Descriptive epidemiology and phylogenetic analysis of highly pathogenic avian influenza H5N1 clade 2.3.4.4b in British Columbia (B.C.) and the Yukon, Canada, September 2022 to June 2023.

Surveillance data from wildlife and poultry was used to describe the spread of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b in British Columbia (B.C.) and the Yukon, Canada from September 2022 - June 2023 compared to the first "wave" of the outbreak in this region, which occurred April - August 2022, after the initial viral introduction. Although the number of HPAI-positive poultry farms and wildlife samples was greater in "Wave 2", cases were more tightly clustered in southwestern B.C. and the most commonly affected species differed, likely due to an influx of overwintering waterfowl in the area. Eight HPAI genetic clusters, representing seven genotypes and two inter-continental viral incursions, were detected, with significant variation in the relative abundance of each cluster between the waves. Phylogenetic data suggests multiple spillover events from wild birds to poultry and mammals but could not rule out transmission among farms and among mammals.

Cocirculation of Genetically Distinct Highly Pathogenic Avian Influenza H5N5 and H5N1 Viruses in Crows, Hokkaido, Japan.

We isolated highly pathogenic avian influenza (HPAI) H5N5 and H5N1 viruses from crows in Hokkaido, Japan, during winter 2023-24. They shared genetic similarity with HPAI H5N5 viruses from northern Europe but differed from those in Asia. Continuous monitoring and rapid information sharing between countries are needed to prevent HPAI virus transmission.

Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b in wild rats in Egypt during 2023.

We detected highly pathogenic avian influenza A(H5N1) virus in wild rats collected from a rural area in Giza, Egypt, near poultry farms, markets, and backyard flocks. Sequence and phylogenetic analyses indicated that the virus from the rats belonged to clade 2.3.4.4b, which has been the predominant virus genotype circulating in Egypt and worldwide since 2021-2022. Active surveillance of avian influenza viruses in wild and domestic mammals is recommended to prevent further spread to mammals and humans.

Phylodynamics of high pathogenicity avian influenza virus in Bangladesh identifying domestic ducks as the amplifying host reservoir.

High pathogenicity avian influenza (HPAI) virus H5N1 first emerged in Bangladesh in 2007. Despite the use of vaccines in chickens since 2012 to control HPAI, HPAI H5Nx viruses have continued to infect poultry, and wild birds, resulting in notable mass mortalities in house crows (Corvus splendens). The first HPAI H5Nx viruses in Bangladesh belonged to clade 2.2.2, followed by clade 2.3.4.2 and 2.3.2.1 viruses in 2011. After the implementation of chicken vaccination in 2012, these viruses were mostly replaced by clade 2.3.2.1a viruses and more recently clade 2.3.4.4b and h viruses. In this study, we reconstruct the phylogenetic history of HPAI H5Nx viruses in Bangladesh to evaluate the role of major host species in the maintenance and evolution of HPAI H5Nx virus in Bangladesh and reveal the role of heavily impacted crows in virus epidemiology. Epizootic waves caused by HPAI H5N1 and H5N6 viruses amongst house crows occurred annually in winter. Bayesian phylodynamic analysis of clade 2.3.2.1a revealed frequent bidirectional viral transitions between domestic ducks, chickens, and house crows that was markedly skewed towards ducks; domestic ducks might be the source, or reservoir, of HPAI H5Nx in Bangladesh, as the number of viral transitions from ducks to chickens and house crows was by far more numerous than the other transitions. Our results suggest viral circulation in domestic birds despite vaccination, with crow epizootics acting as a sentinel. The vaccination strategy needs to be updated to use more effective vaccinations, assess vaccine efficacy, and extension of vaccination to domestic ducks, the key reservoir.

Mortality in sea lions is associated with the introduction of the H5N1 clade 2.3.4.4b virus in Brazil October 2023: whole genome sequencing and phylogenetic analysis.

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 virus was detected in the South American sea lions found dead in Santa Catarina, Brazil, in October 2023. Whole genome sequencing and comparative phylogenetic analysis were conducted to investigate the origin, genetic diversity, and zoonotic potentials of the H5N1 viruses. The H5N1 viruses belonged to the genotype B3.2 of clade 2.3.4.4b H5N1 virus, which was identified in North America and disseminated to South America. They have acquired new amino acid substitutions related to mammalian host affinity. Our study provides insights into the genetic landscape of HPAI H5N1 viruses in Brazil, highlighting the continuous evolutionary processes contributing to their possible adaptation to mammalian hosts.

Genomic characterization of highly pathogenic avian influenza A H5N1 virus newly emerged in dairy cattle.

In March 2024, the emergence of highly pathogenic avian influenza (HPAI) A (H5N1) infections in dairy cattle was detected in the United Sates for the first time. We genetically characterize HPAI viruses from dairy cattle showing an abrupt drop in milk production, as well as from two cats, six wild birds, and one skunk. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627 K mutation in a human case associated with cattle underscores the potential for rapid evolution post infection, highlighting the need for continued surveillance to monitor public health threats.

Detection and characterization of H5N1 HPAIV in environmental samples from a dairy farm.

The recent expansion of HPAIV H5N1 infections in terrestrial mammals in the Americas, most recently including the outbreak in dairy cattle, emphasizes the critical need for better epidemiological monitoring of zoonotic diseases. In this work, we detected, isolated, and characterized the HPAIV H5N1 from environmental swab samples collected from a dairy farm in the state of Kansas, USA. Genomic sequencing of these samples uncovered two distinctive substitutions in the PB2 (E249G) and NS1 (R21Q) genes which are rare and absent in recent 2024 isolates of H5N1 circulating in the mammalian and avian species. Additionally, approximately 1.7% of the sequence reads indicated a PB2 (E627K) substitution, commonly associated with virus adaptation to mammalian hosts. Phylogenetic analyses of the PB2 and NS genes demonstrated more genetic identity between this environmental isolate and the 2024 human isolate (A/Texas/37/2024) of H5N1. Conversely, HA and NA gene analyses revealed a closer relationship between our isolate and those found in other dairy cattle with almost 100% identity, sharing a common phylogenetic subtree. These findings underscore the rapid evolutionary progression of HPAIV H5N1 among dairy cattle and reinforces the need for more epidemiological monitoring which can be done using environmental sampling.

Novel Genotypes of Highly Pathogenic Avian Influenza H5N1 Clade 2.3.4.4b Viruses, Germany, November 2023.

Several subtypes and many different genotypes of highly pathogenic avian influenza viruses of subtype H5 clade 2.3.4.4b have repeatedly caused outbreaks in Germany. Four new highly pathogenic avian influenza genotypes emerged in November 2023 after reassortment with low pathogenicity precursors, replacing genotype BB, which had dominated in Europe since 2022.

Multiplex Dual-Target Reverse Transcription PCR for Subtyping Avian Influenza A(H5) Virus.

An increased risk for human infection with avian influenza A(H5N1) viruses is of concern. We developed an internally controlled, dual-target reverse transcription PCR for influenza A(H5) subtyping. This test could be used to detect influenza A(H5) in clinical samples.

Avian influenza viruses in wild birds in Canada following incursions of highly pathogenic H5N1 virus from Eurasia in 2021-2022.

Following the detection of novel highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in Newfoundland, Canada, in late 2021, avian influenza virus (AIV) surveillance in wild birds was scaled up across Canada. Herein, we present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds during the first year (November 2021-November 2022) following the incursions of HPAIV from Eurasia. The key objectives of the surveillance program were to (i) identify the presence, distribution, and spread of HPAIV and other AIVs; (ii) identify wild bird morbidity and mortality associated with HPAIV; (iii) identify the range of wild bird species infected by HPAIV; and (iv) genetically characterize detected AIV. A total of 6,246 sick and dead wild birds were tested, of which 27.4% were HPAIV positive across 12 taxonomic orders and 80 species. Geographically, HPAIV detections occurred in all Canadian provinces and territories, with the highest numbers in the Atlantic and Central Flyways. Temporally, peak detections differed across flyways, though the national peak occurred in April 2022. In an additional 11,295 asymptomatic harvested or live-captured wild birds, 5.2% were HPAIV positive across 3 taxonomic orders and 19 species. Whole-genome sequencing identified HPAIV of Eurasian origin as most prevalent in the Atlantic Flyway, along with multiple reassortants of mixed Eurasian and North American origins distributed across Canada, with moderate structuring at the flyway scale. Wild birds were victims and reservoirs of HPAIV H5N1 2.3.4.4b, underscoring the importance of surveillance encompassing samples from sick and dead, as well as live and harvested birds, to provide insights into the dynamics and potential impacts of the HPAIV H5N1 outbreak. This dramatic shift in the presence and distribution of HPAIV in wild birds in Canada highlights a need for sustained investment in wild bird surveillance and collaboration across interagency partners. IMPORTANCE: We present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds in the year following the first detection of highly pathogenic avian influenza virus (HPAIV) H5N1 on the continent. The surveillance program tested over 17,000 wild birds, both sick and apparently healthy, which revealed spatiotemporal and taxonomic patterns in HPAIV prevalence and mortality across Canada. The significant shift in the presence and distribution of HPAIV in Canada's wild birds underscores the need for sustained investment in wild bird surveillance and collaboration across One Health partners.

Outbreaks of H5N1 High Pathogenicity Avian Influenza in South Africa in 2023 Were Caused by Two Distinct Sub-Genotypes of Clade 2.3.4.4b Viruses.

In 2023, South Africa continued to experience sporadic cases of clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) in coastal seabirds and poultry. Active environmental surveillance determined that H5Nx, H7Nx, H9Nx, H11Nx, H6N2, and H12N2, amongst other unidentified subtypes, circulated in wild birds and ostriches in 2023, but that H5Nx was predominant. Genome sequencing and phylogenetic analysis of confirmed H5N1 HPAI cases determined that only two of the fifteen sub-genotypes that circulated in South Africa in 2021-2022 still persisted in 2023. Sub-genotype SA13 remained restricted to coastal seabirds, with accelerated mutations observed in the neuraminidase protein. SA15 caused the chicken outbreaks, but outbreaks in the Paardeberg and George areas, in the Western Cape province, and the Camperdown region of the KwaZulu-Natal province were unrelated to each other, implicating wild birds as the source. All SA15 viruses contained a truncation in the PB1-F2 gene, but in the Western Cape SA15 chicken viruses, PA-X was putatively expressed as a novel isoform with eight additional amino acids. South African clade 2.3.4.4b H5N1 viruses had comparatively fewer markers of virulence and pathogenicity compared to European strains, a possible reason why no spillover to mammals has occurred here yet.

Phylodynamics of avian influenza A(H5N1) viruses from outbreaks in Brazil.

Our study identified strains of the A/H5N1 virus in analyzed samples of subsistence poultry, wild birds, and mammals, belonging to clade 2.3.4.4b, genotype B3.2, with very high genetic similarity to strains from Chile, Uruguay, and Argentina. This suggests a migratory route for wild birds across the Pacific, explaining the phylogenetic relatedness. The Brazilian samples displayed similarity to strains that had already been previously detected in South America. Phylogeographic analysis suggests transmission of US viruses from Europe and Asia, co-circulating with other lineages in the American continent. As mutations can influence virulence and host specificity, genomic surveillance is essential to detect those changes, especially in critical regions, such as hot spots in the HA, NA, and PB2 sequences. Mutations in the PB2 gene (D701N and Q591K) associated with adaptation and transmission in mammals were detected suggesting a potential zoonotic risk. Nonetheless, resistance to neuraminidase inhibitors (NAIs) was not identified, however, continued surveillance is crucial to detect potential resistance. Our study also mapped the spread of the virus in the Southern hemisphere, identifying possible entry routes and highlighting the importance of surveillance to prevent outbreaks and protect both human and animal populations.

Genetic diversity of H5N1 and H5N2 high pathogenicity avian influenza viruses isolated from poultry in Japan during the winter of 2022-2023.

High pathogenicity avian influenza viruses (HPAIVs) of the H5N1 and H5N2 subtypes were responsible for 84 HPAI outbreaks on poultry premises in Japan during October 2022-April 2023. The number of outbreaks during the winter of 2022-2023 is the largest ever reported in Japan. In this study, we performed phylogenetic analyses using the full genetic sequences of HPAIVs isolated in Japan during 2022-2023 and those obtained from a public database to identify their genetic origin. Based on the hemagglutinin genes, these HPAIVs were classified into the G2 group of clade 2.3.4.4b, whose ancestors were H5 HPAIVs that circulated in Europe in late 2020, and were then further divided into three subgroups (G2b, G2d, and G2c). Approximately one-third of these viruses were classified into the G2b and G2d groups, which also included H5N1 HPAIVs detected in Japan during 2021-2022. In contrast, the remaining two-thirds were classified into the G2c group, which originated from H5N1 HPAIVs isolated in Asian countries and Russia during the winter of 2021-2022. Unlike the G2b and G2d viruses, the G2c viruses were first detected in Japan in the fall of 2022. Importantly, G2c viruses caused the largest number of outbreaks throughout Japan over the longest period during the season. Phylogenetic analyses using eight segment genes revealed that G2b, G2d, and G2c viruses were divided into 2, 4, and 11 genotypes, respectively, because they have various internal genes closely related to those of avian influenza viruses detected in wild birds in recent years in Asia, Russia, and North America, respectively. These results suggest that HPAIVs were disseminated among migratory birds, which may have generated numerous reassortant viruses with various gene constellations, resulting in a considerable number of outbreaks during the winter of 2022-2023.

Molecular epidemiology and genetic evolution of avian influenza H5N1 subtype in Nigeria, 2006 to 2021.

Nigeria recorded one of the earliest outbreaks of the Highly Pathogenic Avian Influenza (HPAI) virus H5N1 in 2006, which spread to other African countries. In 2023, 18 countries reported outbreaks of H5N1 in poultry, with human cases documented in Egypt, Nigeria, and Djibouti. There is limited information on the molecular epidemiology of HPAI H5N1 in Nigeria. We determined the molecular epidemiology and genetic evolution of the virus from 2006 to 2021. We investigated the trend and geographical distribution across Nigeria. The evolutionary history of 61 full-length genomes was performed from 13 countries worldwide, and compared with sequences obtained from the early outbreaks in Nigeria up to 2021. MEGA 11 was used to determine the phylogenetic relationships of H5N1 strains, which revealed close ancestry between sequences in Nigeria and those from other African countries. Clade classification was performed using the subspecies classification tool for Bacterial and Viral Bioinformatics Research Center (BV-BRC) version 3.35.5. H5N1 Clade 2.2 was observed in 2006, with 2.3.2, 2.3.2.1f clades observed afterwards and 2.3.4.4b in 2021. Our findings underscore the need for genomics surveillance to track antigenic variation and clades switching to monitor the epidemiological of the virus and safeguard human and animal health.Impacts Specific variations in the hemagglutinin (HA) and neuraminidase (NA) genes of Avian influenza virus are consistent in different geographical regions. H5N1 Clade 2.2 was reported in 2006, with 2.3.2, 2.3.2.1f afterwards and 2.3.4.4b in 2021. Nigeria is an epicentre for avian influenza with three major migratory routes for wild birds transversing the country. It is plausible that the Avian influenza in Northern Nigeria may be linked to wild bird sanctuaries in the region.

Detection of clade 2.3.4.4b highly pathogenic H5N1 influenza virus in New York City.

Highly pathogenic avian influenza viruses of the H5N1 clade 2.3.4.4b were detected in North America in the winter of 2021/2022. These viruses have spread across the Americas, causing morbidity and mortality in both wild and domestic birds as well as some mammalian species, including cattle. Many surveillance programs for wildlife as well as commercial poultry operations have detected these viruses. In this study, we conducted surveillance of avian species in the urban environment in New York City. We detected highly pathogenic H5N1 viruses in six samples from four different bird species and performed whole-genome sequencing. Sequencing analysis showed the presence of multiple different genotypes. Our work highlights that the interface between animals and humans that may give rise to zoonotic infections or even pandemics is not limited to rural environments and commercial poultry operations but extends into the heart of our urban centers.IMPORTANCEWhile surveillance programs for avian influenza viruses are often focused on migratory routes and their associated stop-over locations or commercial poultry operations, many bird species-including migratory birds-frequent or live in urban green spaces and wetlands. This brings them into contact with a highly dense population of humans and pets, providing an extensive urban animal-human interface in which the general public may have little awareness of circulating infectious diseases. This study focuses on virus surveillance of this interface, combined with culturally responsive science education and community outreach.

On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR/Cas12a.

Avian influenza viruses (AIVs) of the H5 subtype rank among the most serious pathogens, leading to significant economic losses in the global poultry industry and posing risks to human health. Therefore, rapid and accurate virus detection is crucial for the prevention and control of H5 AIVs. In this study, we established a novel detection method for H5 viruses by utilizing the precision of CRISPR/Cas12a and the efficiency of RT-RPA technologies. This assay facilitates the direct visualization of detection results through blue light and lateral flow strips, accurately identifying H5 viruses with high specificity and without cross-reactivity against other AIV subtypes, NDV, IBV, and IBDV. With detection thresholds of 1.9 copies/µL (blue light) and 1.9 × 103 copies/µL (lateral flow strips), our method not only competes with but also slightly surpasses RT-qPCR, demonstrating an 80.70% positive detection rate across 81 clinical samples. The RT-RPA/CRISPR-based detection method is characterized by high sensitivity, specificity, and independence from specialized equipment. The immediate field applicability of the RT-RPA/CRISPR approach underscores its importance as an effective tool for the early detection and management of outbreaks caused by the H5 subtype of AIVs.

Phylogeography and gene pool analysis of highly pathogenic avian influenza H5N1 viruses reported in India from 2006 to 2021.

India has reported highly pathogenic avian influenza (HPAI) H5N1 virus outbreaks since 2006, with the first human case reported in 2021. These included viruses belonging to the clades 2.2, 2.2.2, 2.2.2.1, 2.3.2.1a, and 2.3.2.1c. There are currently no data on the gene pool of HPAI H5N1 viruses in India. Molecular clock and phylogeography analysis of the HA and NA genes; and phylogenetic analysis of the internal genes of H5N1 viruses from India were carried out. Sequences reported from 2006 to 2015; and sequences from 2021 that were available in online databases were used in the analysis. Five separate introductions of H5N1 viruses into India were observed, via Indonesia or Korea (2002), Bangladesh (2009), Bhutan (2010), and China (2013, 2018) (clades 2.2, 2.2.2, 2.2.2.1, 2.3.2.1a, 2.3.2.1c, and 2.3.4.4b). Phylogenetic analysis revealed eight reassortant genotypes. The H5N1 virus isolated from the human case showed a unique reassortant genotype. Amino acid markers associated with adaptation to mammals were also present. This is the first report of the spatio-temporal origins and gene pool analysis of H5N1 viruses from India, highlighting the need for increased molecular surveillance.

Isolation and genetic characterization of multiple genotypes of both H5 and H7 avian influenza viruses from environmental water in the Izumi plain, Kagoshima prefecture, Japan during the 2021/22 winter season.

In the 2021/22 winter, one H5N1 and nine H5N8 high pathogenicity avian influenza viruses (HPAIVs) of clade 2.3.3.4b were isolated from the water in crane roosts on the Izumi plain, Japan. Additionally, we isolated low pathogenicity avian influenza viruses (LPAIVs) of five subtypes: H1N1, H4N2, H4N6, H7N7, and H10N4. H5N8 HPAIVs belonging to the G2a group were isolated throughout winter, whereas H5N1 HPAIV belonging to the G2b group were isolated only in early winter. These findings suggest co-circulation of both G2a and G2b HPAIVs in early winter. Although two H7N7 LPAIVs were isolated from cranes' roost water collected on the same day, the gene constellations of the two isolates were clearly different, indicating the contemporary invasion of at least two different genotypes of H7N7 LPAIVs in the Izumi plain. This study underscores the importance of monitoring both HPAIVs and LPAIVs to understand avian influenza virus ecology in migratory waterfowl populations.