Pathogenic Avian Influenza H5N1 Virus Research Articles

Pigs are highly susceptible to but do not transmit mink-derived highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b

Rapid evolution of highly pathogenic avian influenza viruses (HPAIVs) is driven by antigenic drift but also by reassortment, which might result in robust replication in and transmission to mammals. Recently, spillover of clade 2.3.4.4b HPAIV to mammals including humans, and their transmission between mammal species has been reported. This study aimed to evaluate the pathogenicity and transmissibility of a mink-derived clade 2.3.4.4b H5N1 HPAIV isolate from Spain in pigs. Experimental infection caused interstitial pneumonia with necrotizing bronchiolitis with high titers of virus present in the lower respiratory tract and 100% seroconversion. Infected pigs shed limited amount of virus, and importantly, there was no transmission to contact pigs. Notably, critical mammalian-like mutations such as PB2-E627K and HA-Q222L emerged at low frequencies in principal-infected pigs. It is concluded that pigs are highly susceptible to infection with the mink-derived clade 2.3.4.4b H5N1 HPAIV and provide a favorable environment for HPAIV to acquire mammalian-like adaptations.

Concurrent Infection with Clade 2.3.4.4b Highly Pathogenic Avian Influenza H5N6 and H5N1 Viruses, South Korea, 2023.

Highly pathogenic avian influenza H5N6 and H5N1 viruses of clade 2.3.4.4b were simultaneously introduced into South Korea at the end of 2023. An outbreak at a broiler duck farm consisted of concurrent infection by both viruses. Sharing genetic information and international surveillance of such viruses in wild birds and poultry is critical.

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 IndonesiaorKorea (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.

Highly pathogenic avian influenza H5N1 virus infections in pinnipeds and seabirds in Uruguay: Implications for bird-mammal transmission in South America.

The highly pathogenic avian influenza viruses of clade 2.3.4.4b have caused unprecedented deaths in South American wild birds, poultry, and marine mammals. In September 2023, pinnipeds and seabirds appeared dead on the Uruguayan Atlantic coast. Sixteen influenza virus strains were characterized by real-time reverse transcription PCR and genome sequencing in samples from sea lions (Otaria flavescens), fur seals (Arctocephalus australis), and terns (Sterna hirundinacea). Phylogenetic and ancestral reconstruction analysis showed that these strains have pinnipeds most likely as the ancestral host, representing a recent introduction of clade 2.3.4.4b in Uruguay. The Uruguayan and closely related strains from Peru (sea lions) and Chile (sea lions and a human case) carry mammalian adaptative residues 591K and 701N in the viral polymerase basic protein 2 (PB2). Our findings suggest that clade 2.3.4.4b strains in South America may have spread from mammals to mammals and seabirds, revealing a new transmission route.

Stranding and mass mortality in humboldt penguins (Spheniscus humboldti), associated to HPAIV H5N1 outbreak in Chile

The highly pathogenic Avian Influenza virus (HPAIV) H5N1 has caused a global outbreak affecting both wild and domestic animals, predominantly avian species. To date, cases of the HPAIV H5 Clade 2.3.4.4b in penguins have exclusively been reported in African Penguins. In Chile, the virus was confirmed in pelicans in December 2022 and subsequently spread across the country, affecting several species, including Humboldt penguins. This study aims to provide an overview of the incidents involving stranded and deceased Humboldt penguins and establish a connection between these events and HPAIV H5N1. Historical data about strandings between 2009 and 2023 was collected, and samples from suspected cases in 2023 were obtained to confirm the presence of HPAIV H5N1. Between January and August 2023, 2,788 cases of stranded and deceased penguins were recorded. Out of these, a total of 2,712 penguins deceased, evidencing a significative increase in mortality starting in early 2023 coinciding with the introduction and spreading of HPAIV H5N1 in the country. Thirty-seven events were categorized as mass mortality events, with the number of deceased penguins varying from 11 to 98. Most cases (97 %) were observed in the North of Chile. One hundred and eighty-one specimens were subjected to HPAIV diagnosis, four of which tested positive for HPAIV H5N1. Spatial analysis validates the correlation between mass mortality events and outbreaks of HPAIV in Chile. However, the limited rate of HPAIV H5N1 detection, which can be attributed to the type and quality of the samples, requiring further exploration.

Isolation and Identification of The Highly Pathogenic Avian Influenza H5N8 Virus Isolated From Commercial Layer Chickens in Al-Sharkia Province in Egypt During 2019-2021

Isolation and Identification of The Highly Pathogenic Avian Influenza H5N8 Virus Isolated From Commercial Layer Chickens in Al-Sharkia Province in Egypt During 2019-2021

Spatiotemporal genotype replacement of H5N8 avian influenza viruses contributed to H5N1 emergence in 2021/2022 panzootic.

Since 2020, clade 2.3.4.4b highly pathogenic avian influenza H5N8 and H5N1 viruses have swept through continents, posing serious threats to the world. Through comprehensive analyses of epidemiological, genetic, and bird migration data, we found that the dominant genotype replacement of the H5N8 viruses in 2020 contributed to the H5N1 outbreak in the 2021/2022 wave. The 2020 outbreak of the H5N8 G1 genotype instead of the G0 genotype produced reassortment opportunities and led to the emergence of a new H5N1 virus with G1's HA and MP genes. Despite extensive reassortments in the 2021/2022 wave, the H5N1 virus retained the HA and MP genes, causing a significant outbreak in Europe and North America. Furtherly, through the wild bird migration flyways investigation, we found that the temporal-spatial coincidence between the outbreak of the H5N8 G1 virus and the bird autumn migration may have expanded the H5 viral spread, which may be one of the main drivers of the emergence of the 2020-2022 H5 panzootic.IMPORTANCESince 2020, highly pathogenic avian influenza (HPAI) H5 subtype variants of clade 2.3.4.4b have spread across continents, posing unprecedented threats globally. However, the factors promoting the genesis and spread of H5 HPAI viruses remain unclear. Here, we found that the spatiotemporal genotype replacement of H5N8 HPAI viruses contributed to the emergence of the H5N1 variant that caused the 2021/2022 panzootic, and the viral evolution in poultry of Egypt and surrounding area and autumn bird migration from the Russia-Kazakhstan region to Europe are important drivers of the emergence of the 2020-2022 H5 panzootic. These findings provide important targets for early warning and could help control the current and future HPAI epidemics.

Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b from Peru forms a monophyletic group with Chilean isolates in South America

Highly pathogenic avian Influenza virus (HPAIV) has spread in an unprecedented extent globally in recent years. Despite the large reports of cases in Asia, Europe, and North America, little is known about its circulation in South America. Here, we describe the isolation, and whole genome characterization of HPAIV obtained from sampling 26 wild bird species in Peru, representing one of the largest studies in our region following the latest HPAIV introduction in South America. Out of 147 samples analyzed, 22 were positive for detection of avian influenza virus using a qRT-PCR-based assay. Following inoculation into embryonated chicken eggs, fourteen viral isolates were obtained from which nine isolates were selected for genome characterization, based on their host relevance. Our results identified the presence of HPAIV H5N1 subtype in a highly diverse wild bird species. Phylogenetic analysis revealed that these isolates correspond to the clade 2.3.4.4b, sharing a common ancestor with North American isolates and forming a monophyletic group along with isolates from Chile. Altogether, changes at the amino acid levels compared to their closest relatives indicates the virus is evolving locally, highlighting the need for constant genomic surveillance. This data evidence the chances for spillover events increases as the virus spreads into large populations of immunologically naïve avian species and adding conditions for cross species transmission.

A highly pathogenic avian influenza virus H5N1 clade 2.3.4.4 detected in Samara Oblast, Russian Federation.

Avian influenza (AI) is a global problem impacting birds and mammals, causing economic losses in commercial poultry farms and backyard settings. In 2022, over 8,500 AI cases were reported worldwide, with the H5 subtype being responsible for many outbreaks in wild and domestic birds. In the territory of the Russian Federation, outbreaks of AI have been massively reported since 2020, both among domestic bird species and wild bird species. Wild migratory birds often serve as natural reservoirs for AI viruses, and interactions between bird species can lead to the emergence of new, highly pathogenic variants through genetic recombination between strains. In order to combat the widespread outbreaks of the disease and potential risks of further spread in 2021, monitoring studies were conducted in the Samara Oblast, the southeastern region of European Russian Federation. These studies aimed to diagnose and characterize circulating AI virus variants among wild migratory birds during waterfowl hunting in areas of mass nesting. Among the 98 shot birds, a highly pathogenic A/H5N1 AI virus was detected in a Eurasian Teal from the Bolshechernigovsky district. It was classified into clade 2.3.4.4 based on the cleavage site structure of HA. Phylogenetic analysis showed a high relatedness of the identified strain in the Samara Oblast with field isolates from Russia, Nigeria, Bangladesh, and Benin. The article emphasizes the importance of monitoring AI virus spread in both wild and poultry, highlighting the need for timely information exchange to assess risks. Further comprehensive studies are necessary to understand virus dissemination pathways.

Bivalent Hemagglutinin Cleavage-Site Peptide Vaccines Protect Chickens from Lethal Infections with Highly Pathogenic H5N1 and H5N6 Avian Influenza Viruses.

Outbreaks of highly pathogenic avian influenza viruses cause huge economic losses to the poultry industry worldwide. Vaccines that can protect chickens from infections caused by various variants of highly pathogenic H5Nx avian influenza viruses are needed owing to the continuous emergence of new variants. We previously showed that vaccines containing the H5 cleavage-site peptide from clade 2.3.4.4. H5N6 avian influenza virus protects chickens from infection with homologous clade 2.3.4.4. H5N6 avian influenza virus, but not from infection with the heterologous clade 1 H5N1 avian influenza virus. Therefore, we developed bivalent peptide vaccines containing H5 cleavage sites of viruses from both clades to protect chickens from both H5N1 and H5N6 avian influenza viruses. Chickens were vaccinated with two doses of a combined peptide vaccine containing cleavage-site peptides from clade 1 and clade 2.3.4.4. highly pathogenic H5N1 and H5N6 avian influenza viruses and then challenged with both viruses. The infected chickens were monitored for survival and their tracheae and cloacae were sampled to check for viral shedding based on the median tissue culture infectious dose of 50 (log10TCID50/mL) in Madin-Darby canine kidney cells. Antibody production was induced at similar levels in the sera of chickens immunized with two doses of the combined peptide vaccines containing cleavage-site peptides from highly pathogenic H5N1 and H5N6 avian influenza viruses. The immunized chickens were protected from infection with both H5N1 and H5N6 avian influenza viruses without viral shedding in the tracheae and cloacae. Dual-peptide vaccines containing cleavage-site peptides of both clades can protect chickens from highly pathogenic avian influenza virus infections.

HA N193D substitution in the HPAI H5N1 virus alters receptor binding affinity and enhances virulence in mammalian hosts

ABSTRACT During the 2021/2022 winter season, we isolated highly pathogenic avian influenza (HPAI) H5N1 viruses harbouring an amino acid substitution from Asparagine(N) to Aspartic acid (D) at residue 193 of the hemagglutinin (HA) receptor binding domain (RBD) from migratory birds in South Korea. Herein, we investigated the characteristics of the N193D HA-RBD substitution in the A/CommonTeal/Korea/W811/2021[CT/W811] virus by using recombinant viruses engineered via reverse genetics (RG). A receptor affinity assay revealed that the N193D HA-RBD substitution in CT/W811 increases α2,6 sialic acid receptor binding affinity. The rCT/W811-HA193N virus caused rapid lethality with high virus titres in chickens compared with the rCT/W811-HA193D virus, while the rCT/W811-HA193D virus exhibited enhanced virulence in mammalian hosts with multiple tissue tropism. Surprisingly, a ferret-to-ferret transmission assay revealed that rCT/W811-HA193D virus replicates well in the respiratory tract, at a rate about 10 times higher than that of rCT/W811-HA193N, and all rCT/W811-HA193D direct contact ferrets were seroconverted at 10 days post-contact. Further, competition transmission assay of the two viruses revealed that rCT/W811-HA193D has enhanced growth kinetics compared with the rCT/W811-HA193N, eventually becoming the dominant strain in nasal turbinates. Further, rCT/W811-HA193D exhibits high infectivity in primary human bronchial epithelial (HBE) cells, suggesting the potential for human infection. Taken together, the HA-193D containing HPAI H5N1 virus from migratory birds showed enhanced virulence in mammalian hosts, but not in avian hosts, with multi-organ replication and ferret-to-ferret transmission. Thus, this suggests that HA-193D change increases the probability of HPAI H5N1 infection and transmission in humans.

Highly pathogenic avian influenza virus H5N1 infection in skua and gulls in the United Kingdom, 2022.

The reemergence of the highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in the United Kingdom in 2021-2022 has caused unprecedented epizootic events in wild birds and poultry. During the summer of 2022, there was a shift in virus transmission dynamics resulting in increased HPAIV infection in seabirds, and consequently, a profound impact on seabird populations. To understand the pathological impact of HPAIV in seabirds, we evaluated the virus antigen distribution and associated pathological changes in the tissues of great skua (Stercorarius skua, n = 8), long-tailed skua (Stercorarius longicaudus, n = 1), European herring gull (Larus argentatus, n = 5), and black-headed gull (Chroicocephalus ridibundus, n = 4), which succumbed to natural infection of HPAIV during the summer of 2022. Cases were collected from Shetland, including Scatness (mainland), No Ness (mainland), Clumlie (mainland), Hermaness (island), Fair Isle (island), Noss (island), and the West Midlands, South East, and South West of England. Grossly, gizzard ulceration was observed in one great skua and pancreatic necrosis was observed in 4 herring gulls, with intralesional viral antigen detected subsequently. Microscopical analysis revealed neuro-, pneumo-, lymphoid-, and cardiomyotropism of HPAIV H5N1, with the most common virus-associated pathological changes being pancreatic and splenic necrosis. Examination of the reproductive tract of the great skua revealed HPAIV-associated oophoritis and salpingitis, and virus replication within the oviductal epithelium. The emergence of HPAIV in seabirds Stercorariidae and Laridae, particularly during summer 2022, has challenged the dogma of HPAIV dynamics, posing a significant threat to wild bird life with potential implications for the reproductive performance of seabirds of conservation importance.

Influenza virus immune imprinting dictates the clinical outcomes in ferrets challenged with highly pathogenic avian influenza virus H5N1.

Zoonotic transmission of H5N1 highly pathogenic avian influenza virus (HPAIV) into the human population is an increasing global threat. The recent 2022 HPAIV outbreak significantly highlighted this possibility, increasing concern in the general population. The clinical outcomes of H5N1 influenza virus exposure can be determined by an individual's primary influenza virus infection (imprinting) or vaccination status. Immunological imprinting with Group 1 - (H1N1, H2N2, and H2N3) increases survival rates following H5N1 viral infection compared to Group 2 - (H3N2) imprinted individuals. Vaccination against H5N1 influenza viruses can offer protection to at-risk populations; however, stockpiled inactivated H5N1 influenza vaccines are not readily available to the public. We hypothesize that the immunological response to vaccination and subsequent clinical outcome following H5N1 influenza virus infection is correlated with the immunological imprinting status of an individual. To test this hypothesis, our lab established a ferret pre-immune model of disease. Naïve ferrets were intranasally inoculated with seasonal influenza viruses and allowed to recover for 84 days prior to H5N1 virus infection. Ferrets imprinted following H1N1 and H2N3 virus infections were completely protected against lethal H5N1 influenza virus challenge (100% survival), with few to no clinical symptoms. In comparison, H3N2 influenza virus-imprinted ferrets had severe clinical symptoms, delayed disease progression, and a sublethal phenotype (40% mortality). Consecutive infections with H1N1 influenza viruses followed by an H3N2 influenza virus infection did not abrogate the immune protection induced by the original H1N1 influenza virus infection. In addition, ferrets consecutively infected with H1N1 and H2N3 viruses had no clinical symptoms or weight loss. H3N2 pre-immune ferrets were vaccinated with a broadly reactive H5 HA-based or H1 NA-based vaccine (Hu-CO 2). These ferrets were protected against H5N1 influenza virus challenge, whereas ferrets vaccinated with the H1N1 wild-type CA/09 rHA vaccine had similar phenotypes as non-vaccinated H3N2-imprinted ferrets with 40% survival. Overall, Group 2 imprinted ferrets, which were vaccinated with heterologous Group 1 HA vaccines, had redirected immune responses to Group 1 influenza viral antigens and rescued a sublethal phenotype to complete protection.

Phosphorylation of PB2 at serine 181 restricts viral replication and virulence of the highly pathogenic H5N1 avian influenza virus in mice

Influenza A virus (IAV) continues to pose a pandemic threat to public health, resulting a high mortality rate annually and during pandemic years. Posttranslational modification of viral protein plays a substantial role in regulating IAV infection. Here, based on immunoprecipitation (IP)-based mass spectrometry (MS) and purified virus-coupled MS, a total of 89 phosphorylation sites distributed among 10 encoded viral proteins of IAV were identified, including 60 novel phosphorylation sites. Additionally, for the first time, we provide evidence that PB2 can also be acetylated at site K187. Notably, the PB2 S181 phosphorylation site was consistently identified in both IP-based MS and purified virus-based MS. Both S181 and K187 are exposed on the surface of the PB2 protein and are highly conserved in various IAV strains, suggesting their fundamental importance in the IAV life cycle. Bioinformatic analysis results demonstrated that S181E/A and K187Q/R mimic mutations do not significantly alter the PB2 protein structure. While continuous phosphorylation mimicked by the PB2 S181E mutation substantially decreases viral fitness in mice, PB2 K187Q mimetic acetylation slightly enhances viral virulence in mice. Mechanistically, PB2 S181E substantially impairs viral polymerase activity and viral replication, remarkably dampens protein stability and nuclear accumulation of PB2, and significantly weakens IAV-induced inflammatory responses. Therefore, our study further enriches the database of phosphorylation and acetylation sites of influenza viral proteins, laying a foundation for subsequent mechanistic studies. Meanwhile, the unraveled antiviral effect of PB2 S181E mimetic phosphorylation may provide a new target for the subsequent study of antiviral drugs.

Low Pathogenic Avian Influenza H9N2 Viruses in Morocco: Antigenic and Molecular Evolution from 2021 to 2023.

Avian influenza viruses pose significant threats to both the poultry industry and public health worldwide. Among them, the H9N2 subtype has gained substantial attention due to its high prevalence, especially in Asia, the Middle East, and Africa; its ability to reassort with other influenza viruses; and its potential to infect humans. This study presents a comprehensive phylogenetic and molecular analysis of H9N2 avian influenza viruses circulating in Morocco from 2021 to 2023. Through an active epidemiological survey, a total of 1140 samples (trachea and lungs) and oropharyngeal swabs pooled into 283 pools, collected from 205 farms located in 7 regions of Morocco known for having a high density of poultry farms, were analyzed. Various poultry farms were investigated (159 broiler farms, 24 layer farms, 10 breeder farms, and 12 turkey breeder farms). A total of 21 AI H9N2 strains were isolated, and in order to understand the molecular evolution of the H9N2 avian influenza virus, their genetic sequences were determined using the Sanger sequencing technique. Phylogenetic analysis was performed using a dataset comprising global H9N2 sequences to determine the genetic relatedness and evolutionary dynamics of the Moroccan strains. The results revealed the continued circulation and diversification of H9N2 avian influenza viruses in Morocco during the study period. Real-time RT-PCR showed a positivity rate of 35.6% (73/205), with cycle threshold values ranging from 19.2 to 34.9. The phylogenetic analysis indicated that all Moroccan strains belonged to a G1-like lineage and regrouped into two distinct clusters. Our newly detected isolates aggregated distinctly from the genotypes previously isolated in Morocco, North and West Africa, and the Middle East. This indicats the potential of virus evolution resulting from both national circulation and cross-border transmission. A high genetic diversity at both nucleotide and amino-acid levels was observed among all the strains isolated in this study, as compared to H9N2 strains isolated in Morocco since 2016, which suggests the co-circulation of genetically diverse H9N2 variants. Newly discovered mutations were detected in hemagglutinin positions 226, 227, and 193 (H3 numbering), which highlights the genetic evolution of the H9N2 AIVs. These findings contribute to our understanding of the evolution and epidemiology of H9N2 in the region and provide valuable insights for the development of effective prevention and control strategies against this emerging avian influenza subtype.

Investigation of risk factors for the introduction of highly pathogenic avian influenza H5N1 virus onto table egg farms in the United States, 2022: A case-control study. Proceeding of The First International Avian Influenza Summit, University of Arkansas- October 16-17, 2023”

Introduction: The 2022–2023 highly pathogenic avian influenza (HPAI) H5N1 outbreak in the United States (U.S.) is the most geographically extensive and costly animal health event in U.S. history. In 2022 alone, over 57 million commercial and backyard poultry in 47 U.S. states were affected. Over 75% of the affected poultry were part of the commercial table egg production sector. Methods: We conducted a case-control study to identify potential risk factors for the introduction of the HPAI virus onto commercial table egg operations. Univariate and multivariable analyses were conducted to compare farm characteristics, management, and biosecurity factors on case and control farms. Results: Factors associated with increased risk of infection included being in an existing control zone, sightings of wild waterfowl, mowing or bush hogging vegetation less than 4 times a month, having an off-site method of daily mortality disposal (off-site composting or burial, rendering, or landfill), and wild bird access to feed/feed ingredients at least some of the time. Protective factors included a high level of vehicle washing for trucks and trailers entering the farm (a composite variable that included having a permanent wash station), having designated personnel assigned to specific barns, having a farm entrance gate, and requiring a change of clothing for workers entering poultry barns. Discussion: Study results improve our understanding of risk factors for HPAI infection and control measures for preventing HPAI on commercial U.S. table egg farms.

Wild bird origin Influenza A subtype diversity and reassortment events during the HPAI H5N1 outbreak in Chile, 2022-2023. Proceeding of The First International Avian Influenza Summit, University of Arkansas- October 16-17, 2023"

Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to pose a significant threat to animal and human health worldwide. Since the onset of the HPAI H5N1 outbreak in South America, our research group has been conducting environmental influenza A virus (IAV) surveillance in different wetlands throughout Chile to detect and characterize circulating IAV strains. Between November 2022 and June 2023, we collected and screened 3275 environmental samples for IAV by RT-qPCR targeting the M gene. Of these, 133 were IAV-positive (4.1\%). Further testing by H5-specific RT-qPCR revealed that 14 (10.5\%) of the positive samples corresponded to HPAI H5 2.3.4.4b, collected during the month of November (2), December (8 positives), January (1 positive), March (1 Positive) and June (1 positive). Samples obtained during spring (November and December) were obtained at the Lluta River estuary in the extreme north of Chile. Samples obtained during Summer (January and March) were obtained in Lluta and at Punta Arenas, located in the extreme South of Chile. The sample obtained in Winter (June) was obtained in Maipo, located in Central Chile. Sequence analysis of 6 fully sequenced H5N1 viruses revealed that all segments were closely related to other H5N1 2.3.4.4b viruses circulating at the time in South America. Interestingly, we also obtained several other subtypes of low pathogenic avian influenza viruses (LPAI) during the sampling period: H13N9, H13N8, H12N5, H8N4, H6N8, H6N5, H5N7, H4N6, H2N8 and H2N2. Phylogenetic analysis of these viruses revealed that locally circulating LPAI H12N5, H6N8, and H5N7 subtype viruses, additionally to 4 more viruses without subtype information, had reassorted with HPAI viruses in at least one of the PA and NP segments. The role that these novel reassortant viruses play in the ecology of IAV in Chile and their pathogenic potential is unknown; however, in light of these reassortment events, further surveillance is warranted to detect potentially novel HPAI H5Nx viruses circulating in wild birds in Chile that could have unknown pathogenic and transmission potential.

Avian sarcoma/leukosis virus (RCAS)-mediated over-expression of IFITM3 protects chicks from highly pathogenic avian influenza virus subtype H5N1

Broad-spectrum antiviral activities of interferon-induced transmembrane proteins (IFITMs) are primarily attributed to in vitro inhibition of viral entry. Here, we used an avian sarcoma-leukosis virus (RCAS)-based gene transfer system and successfully generated chicks that constitutively express chicken IFITM3 (chIFITM3). The chIFITM3-overexpressing chicks showed significant protection and disease tolerance against highly pathogenic avian influenza virus (HPAIV) H5N1 (Clade 2.2.1.2). The chicks, overexpressing chIFITM3, also showed delayed onset of clinical symptoms, reduced viral shedding, and alleviated histopathologic alterations compared to control and challenged chicks. These findings highlight that overexpression of chIFITM3 provide a substantial defense against zoonotic H5N1 in vivo.

Highly pathogenic avian influenza A virus (HPAIV) H5N1 infection in two European grey seals (Halichoerus grypus) with encephalitis

ABSTRACT Recent reports documenting sporadic infections in carnivorous mammals worldwide with highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b have raised concerns about the potential risk of adaptation to sustained transmission in mammals, including humans. We report H5N1 clade 2.3.4.4b infection of two grey seals (Halichoerus grypus) from coastal waters of The Netherlands and Germany in December 2022 and February 2023, respectively. Histological and immunohistochemical investigations showed in both animals a non-suppurative and necrotising encephalitis with viral antigen restricted to the neuroparenchyma. Whole genome sequencing showed the presence of HPAIV H5N1 clade 2.3.4.4b strains in brain tissue, which were closely related to sympatric avian influenza viruses. Viral RNA was also detected in the lung of the seal from Germany by real-time quantitative PCR. No other organs tested positive. The mammalian adaptation PB2-E627K mutation was identified in approximately 40% of the virus population present in the brain tissue of the German seal. Retrospective screening for nucleoprotein-specific antibodies, of sera collected from 251 seals sampled in this region from 2020 to 2023, did not show evidence of influenza A virus-specific antibodies. Similarly, screening by reverse transcription PCR of tissues of 101 seals that had died along the Dutch coast in the period 2020–2021, did not show evidence of influenza virus infection. Collectively, these results indicate that individual seals are sporadically infected with HPAIV-H5N1 clade 2.3.4.4b, resulting in an encephalitis in the absence of a systemic infection, and with no evidence thus far of onward spread between seals.

Emergence of a new genotype of clade 2.3.4.4b H5N1 highly pathogenic avian influenza A viruses in Bangladesh

ABSTRACT Influenza virological surveillance was conducted in Bangladesh from January to December 2021 in live poultry markets (LPMs) and in Tanguar Haor, a wetland region where domestic ducks have frequent contact with migratory birds. The predominant viruses circulating in LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses. Additional LPAIs were found in both LPM (H4N6) and Tanguar Haor wetlands (H7N7). Genetic analyses of these LPAIs strongly suggested long-distance movement of viruses along the Central Asian migratory bird flyway. We also detected a novel clade 2.3.4.4b H5N1 virus from ducks in free-range farms in Tanguar Haor that was similar to viruses first detected in October 2020 in The Netherlands but with a different PB2. Identification of clade 2.3.4.4b HPAI H5N1 viruses in Tanguar Haor provides continued support of the role of migratory birds in transboundary movement of influenza A viruses (IAV), including HPAI viruses. Domestic ducks in free range farm in wetland areas, like Tangua Haor, serve as a conduit for the introduction of LPAI and HPAI viruses into Bangladesh. Clade 2.3.4.4b viruses have dominated in many regions of the world since mid-2021, and it remains to be seen if these viruses will replace the endemic clade 2.3.2.1a H5N1 viruses in Bangladesh.