reassortants helps to better understand their complex nature. The eruption of reports of each H5Nx reassortant through time demonstrates that it could persist beyond its usual seasonal activity, intensifying the possibility of these emerging viruses' pandemic potential. This review paper provides an overview of the emergence of each novel HPAI H5Nx virus as well as its current epidemiological distribution.

Comparative micro-epidemiology of pathogenic avian influenza virus outbreaks in a wild bird population.

Understanding the epidemiological dynamics of highly pathogenic avian influenza virus (HPAIV) in wild birds is crucial for guiding effective surveillance and control measures. The spread of H5 HPAIV has been well characterized over large geographical and temporal scales. However, information about the detailed dynamics and demographics of individual outbreaks in wild birds is rare and important epidemiological parameters remain unknown. We present data from a wild population of long-lived birds (mute swans; Cygnus olor) that has experienced three outbreaks of related H5 HPAIVs in the past decade, specifically, H5N1 (2007), H5N8 (2016) and H5N6 (2017). Detailed demographic data were available and intense sampling was conducted before and after the outbreaks; hence the population is unusually suitable for exploring the natural epidemiology, evolution and ecology of HPAIV in wild birds. We show that key epidemiological features remain remarkably consistent across multiple outbreaks, including the timing of virus incursion and outbreak duration, and the presence of a strong age-structure in morbidity that likely arises from an equivalent age-structure in immunological responses. The predictability of these features across a series of outbreaks in a complex natural population is striking and contributes to our understanding of HPAIV in wild birds. This article is part of the theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: approaches and important themes'. This issue is linked with the subsequent theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: epidemic forecasting and control'.