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.

Genetic Diversity of Avian Influenza Viruses Detected in Waterbirds in Northeast Italy Using Two Different Sampling Strategies.

Avian influenza viruses (AIVs), which circulate endemically in wild aquatic birds, pose a significant threat to poultry and raise concerns for their zoonotic potential. From August 2021 to April 2022, a multi-site cross-sectional study involving active AIV epidemiological monitoring was conducted in wetlands of the Emilia-Romagna region, northern Italy, adjacent to densely populated poultry areas. A total of 129 cloacal swab samples (CSs) and 407 avian faecal droppings samples (FDs) were collected, with 7 CSs (5.4%) and 4 FDs (1%) testing positive for the AIV matrix gene through rRT-PCR. A COI-barcoding protocol was applied to recognize the species of origin of AIV-positive FDs. Multiple low-pathogenic AIV subtypes were identified, and five of these were isolated, including an H5N3, an H1N1, and three H9N2 in wild ducks. Following whole-genome sequencing, phylogenetic analyses of the hereby obtained strains showed close genetic relationships with AIVs detected in countries along the Black Sea/Mediterranean migratory flyway. Notably, none of the analyzed gene segments were genetically related to HPAI H5N1 viruses of clade 2.3.4.4b isolated from Italian poultry during the concurrent 2021-2022 epidemic. Overall, the detected AIV genetic diversity emphasizes the necessity for ongoing monitoring in wild hosts using diverse sampling strategies and whole-genome sequencing.

Emergence and Persistent Circulation of Highly Pathogenic Avian Influenza Virus A (H5N8) in Kosovo, May 2021-May 2022.

In this study, we report the first outbreak of highly pathogenic avian influenza (HPAI) A H5N8, clade 2.3.4.4b in Kosovo on 19 May 2021. The outbreak consisted of three phases: May-June 2021, September-November 2021, and January-May 2022. In total, 32 backyards and 10 commercial holdings tested positive for the virus. Interestingly, the third and last phase of the outbreak coincided with the massive H5N1 clade 2.3.4.4b epidemic in Europe. Phylogenetic analyses of 28 viral strains from Kosovo revealed that they were closely related to the H5N8 clade 2.3.4.4.b viruses that had been circulating in Albania, Bulgaria, Croatia, Hungary, and Russia in early 2021. Whole genome sequencing of the 25 and partial sequencing of three H5N8 viruses from Kosovo showed high nucleotide identity, forming a distinctive cluster and suggesting a single introduction. The results of the network analysis were in accordance with the three epidemic waves and suggested that the viral diffusion could have been caused by secondary spreads among farms and/or different introductions of the same virus from wild birds. The persistent circulation of the same virus over a one-year period highlights the potential risk of the virus becoming endemic, especially in settings with non-adequate biosecurity.

Outbreak of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in cats, Poland, June to July 2023.

BackgroundOver a 3-week period in late June/early July 2023, Poland experienced an outbreak caused by highly pathogenic avian influenza (HPAI) A(H5N1) virus in cats.AimThis study aimed to characterise the identified virus and investigate possible sources of infection.MethodsWe performed next generation sequencing and phylogenetic analysis of detected viruses in cats.ResultsWe sampled 46 cats, and 25 tested positive for avian influenza virus. The identified viruses belong to clade 2.3.4.4b, genotype CH (H5N1 A/Eurasian wigeon/Netherlands/3/2022-like). In Poland, this genotype was responsible for several poultry outbreaks between December 2022 and January 2023 and has been identified only sporadically since February 2023. Viruses from cats were very similar to each other, indicating one common source of infection. In addition, the most closely related virus was detected in a dead white stork in early June. Influenza A(H5N1) viruses from cats possessed two amino acid substitutions in the PB2 protein (526R and 627K) which are two molecular markers of virus adaptation in mammals. The virus detected in the white stork presented one of those mutations (627K), which suggests that the virus that had spilled over to cats was already partially adapted to mammalian species.ConclusionThe scale of HPAI H5N1 virus infection in cats in Poland is worrying. One of the possible sources seems to be poultry meat, but to date no such meat has been identified with certainty. Surveillance should be stepped up on poultry, but also on certain species of farmed mammals kept close to infected poultry farms.

Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus.

Starting from October 2021, several outbreaks of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 were reported in wild and domestic birds in Italy. Following the detection of an HPAIV in a free-ranging poultry farm in Ostia, province of Rome, despite the lack of clinical signs, additional virological and serological analyses were conducted on samples collected from free-ranging pigs, reared in the same holding, due to their direct contact with the infected poultry. While the swine nasal swabs were all RT-PCR negative for the influenza type A matrix (M) gene, the majority (%) of the tested pigs resulted serologically positive for the hemagglutination inhibition test and microneutralization assay, using an H5N1 strain considered to be homologous to the virus detected in the farm. These results provide further evidence of the worrisome replicative fitness that HPAI H5Nx viruses of the 2.3.4.4b clade have in mammalian species. Moreover, our report calls for additional active surveillance, to promptly intercept occasional spillover transmissions to domestic mammals in close contact with HPAI affected birds. Strengthened biosecurity measures and efficient separation should be prioritized in mixed-species farms in areas at risk of HPAI introduction.

The Evolution of Highly Pathogenic Avian Influenza A (H5) in Poultry in Nigeria, 2021-2022.

In 2021, amidst the COVID-19 pandemic and global food insecurity, the Nigerian poultry sector was exposed to the highly pathogenic avian influenza (HPAI) virus and its economic challenges. Between 2021 and 2022, HPAI caused 467 outbreaks reported in 31 of the 37 administrative regions in Nigeria. In this study, we characterized the genomes of 97 influenza A viruses of the subtypes H5N1, H5N2, and H5N8, which were identified in different agro-ecological zones and farms during the 2021-2022 epidemic. The phylogenetic analysis of the HA genes showed a widespread distribution of the H5Nx clade 2.3.4.4b and similarity with the HPAI H5Nx viruses that have been detected in Europe since late 2020. The topology of the phylogenetic trees indicated the occurrence of several independent introductions of the virus into the country, followed by a regional evolution of the virus that was most probably linked to its persistent circulation in West African territories. Additional evidence of the evolutionary potential of the HPAI viruses circulating in this region is the identification in this study of a putative H5N1/H9N2 reassortant virus in a mixed-species commercial poultry farm. Our data confirm Nigeria as a crucial hotspot for HPAI virus introduction from the Eurasian territories and reveal a dynamic pattern of avian influenza virus evolution within the Nigerian poultry population.

Highly pathogenic avian influenza A (H5N1) virus infections in wild carnivores connected to mass mortalities of pheasants in Finland.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe during 2020-2022. Virus types H5N8 and H5N1 have dominated the epidemic. Isolated spill-over infections in mammals started to emerge as the epidemic continued. In autumn 2021, HPAI H5N1 caused a series of mass mortality events in farmed and released pheasants (Phasianus colchicus) in a restricted area in southern Finland. Later, in the same area, an otter (Lutra lutra), two red foxes (Vulpes vulpes) and a lynx (Lynx lynx) were found moribund or dead and infected with H5N1 HPAI virus. Phylogenetically, H5N1 strains from pheasants and mammals clustered together. Molecular analyses of the four mammalian virus strains revealed mutations in the PB2 gene segment (PB2-E627K and PB2-D701N) that are known to facilitate viral replication in mammals. This study revealed that avian influenza cases in mammals were spatially and temporally connected with avian mass mortalities suggesting increased infection pressure from birds to mammals.

Integration of Epidemiological and Genomic Data to Investigate H5N1 HPAI Outbreaks in Northern Italy in 2021-2022.

Between October 2021 and April 2022, 317 outbreaks caused by highly pathogenic avian influenza (HPAI) H5N1 viruses were notified in poultry farms in the northeastern Italian regions. The complete genomes of 214 strains were used to estimate the genetic network based on the similarity of the viruses. An exponential random graph model (ERGM) was used to assess the effect of 'at-risk contacts', 'same owners', 'in-bound/out-bound risk windows overlap', 'genetic differences', 'geographic distances', 'same species', and 'poultry company' on the probability of observing a link within the genetic network, which can be interpreted as the potential propagation of the epidemic via lateral spread or a common source of infection. The variables 'same poultry company' (Est. = 0.548, C.I. = [0.179; 0.918]) and 'risk windows overlap' (Est. = 0.339, C.I. = [0.309; 0.368]) were associated with a higher probability of link formation, while the 'genetic differences' (Est. = -0.563, C.I. = [-0.640; -0.486]) and 'geographic distances' (Est. = -0.058, C.I. = [-0.078; -0.038]) indicated a reduced probability. The integration of epidemiological data with genomic analyses allows us to monitor the epidemic evolution and helps to explain the dynamics of lateral spreads casting light on the potential diffusion routes. The 2021-2022 epidemic stresses the need to further strengthen the biosecurity measures, and to encourage the reorganization of the poultry production sector to minimize the impact of future epidemics.

Highly pathogenic avian influenza A(H5N1) virus infection in farmed minks, Spain, October 2022.

In October 2022, an outbreak in Europe of highly pathogenic avian influenza (HPAI) A(H5N1) in intensively farmed minks occurred in northwest Spain. A single mink farm hosting more than 50,000 minks was involved. The identified viruses belong to clade 2.3.4.4b, which is responsible of the ongoing epizootic in Europe. An uncommon mutation (T271A) in the PB2 gene with potential public health implications was found. Our investigations indicate onward mink transmission of the virus may have occurred in the affected farm.

Emergence of a Reassortant 2.3.4.4b Highly Pathogenic H5N1 Avian Influenza Virus Containing H9N2 PA Gene in Burkina Faso, West Africa, in 2021.

Since 2006, the poultry population in Burkina Faso has been seriously hit by different waves of Highly Pathogenic Avian Influenza (HPAI) H5N1 epizootics. In December 2021, three distinct regions of Burkina Faso, namely, Gomboussougou, Bonyollo, and Koubri, detected HPAI H5N1 viruses in poultry. Whole genome characterization and statistical phylogenetic approaches were applied to shed light on the potential origin of these viruses and estimate the time of virus emergence. Our results revealed that the HPAI H5N1 viruses reported in the three affected regions of Burkina Faso cluster together within clade 2.3.4.4b, and are closely related to HPAI H5N1 viruses identified in Nigeria and Niger in the period 2021-2022, except for the PA gene, which clusters with H9N2 viruses of the zoonotic G1 lineage collected in West Africa between 2017 and 2020. These reassortant viruses possess several mutations that may be associated with an increased zoonotic potential. Although it is difficult to ascertain where and when the reassortment event occurred, the emergence of a H5N1/H9N2 reassortant virus in a vulnerable region, such as West Africa, raises concerns about its possible impact on animal and human health. These findings also highlight the risk that West Africa may become a new hotspot for the emergence of new genotypes of HPAI viruses.

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.

Spatiotemporal Dynamics, Evolutionary History and Zoonotic Potential of Moroccan H9N2 Avian Influenza Viruses from 2016 to 2021.

The H9N2 virus continues to spread in wild birds and poultry worldwide. At the beginning of 2016, the H9N2 Avian influenza virus (AIV) was detected in Morocco for the first time; despite the implementation of vaccination strategies to control the disease, the virus has become endemic in poultry in the country. The present study was carried out to investigate the origins, zoonotic potential, as well as the impact of vaccination on the molecular evolution of Moroccan H9N2 viruses. Twenty-eight (28) H9N2 viruses collected from 2016 to 2021 in Moroccan poultry flocks were isolated and their whole genomes sequenced. Phylogenetic and evolutionary analyses showed that Moroccan H9N2 viruses belong to the G1-like lineage and are closely related to viruses isolated in Africa and the Middle East. A high similarity among all the 2016-2017 hemagglutinin sequences was observed, while the viruses identified in 2018-2019 and 2020-2021 were separated from their 2016-2017 ancestors by long branches. Mutations in the HA protein associated with antigenic drift and increased zoonotic potential were also found. The Bayesian phylogeographic analyses revealed the Middle East as being the region where the Moroccan H9N2 virus may have originated, before spreading to the other African countries. Our study is the first comprehensive analysis of the evolutionary history of the H9N2 viruses in the country, highlighting their zoonotic potential and pointing out the importance of implementing effective monitoring systems.

Genetic characterization of highly pathogenic avian Influenza H5Nx clade 2.3.4.4b reveals independent introductions in nigeria.

Among recurrent sanitary emergencies able to spread rapidly worldwide, avian influenza is one of the main constraints for animal health and food security. In West Africa, Nigeria has been experiencing repeated outbreaks of different strains of avian influenza virus (AIV) since 2006 and is also recognized as a hot spot in the region for the introduction of emerging strains by migratory wild birds. Here, we generated complete genomes of 20 highly pathogenic avian influenza (HPAI) H5N8 viruses collected during active surveillance in Nigerian live bird markets (LBM) and from outbreaks reported in the country between 2016 and 2019. Phylogenetic analysis reveals that the Nigerian viruses cluster into four separate genetic groups within HPAI H5 clade 2.3.4.4b. The first group includes 2016-2017 Nigerian viruses with high genetic similarity to H5N8 viruses detected in Central African countries, while the second includes Nigerian viruses collected both in LBM and poultry farms (2018-2019), as well as in Cameroon, Egypt and Siberia. A natural reassortant strain identified in 2019 represents the third group: H5N8 viruses with the same gene constellation were identified in 2018 in South Africa. Finally, the fourth introduction represents the first detection in the African continent of the H5N6 subtype, which is related to European viruses. Bayesian phylogeographic analyses confirmed that the four introductions originated from different sources and provide evidence of the virus spread within Nigeria, as well as diffusion beyond its borders. The multiple epidemiological links between Nigeria, Central and Southern African countries highlight the need for harmonized and coordinated surveillance system to control AIV impact. Improved surveillance at the Wetlands, LBMs and early warning of outbreaks are crucial for prevention and control of AIV, which can be potentially zoonotic and be a threat to human health.

Intercontinental Spread of Eurasian Highly Pathogenic Avian Influenza A(H5N1) to Senegal.

In January 2021, Senegal reported the emergence of highly pathogenic avian influenza virus A(H5N1), which was detected on a poultry farm in Thies, Senegal, and in great white pelicans in the Djoudj National Bird Sanctuary. We report evidence of new transcontinental spread of H5N1 from Europe toward Africa.

Active Surveillance for Highly Pathogenic Avian Influenza Viruses in Wintering Waterbirds in Northeast Italy, 2020-2021.

The increasing involvement of wild waterfowl in H5 Highly Pathogenic Avian Influenza Virus (HPAIV) circulation continues to pose a threat to animal and public health worldwide. In winter 2020-2021, two field surveillance activities were carried out on a weekly basis, through virological and serological analyses, in 823 hunted and 521 trapped migratory aquatic birds in northeast Italy. Sixty Eurasian teals were recaptured several times, which allowed us to follow the progression of the HPAI H5 infection in naturally infected wild waterfowl. Oropharyngeal, cloacal, and feather swabs (OS, CS and FS) were collected from each duck and tested by real time rRT-PCR Type A influenza. The identified viruses were characterized and pathotyped by sequencing. Several viruses belonging to three different HPAI H5 subtypes were detected: H5N8, H5N5, and H5N1. High prevalence of infection with HPAI H5 clade 2.3.4.4b during November-December 2020 (up to 27.1%) was observed in captured Eurasian teals, while infection rates in hunted dabbling ducks, mainly Eurasian wigeons, showed the highest prevalence of infection in November 2020 (8.9%) and January 2021 (10.2%). All HPAI positive birds were also clinically healthy when recaptured weeks apart. The OS and FS showed the highest detection efficiency of HPAIV. Our results highlight that HPAI passive surveillance should be complemented by a targeted active surveillance to more efficiently detect novel HPAI viruses.

Evolutionary Dynamics of H5 Highly Pathogenic Avian Influenza Viruses (Clade 2.3.4.4B) Circulating in Bulgaria in 2019-2021.

The first detection of a Highly Pathogenic Avian Influenza (HPAI) H5N8 virus in Bulgaria dates back to December 2016. Since then, many outbreaks caused by HPAI H5 viruses from clade 2.3.4.4B have been reported in both domestic and wild birds in different regions of the country. In this study, we characterized the complete genome of sixteen H5 viruses collected in Bulgaria between 2019 and 2021. Phylogenetic analyses revealed a persistent circulation of the H5N8 strain for four consecutive years (December 2016-June 2020) and the emergence in 2020 of a novel reassortant H5N2 subtype, likely in a duck farm. Estimation of the time to the most recent common ancestor indicates that this reassortment event may have occurred between May 2019 and January 2020. At the beginning of 2021, Bulgaria experienced a new virus introduction in the poultry sector, namely a HPAI H5N8 that had been circulating in Europe since October 2020. The periodical identification in domestic birds of H5 viruses related to the 2016 epidemic as well as a reassortant strain might indicate undetected circulation of the virus in resident wild birds or in the poultry sector. To avoid the concealed circulation and evolution of viruses, and the risk of emergence of strains with pandemic potential, the implementation of control measures is of utmost importance, particularly in duck farms where birds display no clinical signs.

Live Bird Markets in Nigeria: A Potential Reservoir for H9N2 Avian Influenza Viruses.

Since 2006, multiple outbreaks of avian influenza (AI) have been reported in Nigeria involving different subtypes. Surveillance and molecular epidemiology have revealed the vital role of live bird markets (LBMs) in the dissemination of AI virus to commercial poultry farms. To better understand the ecology and epidemiology of AI in Nigeria, we performed whole-genome sequencing of nineteen H9N2 viruses recovered, from apparently healthy poultry species, during active surveillance conducted in nine LBMs across Nigeria in 2019. Analyses of the HA gene segment of these viruses showed that the H9N2 strains belong to the G1 lineage, which has zoonotic potential, and are clustered with contemporary H9N2 identified in Africa between 2016 and 2020. We observed two distinct clusters of H9N2 viruses in Nigeria, suggesting different introductions into the country. In view of the zoonotic potential of H9N2 and the co-circulation of multiple subtypes of AI virus in Nigeria, continuous monitoring of the LBMs across the country and molecular characterization of AIVs identified is advocated to mitigate economic losses and public health threats.

A New Variant Among Newcastle Disease Viruses Isolated in the Democratic Republic of the Congo in 2018 and 2019.

Newcastle disease (ND) is a highly transmissible and devastating disease that affects poultry and wild birds worldwide. Comprehensive knowledge regarding the characteristics and epidemiological factors of the ND virus (NDV) is critical for the control and prevention of ND. Effective vaccinations can prevent and control the spread of the NDV in poultry populations. For decades, the Democratic Republic of the Congo (DRC) has reported the impacts of ND on commercial and traditional poultry farming systems. The reports were preliminary clinical observations, and few cases were confirmed in the laboratory. However, data on the phylogenetic, genetic, and virological characteristics of NDVs circulating in the DRC are not available. In this study, the whole-genome sequences of three NDV isolates obtained using the next-generation sequencing method revealed two isolates that were a new variant of NDV, and one isolate that was clustered in the subgenotype VII.2. All DRC isolates were velogenic and were antigenically closely related to the vaccine strains. Our findings reveal that despite the circulation of the new variant, ND can be controlled in the DRC using the current vaccine. However, epidemiological studies should be conducted to elucidate the endemicity of the disease so that better control strategies can be implemented.

Spatiotemporal reconstruction and transmission dynamics during the 2016-17 H5N8 highly pathogenic avian influenza epidemic in Italy.

Effective control of avian diseases in domestic populations requires understanding of the transmission dynamics facilitating viral emergence and spread. In 2016-17, Italy experienced a significant avian influenza epidemic caused by a highly pathogenic A(H5N8) virus, which affected domestic premises housing around 2.7 million birds, primarily in the north-eastern regions with the highest density of poultry farms (Lombardy, Emilia-Romagna and Veneto). We perform integrated analyses of genetic, spatiotemporal and host data within a Bayesian phylogenetic framework. Using continuous and discrete phylogeography, we estimate the locations of movements responsible for the spread and persistence of the epidemic. The information derived from these analyses on rates of transmission between regions through time can be used to assess the success of control measures. Using an approach based on phylogenetic-temporal distances between domestic cases, we infer the presence of cryptic wild bird-mediated transmission, information that can be used to complement existing epidemiological methods for distinguishing transmission within the domestic population from incursions across the wildlife-domestic interface, a common challenge in veterinary epidemiology. Spatiotemporal reconstruction of the epidemic reveals a highly skewed distribution of virus movements with a high proportion of shorter distance local movements interspersed with occasional long-distance dispersal events associated with wild birds. We also show how such inference be used to identify possible instances of human-mediated movements where distances between phylogenetically linked domestic cases are unusually high.