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.

Avian influenza overview December 2023-March 2024.

Between 2 December 2023 and 15 March 2024, highly pathogenic avian influenza (HPAI) A(H5) outbreaks were reported in domestic (227) and wild (414) birds across 26 countries in Europe. Compared to previous years, although still widespread, the overall number of HPAI virus detections in birds was significantly lower, among other reasons, possibly due to some level of flock immunity in previously affected wild bird species, resulting in reduced contamination of the environment, and a different composition of circulating A(H5N1) genotypes. Most HPAI outbreaks reported in poultry were primary outbreaks following the introduction of the virus by wild birds. Outside Europe, the majority of outbreaks in poultry were still clustered in North America, while the spread of A(H5) to more naïve wild bird populations on mainland Antarctica is of particular concern. For mammals, A(H5N5) was reported for the first time in Europe, while goat kids in the United States of America represented the first natural A(H5N1) infection in ruminants. Since the last report and as of 12 March 2024, five human avian influenza A(H5N1) infections, including one death, three of which were clade 2.3.2.1c viruses, have been reported by Cambodia. China has reported two human infections, including one fatal case, with avian influenza A(H5N6), four human infections with avian influenza A(H9N2) and one fatal case with co-infection of seasonal influenza A(H3N2) and avian influenza A(H10N5). The latter case was the first documented human infection with avian influenza A(H10N5). Human infections with avian influenza remain rare and no sustained human-to-human infection has been observed. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA. The risk of infection remains low to moderate for those occupationally or otherwise exposed to infected animals.

Detection of clade 2.3.4.4 highly pathogenic avian influenza H5 viruses in healthy wild birds in the Hadeji-Nguru wetland, Nigeria 2022.

Background: The introduction of multiple avian influenza virus (AIV) subtypes into Nigeria has resulted in several poultry outbreaks purportedly linked to trade and wild birds. The role of wild birds in perpetuating AIV in Nigeria was, therefore, elucidated. Methods: A cross-sectional study was conducted among wild aquatic bird species at the Hadejia-Nguru wetlands in Northeastern Nigeria between March and April 2022. A total of 452 swabs (226 cloacae and 226 oropharyngeal) were collected using a mist net to capture the birds. These samples were tested by RT-qPCR, followed by sequencing. Results: Highly pathogenic AIV of the H5N1 subtype was identified in clinically healthy wild bird species, namely, African jacana, ruff, spur-winged goose, squared-tailed nightjar, white-faced whistling ducks, and white stork. A prevalence of 11.1% (25/226) was recorded. Phylogenetic analysis of the complete HA gene segment indicated the presence of clade 2.3.4.4b. However, these H5N1 viruses characterized from these wild birds cluster separately from the H5N1 viruses characterized in Nigerian poultry since early 2021. Specifically, the viruses form two distinct genetic groups both linked with the Eurasian H5N1 gene pool but likely resulting from two distinct introductions of the virus in the region. Whole-genome characterization of the viruses reveals the presence of mammalian adaptive marker E627K in two Afro-tropical resident aquatic ducks. This has zoonotic potential. Conclusion: Our findings highlight the key role of surveillance in wild birds to monitor the diversity of viruses in this area, provide the foundations of epidemiological understanding, and facilitate risk assessment.

Avian influenza overview September-December 2023.

Between 2 September and 1 December 2023, highly pathogenic avian influenza (HPAI) A(H5) outbreaks were reported in domestic (88) and wild (175) birds across 23 countries in Europe. Compared to previous years, the increase in the number of HPAI virus detections in waterfowl has been delayed, possibly due to a later start of the autumn migration of several wild bird species. Common cranes were the most frequently affected species during this reporting period with mortality events being described in several European countries. Most HPAI outbreaks reported in poultry were primary outbreaks following the introduction of the virus by wild birds, with the exception of Hungary, where two clusters involving secondary spread occurred. HPAI viruses identified in Europe belonged to eleven different genotypes, seven of which were new. With regard to mammals, the serological survey conducted in all fur farms in Finland revealed 29 additional serologically positive farms during this reporting period. Wild mammals continued to be affected mostly in the Americas, from where further spread into wild birds and mammals in the Antarctic region was described for the first time. Since the last report and as of 1 December 2023, three fatal and one severe human A(H5N1) infection with clade 2.3.2.1c viruses have been reported by Cambodia, and one A(H9N2) infection was reported from China. No human infections related to the avian influenza detections in animals in fur farms in Finland have been reported, and human infections with avian influenza remain a rare event. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA. The risk of infection remains low to moderate for occupationally or otherwise exposed people to infected birds or mammals (wild or domesticated); this assessment covers different situations that depend on the level of exposure.

Tracking the Selective Pressure Profile and Gene Flow of SARS-CoV-2 Delta Variant in Italy from April to October 2021 and Frequencies of Key Mutations from Three Representative Italian Regions.

The SARS-CoV-2 Delta variant of concern (VOC) was often associated with serious clinical course of the COVID-19 disease. Herein, we investigated the selective pressure, gene flow and evaluation on the frequencies of mutations causing amino acid substitutions in the Delta variant in three Italian regions. A total of 1500 SARS-CoV-2 Delta genomes, collected in Italy from April to October 2021 were investigated, including a subset of 596 from three Italian regions. The selective pressure and the frequency of amino acid substitutions and the prediction of their possible impact on the stability of the proteins were investigated. Delta variant dataset, in this study, identified 68 sites under positive selection: 16 in the spike (23.5%), 11 in nsp2 (16.2%) and 10 in nsp12 (14.7%) genes. Three of the positive sites in the spike were located in the receptor-binding domain (RBD). In Delta genomes from the three regions, 6 changes were identified as very common (>83.7%), 4 as common (>64.0%), 21 at low frequency (2.1%-25.0%) and 29 rare (≤2.0%). The detection of positive selection on key mutations may represent a model to identify recurrent signature mutations of the virus.

Avian influenza overview June-September 2023.

Between 24 June and 1 September 2023, highly pathogenic avian influenza (HPAI) A(H5) outbreaks were reported in domestic (25) and wild (482) birds across 21 countries in Europe. Most of these outbreaks appeared to be clustered along coastlines with only few HPAI virus detections inland. In poultry, all HPAI outbreaks were primary and sporadic with most of them occurring in the United Kingdom. In wild birds, colony-breeding seabirds continued to be most heavily affected, but an increasing number of HPAI virus detections in waterfowl is expected in the coming weeks. The current epidemic in wild birds has already surpassed the one of the previous epidemiological year in terms of total number of HPAI virus detections. As regards mammals, A(H5N1) virus was identified in 26 fur animal farms in Finland. Affected species included American mink, red and Arctic fox, and common raccoon dog. The most likely source of introduction was contact with gulls. Wild mammals continued to be affected worldwide, mostly red foxes and different seal species. Since the last report and as of 28 September 2023, two A(H5N1) clade 2.3.4.4b virus detections in humans have been reported by the United Kingdom, and three human infections with A(H5N6) and two with A(H9N2) were reported from China, respectively. No human infection related to the avian influenza detections in animals on fur farms in Finland or in cats in Poland have been reported, and human infections with avian influenza remain a rare event. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA. The risk of infection remains low to moderate for occupationally or otherwise exposed people to infected birds or mammals (wild or domesticated); this assessment covers different situations that depend on the level of exposure.

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.

Asymptomatic infection with clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) in carnivore pets, Italy, April 2023.

In April 2023, an outbreak of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses carrying the T271A mammalian adaptive mutation in the PB2 protein was detected in a backyard poultry farm in Italy. Five domestic dogs and one cat living on the premises had seroconverted in the absence of clinical signs. Virological and serological monitoring of individuals exposed to the virus proved the absence of human transmission, however, asymptomatic influenza A(H5N1) infections in mammalian pets may have important public health implications.

Avian influenza overview April - June 2023.

Between 29 April and 23 June 2023, highly pathogenic avian influenza (HPAI) A(H5N1) virus (clade 2.3.4.4b) outbreaks were reported in domestic (98) and wild (634) birds across 25 countries in Europe. A cluster of outbreaks in mulard ducks for foie gras production was concentrated in Southwest France, whereas the overall A(H5N1) situation in poultry in Europe and worldwide has eased. In wild birds, black-headed gulls and several new seabird species, mostly gulls and terns (e.g. sandwich terns), were heavily affected, with increased mortality being observed in both adults and juveniles after hatching. Compared to the same period last year, dead seabirds have been increasingly found inland and not only along European coastlines. As regards mammals, A(H5N1) virus was identified in 24 domestic cats and one caracal in Poland between 10 and 30 June 2023. Affected animals showed neurological and respiratory signs, sometimes mortality, and were widely scattered across nine voivodeships in the country. All cases are genetically closely related and identified viruses cluster with viruses detected in poultry (since October 2022, but now only sporadic) and wild birds (December 2022-January 2023) in the past. Uncertainties still exist around their possible source of infection, with no feline-to-feline or feline-to-human transmission reported so far. Since 10 May 2023 and as of 4 July 2023, two A(H5N1) clade 2.3.4.4b virus detections in humans were reported from the United Kingdom, and two A(H9N2) and one A(H5N6) human infections in China. In addition, one person infected with A(H3N8) in China has died. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA, low to moderate for occupationally or otherwise exposed people to infected birds or mammals (wild or domesticated).

Avian influenza overview March - April 2023.

Between 2 March and 28 April 2023, highly pathogenic avian influenza (HPAI) A(H5Nx) virus, clade 2.3.4.4b, outbreaks were reported in domestic (106) and wild (610) birds across 24 countries in Europe. Poultry outbreaks occurred less frequently compared to the previous reporting period and compared to spring 2022. Most of these outbreaks were classified as primary outbreaks without secondary spread and some of them associated with atypical disease presentation, in particular low mortality. In wild birds, black-headed gulls continued to be heavily affected, while also other threatened wild bird species, such as the peregrine falcon, showed increased mortality. The ongoing epidemic in black-headed gulls, many of which breed inland, may increase the risk for poultry, especially in July-August, when first-year birds disperse from the breeding colonies. HPAI A(H5N1) virus also continued to expand in the Americas, including in mammalian species, and is expected to reach the Antarctic in the near future. HPAI virus infections were detected in six mammal species, particularly in marine mammals and mustelids, for the first time, while the viruses currently circulating in Europe retain a preferential binding for avian-like receptors. Since 13 March 2022 and as of 10 May 2023, two A(H5N1) clade 2.3.4.4b virus detections in humans were reported from China (1), and Chile (1), as well as three A(H9N2) and one A(H3N8) human infections in China. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe remains low for the general population in the EU/EEA, and low to moderate for occupationally or otherwise exposed people.

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.

Genomic Analysis of Infectious Bursal Disease Virus in Nigeria: Identification of Unique Mutations of Yet Unknown Biological Functions in Both Segments A and B.

Infectious bursal disease (IBD) is a viral poultry disease known worldwide for impacting the economy and food security. The disease is endemic in Nigeria, with reported outbreaks in vaccinated poultry flocks. To gain insight into the dynamics of infectious bursal disease virus (IBDV) evolution in Nigeria, near-complete genomes of four IBDVs were evaluated. Amino acid sequences in the hypervariable region of the VP2 revealed conserved markers (222A, 242I, 256I, 294I and 299S) associated with very virulent (vv) IBDV, including the serine-rich heptapeptide motif (SWSASGS). Based on the newly proposed classification for segments A and B, the IBDVs clustered in the A3B5 group (where A3 are IBDVs with vvIBDV-like segment A, and where B5 are from non-vvIBDV-like segment B) form a monophyletic subcluster. Unique amino acid mutations with yet-to-be-determined biological functions have been observed in both segments. Amino acid sequences of the Nigerian IBDVs showed that they are reassortant viruses. Circulation of reassortant IBDVs may be responsible for the vaccination failures observed in the Nigerian poultry population. Close monitoring of changes in the IBDV genome is recommended to nip deleterious changes in the bud through the identification and introduction of the most appropriate vaccine candidates and advocacy/extension programs for properly implementing disease control.

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.

Avian influenza overview December 2022 - March 2023.

Between 3 December 2022 and 1 March 2023 highly pathogenic avian influenza (HPAI) A(H5N1) virus, clade 2.3.4.4b, was reported in Europe in domestic (522) and wild (1,138) birds over 24 countries. An unexpected number of HPAI virus detections in sea birds were observed, mainly in gull species and particularly in black-headed gulls (large mortality events were observed in France, Belgium, the Netherlands, and Italy). The close genetic relationship among viruses collected from black-headed gulls suggests a southward spread of the virus. Moreover, the genetic analyses indicate that the virus persisted in Europe in residential wild birds during and after the summer months. Although the virus retained a preferential binding for avian-like receptors, several mutations associated to increased zoonotic potential were detected. The risk of HPAI virus infection for poultry due to the virus circulating in black-headed gulls and other gull species might increase during the coming months, as breeding bird colonies move inland with possible overlap with poultry production areas. Worldwide, HPAI A(H5N1) virus continued to spread southward in the Americas, from Mexico to southern Chile. The Peruvian pelican was the most frequently reported infected species with thousands of deaths being reported. The reporting of HPAI A(H5N1) in mammals also continued probably linked to feeding on infected wild birds. In Peru, a mass mortality event of sea lions was observed in January and February 2023. Since October 2022, six A(H5N1) detections in humans were reported from Cambodia (a family cluster with 2 people, clade 2.3.2.1c), China (2, clade 2.3.4.4b), Ecuador (1, clade 2.3.4.4b), and Vietnam (1, unspecified clade), as well as two A(H5N6) human infections from China. The risk of infection with currently circulating avian H5 influenza viruses of clade 2.3.4.4b in Europe is assessed as low for the general population in the EU/EEA, and low to moderate for occupationally or otherwise exposed people.

Avian influenza overview September - December 2022.

Between October 2021 and September 2022 Europe has suffered the most devastating highly pathogenic avian influenza (HPAI) epidemic with a total of 2,520 outbreaks in poultry, 227 outbreaks in captive birds, and 3,867 HPAI virus detections in wild birds. The unprecedent geographical extent (37 European countries affected) resulted in 50 million birds culled in affected establishments. In the current reporting period, between 10 September and 2 December 2022, 1,163 HPAI virus detections were reported in 27 European countries in poultry (398), captive (151) and wild birds (613). A decrease in HPAI virus detections in colony-breeding seabirds species and an increase in the number of detections in waterfowl has been observed. The continuous circulation of the virus in the wild reservoir has led to the frequent introduction of the virus into poultry populations. It is suspected that waterfowl might be more involved than seabirds in the incursion of HPAI virus into poultry establishments. In the coming months, the increasing infection pressure on poultry establishments might increase the risk of incursions in poultry, with potential further spread, primarily in areas with high poultry densities. The viruses detected since September 2022 (clade 2.3.4.4b) belong to eleven genotypes, three of which have circulated in Europe during the summer months, while eight represent new genotypes. HPAI viruses were also detected in wild and farmed mammal species in Europe and North America, showing genetic markers of adaptation to replication in mammals. Since the last report, two A(H5N1) detections in humans in Spain, one A(H5N1), one A(H5N6) and one A(H9N2) human infection in China as well as one A(H5) infection without NA-type result in Vietnam were reported, respectively. The risk of infection is assessed as low for the general population in the EU/EEA, and low to medium for occupationally exposed people.

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.

Rapid spread of a new West Nile virus lineage 1 associated with increased risk of neuroinvasive disease during a large outbreak in northern Italy, 2022: One Health analysis.

BACKGROUND: A new strain of WNV lineage 1 (WNV - 1) emerged in the Veneto Region, northern Italy, in 2021, eight years after the last outbreak of WNV - 1 in Italy. The virus, which co-circulates with WNV-2, has become endemic in the Region, where, in 2022, most human cases of neuroinvasive disease (WNND) reported in Europe have occurred. METHODS: Comparative analysis of the epidemiology and clinical presentation of WNV-1 and WNV-2 infection in humans, as well as the temporal and geographic distribution of WNV-1 and WNV-2 among wild birds and Culex pipiens mosquitoes in Veneto, from May 16th to August 21st, 2022, to determine if the high number of WNND cases was associated with WNV-1. RESULTS: As of August 21st, 2022, 222 human cases of WNV infection were confirmed by molecular testing, including 103 with fever (WNF) and 119 with WNND. WNV lineage was determined in 201 (90.5%) cases, comprising 138 WNV-1 and 63 WNV-2 infections. During the same period, 35 blood donors tested positive, including 30 in whom WNV lineage was determined (13 WNV-1 and 17 WNV-2). Comparative analysis of the distribution of WNV-1 and WNV-2 infections among WNND cases, WNF cases and WNV-positive blood donors showed that patients with WNND were more likely to have WNV-1 infection than blood donors (odds ratio 3.44; 95% CI 95% 1.54 to 8.24; p = 0.0043). As observed in humans, in wild birds WNV-1 had higher infectious rate (IR) and showed a more rapid expansion than WNV-2. At variance, the distribution of the two lineages was more even in mosquitoes, but with a trend of rapid increase of WNV-1 IR over WNV-2. CONCLUSIONS: Comparative analysis of WNV-1 vs WNV-2 infection in humans, wild birds, and mosquitos showed a rapid expansion of WNV-1 and suggested that WNV-1 infected patients might have an increased risk to develop severe disease.