H5N1 Highly Pathogenic Avian Influenza 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.

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.

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.

Evolutional dynamics of highly pathogenic avian influenza H5N8 genotypes in wintering bird habitats: Insights from South Korea's 2020–2021 season

The winter of 2020–2021 in South Korea witnessed severe outbreaks of Highly Pathogenic Avian Influenza (HPAI) viruses, specifically multiple genotypes of the H5N8 subtype. These outbreaks prompted an extensive investigation into the genetic characteristics and evolutionary dynamics of these viruses. Under the auspices of the National Institute of Wildlife Disease Control and Prevention (NIWDC), we conducted a nationwide surveillance program, collecting 7588 specimens from diverse wild bird habitats. Influenza A viruses were isolated at a rate of 5.0%, with HPAI H5N8 viruses accounting for 38.5% of isolates, predominantly found in wild bird carcasses (97.3%). Genetic analysis revealed the emergence of novel HPAI genotypes due to genetic reassortment events. G1 and G2 viruses were separately introduced into Korea, with G1 viruses displaying dynamic behavior, resulting in diverse sub-genotypes (G1–1 to G1–5) and mainly isolated from clinical specimens. Conversely, the G2 virus, introduced later, became the dominant strain consistently isolated mainly from bird carcasses (88.9%). These findings underscore the emergence of numerous novel HPAI genotypes shaped by multiple reassortment events in high-density wintering grounds of migratory birds. These sites act as hotspots for genetic exchanges, significantly influencing avian ecology, including resident bird species, and contributing to HPAI H5N8 evolution. The genetic diversity and ongoing evolution of these viruses highlight the need for vigilant surveillance and adaptive control measures. Recognizing the potential spillover to human populations, a One Health approach is essential to mitigate the evolving threats posed by avian influenza.

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.

TRIM21 Promotes Oxidative Stress and Ferroptosis through the SQSTM1-NRF2-KEAP1 Axis to Increase the Titers of H5N1 Highly Pathogenic Avian Influenza Virus.

Tripartite motif-containing protein 21 (TRIM21) is involved in signal transduction and antiviral responses through the ubiquitination of protein targets. TRIM21 was reported to be related to the imbalance of host cell homeostasis caused by viral infection. Our studies indicated that H5N1 highly pathogenic avian influenza virus (HPAIV) infection up-regulated TRIM21 expression in A549 cells. Western blot and qPCR results showed that knockdown of TRIM21 alleviated oxidative stress and ferroptosis induced by H5N1 HPAIV and promoted the activation of antioxidant pathways. Co-IP results showed that TRIM21 promoted oxidative stress and ferroptosis by regulating the SQSTM1-NRF2-KEAP1 axis by increasing SQSTM1 K63-linked polyubiquitination under the condition of HPAIV infection. In addition, TRIM21 attenuated the inhibitory effect of antioxidant NAC on HPAIV titers and enhanced the promoting effect of ferroptosis agonist Erastin on HPAIV titers. Our findings provide new insight into the role of TRIM21 in oxidative stress and ferroptosis induced by viral infection.

Analysis of H5N8 influenza virus infection in chicken with mApple reporter genes in vivo and in vitro

H5N8 highly pathogenic avian influenza virus (HPAIV) has caused huge losses to the global poultry industry and critically threatens public health. Chickens are the important host for the transmission. However, the distribution of H5N8 avian influenza virus (AIV) in chicken and the infected cell types are limitedly studied. Therefore, in this study, we detected viral replication and infection by generating recombinant H5N8 AIV expressing an easily tracked mApple fluorescent reporter. The results showed that recombinant viruses passaged four times in chicken embryos successfully expressed mApple proteins detected by fluorescence microscopy and WB, which verified that the constructed recombinant viruses were stable. Compared to parental virus, although recombinant virus attenuated for replication in MDCK cells, it can still replicate effectively, and form visible plaques. Importantly, the experiments on infection of chicken PBMCs in vitro showed a strong correlation between mApple positivity rate and NP positivity rate (r = 0.7594, P =0.0176), demonstrating that mApple reporter could be used as an indicator to accurately reflect AIV infection. Then we infected monocytes/macrophages in PBMCs in vitro and detected the mApple positive percentage was 55.1%-80.4%, which confirmed the chicken primary monocytic/macrophages are important target cells for avian influenza virus infection. In chicken, compared with parental virus, the recombinant virus-infected chickens had lower viral titers in oropharyngeal cloacal and organs, but it can cause significant pathogenicity in chicken and the mortality rate was approximately 66%. In addition, the results of bioluminescent imaging showed that the fluorescence in the lungs was strongest at 5 days post-infection (DPI). Finally, we discovered the mApple positive expression in chicken lung immune cells (CD45+ cells), especially some T cells (CD4 and CD8 T cells) also carrying mApple, which indicates that the H5N8 AIV showed a tropism for immune cells including chicken T cells causing potentially aggressive against cellular immunity. We have provided a simple visualization for further exploration of H5N8 AIV infected chicken immune cells, which contributes to further understanding pathogenic mechanism of H5N8 AIV infection in chicken.

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.

Transmission dynamics and pathogenesis differ between pheasants and partridges infected with clade 2.3.4.4b H5N8 and H5N1 high-pathogenicity avian influenza viruses.

During the UK 2020-2021 epizootic of H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs), high mortality occurred during incursions in commercially farmed common pheasants (Phasianus colchicus). Two pheasant farms, affected separately by H5N8 and H5N1 subtypes, included adjacently housed red-legged partridges (Alectoris rufa), which appeared to be unaffected. Despite extensive ongoing epizootics, H5Nx HPAIV partridge outbreaks were not reported during 2020-2021 and 2021-2022 in the UK, so it is postulated that partridges are more resistant to HPAIV infection than other gamebirds. To assess this, pathogenesis and both intra- and inter-species transmission of UK pheasant-origin H5N8-2021 and H5N1-2021 HPAIVs were investigated. Onward transmission to chickens was also assessed to better understand the risk of spread from gamebirds to other commercial poultry sectors. A lower infectious dose was required to infect pheasants with H5N8-2021 compared to H5N1-2021. However, HPAIV systemic dissemination to multiple organs within pheasants was more rapid following infection with H5N1-2021 than H5N8-2021, with the former attaining generally higher viral RNA levels in tissues. Intraspecies transmission to contact pheasants was successful for both viruses and associated with viral environmental contamination, while interspecies transmission to a first chicken-contact group was also efficient. However, further onward transmission to additional chicken contacts was only achieved with H5N1-2021. Intra-partridge transmission was only successful when high-dose H5N1-2021 was administered, while partridges inoculated with H5N8-2021 failed to shed and transmit, although extensive tissue tropism was observed for both viruses. Mortalities among infected partridges featured a longer incubation period compared to that in pheasants, for both viruses. Therefore, the susceptibility of different gamebird species and pathogenicity outcomes to the ongoing H5Nx clade 2.3.4.4b HPAIVs varies, but pheasants represent a greater likelihood of H5Nx HPAIV introduction into galliforme poultry settings. Consequently, viral maintenance within gamebird populations and risks to poultry species warrant enhanced investigation.

Enhanced Downstream Processing for a Cell-Based Avian Influenza (H5N1) Vaccine.

H5N1 highly pathogenic avian influenza virus (HPAIV) infections pose a significant threat to human health, with a mortality rate of around 50%. Limited global approval of H5N1 HPAIV vaccines, excluding China, prompted the need to address safety concerns related to MDCK cell tumorigenicity. Our objective was to improve vaccine safety by minimizing residual DNA and host cell protein (HCP). We developed a downstream processing method for the cell-based H5N1 HPAIV vaccine, employing CaptoTM Core 700, a multimodal resin, for polishing. Hydrophobic-interaction chromatography (HIC) with polypropylene glycol as a functional group facilitated the reversible binding of virus particles for capture. Following the two-step chromatographic process, virus recovery reached 68.16%. Additionally, HCP and DNA levels were reduced to 2112.60 ng/mL and 6.4 ng/mL, respectively. Western blot, high-performance liquid chromatography (HPLC), and transmission electron microscopy (TEM) confirmed the presence of the required antigen with a spherical shape and appropriate particle size. Overall, our presented two-step downstream process demonstrates potential as an efficient and cost-effective platform technology for cell-based influenza (H5N1 HPAIV) vaccines.

Genetic Analysis of H5N1 High-Pathogenicity Avian Influenza Virus following a Mass Mortality Event in Wild Geese on the Solway Firth.

The United Kingdom (UK) and Europe have seen successive outbreaks of H5N1 clade 2.3.4.4b high-pathogenicity avian influenza virus (HPAIV) since 2020 peaking in the autumn/winter periods. During the 2021/22 season, a mass die-off event of Svalbard Barnacle Geese (Branta leucopsis) was observed on the Solway Firth, a body of water on the west coast border between England and Scotland. This area is used annually by Barnacle Geese to over-winter, before returning to Svalbard to breed. Following initial identification of HPAIV in a Barnacle Goose on 8 November 2021, up to 32% of the total Barnacle Goose population may have succumbed to disease by the end of March 2022, along with other wild bird species in the area. Potential adaptation of the HPAIV to the Barnacle Goose population within this event was evaluated. Whole-genome sequencing of thirty-three HPAIV isolates from wild bird species demonstrated that there had been two distinct incursions of the virus, but the two viruses had remained genetically stable within the population, whilst viruses from infected wild birds were closely related to those from poultry cases occurring in the same region. Analysis of sera from the following year demonstrated that a high percentage (76%) of returning birds had developed antibodies to H5 AIV. This study demonstrates genetic stability of this strain of HPAIV in wild Anseriformes, and that, at the population scale, whilst there is a significant impact on survival, a high proportion of birds recover following infection.

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.

Analysis of the genotypes of Influenza A H5N1 in Bulgaria 2021-2023

Circulating HPAI virus H5N1 can be traced back to the original A/goose/Guangdong/1/96 strain. During the epidemiological years 2022 and 2023, a remarkable number of HPAI H5N1 virus isolates were detected and reported in a diverse range of hosts, including wild and domestic birds, mammals and humans, in more than 28 European countries. Interestingly, the potential for cross-species transmission highlights the complex dynamics of viral spread among these different susceptible species. The persistently emerging outbreaks of HPAI H5N1 in animals and humans demonstrate the need for comprehensive understanding of this virus and its public health implications, as well as for detailed genomic mapping and characterization. With increasing reports of human infections and the potential for devastating economic consequences in industrial livestock production, it is imperative to deepen analyzes of HPAI H5N1 to effectively respond to the continued spread of the virus in animals, prevent future outbreaks and transmission to the people. To provide a comprehensive analysis of the emergence and spread of HPAI H5N1, it is necessary to examine the genetic characteristics, mutations in the viral genome, and the evolution of the virus, and to evaluate the effectiveness of current prevention and control strategies.

ANALYSIS OF THE GENOTYPES OF INFLUENZA A H5N1 IN BULGARIA 2021-2023

Circulating HPAI virus H5N1 can be traced back to the original A/goose/Guangdong/1/96 strain. During the epidemiological years 2022 and 2023, a remarkable number of HPAI H5N1 virus isolates were detected and reported in a diverse range of hosts, including wild and domestic birds, mammals and humans, in more than 28 European countries. Interestingly, the potential for cross-species transmission highlights the complex dynamics of viral spread among these different susceptible species. The persistently emerging outbreaks of HPAI H5N1 in animals and humans demonstrate the need for comprehensive understanding of this virus and its public health implications, as well as for detailed genomic mapping and characterization. With increasing reports of human infections and the potential for devastating economic consequences in industrial livestock production, it is imperative to deepen analyzes of HPAI H5N1 to effectively respond to the continued spread of the virus in animals, prevent future outbreaks and transmission to the people. To provide a comprehensive analysis of the emergence and spread of HPAI H5N1, it is necessary to examine the genetic characteristics, mutations in the viral genome, and the evolution of the virus, and to evaluate the effectiveness of current prevention and control strategies.

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.

A single immunization with H5N1 virus-like particle vaccine protects chickens against divergent H5N1 influenza viruses and vaccine efficacy is determined by adjuvant and dosage

ABSTRACT The H5N1 subtype highly pathogenic avian influenza virus (HPAIV) reveals high variability and threatens poultry production and public health. To prevent the spread of H5N1 HPAIV, we developed an H5N1 virus-like particle (VLP) vaccine based on the insect cell-baculovirus expression system. Single immunization of the H5N1 VLP vaccines induced high levels of HI antibody titres and provided effective protection against homologous virus challenge comparable to the commercial inactivated vaccine. Meanwhile, we assessed the relative efficacy of different adjuvants by carrying out a head-to-head comparison of the adjuvants ISA 201 and ISA 71 and evaluated whether the two adjuvants could induce broadly protective immunity. The ISA 71 adjuvanted vaccine induced significantly higher levels of Th1 and Th2 immune responses and provided superior cross-protection against antigenically divergent H5N1 virus challenge than the ISA 201 adjuvanted vaccine. Importantly, increasing the vaccine dose could further enhance the cross-protective efficacy of H5N1 VLP vaccine and confer completely sterilizing protection against antigenically divergent H5N1 virus challenge, which was mediated by neutralizing antibodies. Our results suggest that the H5N1 VLP vaccine can provide broad-spectrum protection against divergent H5N1 influenza viruses as determined by adjuvant and vaccine dose.

Pathogenicity in Chickens and Turkeys of a 2021 United States H5N1 Highly Pathogenic Avian Influenza Clade 2.3.4.4b Wild Bird Virus Compared to Two Previous H5N8 Clade 2.3.4.4 Viruses.

Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5 of the Gs/GD/96 lineage remain a major threat to poultry due to endemicity in wild birds. H5N1 HPAIVs from this lineage were detected in 2021 in the United States (U.S.) and since then have infected many wild and domestic birds. We evaluated the pathobiology of an early U.S. H5N1 HPAIV (clade 2.3.4.4b, 2021) and two H5N8 HPAIVs from previous outbreaks in the U.S. (clade 2.3.4.4c, 2014) and Europe (clade 2.3.4.4b, 2016) in chickens and turkeys. Differences in clinical signs, mean death times (MDTs), and virus transmissibility were found between chickens and turkeys. The mean bird infective dose (BID50) of the 2021 H5N1 virus was approximately 2.6 log10 50% embryo infective dose (EID50) in chickens and 2.2 log10 EID50 in turkeys, and the virus transmitted to contact-exposed turkeys but not chickens. The BID50 for the 2016 H5N8 virus was also slightly different in chickens and turkeys (4.2 and 4.7 log10 EID50, respectively); however, the BID50 for the 2014 H5N8 virus was higher for chickens than turkeys (3.9 and ~0.9 log10 EID50, respectively). With all viruses, turkeys took longer to die (MDTs of 2.6-8.2 days for turkeys and 1-4 days for chickens), which increased the virus shedding period and facilitated transmission to contacts.

Mortality in Wild Turkey (Meleagris gallopavo) Associated with Natural Infection with H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Subclade 2.3.4.4.

A Eurasian strain of H5N1 highly pathogenic avian influenza virus (HPAIV) was first detected in North America in December 2021 and has since been confirmed in numerous wild and domestic avian species. In April 2022, 41 Wild Turkeys (Meleagris gallopavo) were found dead in Johnson County, Wyoming, USA adjacent to a property with confirmed HPAIV in a backyard poultry flock. Oropharyngeal swabs were collected from 11 of the 41 turkeys and necropsy was performed on seven. Avian influenza virus RNA was detected in all 11 turkeys by real-time reverse-transcription PCR. Acute, multiorgan necrosis was observed grossly and identified in all seven turkeys evaluated by histopathology, most consistently in the lung, spleen, liver, gastrointestinal tract, and gonads. Lesions indicate high virulence of subclade 2.3.4.4b H5N1 HPAIV in Wild Turkeys, with infections presenting as clusters of acute mortality. Although documented cases of HPAIV in Wild Turkeys are rare, these findings signify a risk of spillback from domestic poultry, which may be heightened by the recent rise in backyard poultry ownership and the use of peridomestic habitat by wild birds. Additional research is needed to better understand the risk of disease transmission at the interface of Wild Turkeys and backyard poultry and the potential conservation and management implications of HPAIV in wild gallinaceous birds.