Rapid mortality in captive bush dogs (Speothos venaticus) caused by influenza A of avian origin (H5N1) at a wildlife collection in the United Kingdom.

Europe has suffered unprecedented epizootics of high pathogenicity avian influenza (HPAI) clade 2.3.4.4b H5N1 since Autumn 2021. As well as impacting upon commercial and wild avian species, the virus has also infected mammalian species more than ever observed previously. Mammalian species involved in spill over events have primarily been scavenging terrestrial carnivores and farmed mammalian species although marine mammals have also been affected. Alongside reports of detections of mammalian species found dead through different surveillance schemes, several mass mortality events have been reported in farmed and wild animals. In November 2022, an unusual mortality event was reported in captive bush dogs (Speothos venaticus) with clade 2.3.4.4b H5N1 HPAIV of avian origin being the causative agent. The event involved an enclosure of 15 bush dogs, 10 of which succumbed during a nine-day period with some dogs exhibiting neurological disease. Ingestion of infected meat is proposed as the most likely infection route.

Avian flu: «for whom the bell tolls¼?

The family Orthomyxoviridae consists of 9 genera, including Alphainfluenza virus, which contains avian influenza viruses. In two subtypes H5 and H7 besides common low-virulent strains, a specific type of highly virulent avian virus have been described to cause more than 60% mortality among domestic birds. These variants of influenza virus are usually referred to as «avian influenza virus¼. The difference between high (HPAI) and low (LPAI) virulent influenza viruses is due to the structure of the arginine-containing proteolytic activation site in the hemagglutinin (HA) protein. The highly virulent avian influenza virus H5 was identified more than 100 years ago and during this time they cause outbreaks among wild and domestic birds on all continents and only a few local episodes of the disease in humans have been identified in XXI century. Currently, a sharp increase in the incidence of highly virulent virus of the H5N1 subtype (clade h2.3.4.4b) has been registered in birds on all continents, accompanied by the transmission of the virus to various species of mammals. The recorded global mortality rate among wild, domestic and agricultural birds from H5 subtype is approaching to the level of 1 billion cases. A dangerous epidemic factor is becoming more frequent outbreaks of avian influenza with high mortality among mammals, in particular seals and marine lions in North and South America, minks and fur-bearing animals in Spain and Finland, domestic and street cats in Poland. H5N1 avian influenza clade h2.3.4.4b strains isolated from mammals have genetic signatures of partial adaptation to the human body in the PB2, NP, HA, NA genes, which play a major role in regulating the aerosol transmission and the host range of the virus. The current situation poses a real threat of pre-adaptation of the virus in mammals as intermediate hosts, followed by the transition of the pre-adapted virus into the human population with catastrophic consequences.

Epitopes in the HA and NA of H5 and H7 avian influenza viruses that are important for antigenic drift.

Avian influenza viruses evolve antigenically to evade host immunity. Two influenza A virus surface glycoproteins, the haemagglutinin and neuraminidase, are the major targets of host immunity and undergo antigenic drift in response to host pre-existing humoral and cellular immune responses. Specific sites have been identified as important epitopes in prominent subtypes such as H5 and H7, which are of animal and public health significance due to their panzootic and pandemic potential. The haemagglutinin is the immunodominant immunogen, it has been extensively studied, and the antigenic reactivity is closely monitored to ensure candidate vaccine viruses are protective. More recently, the neuraminidase has received increasing attention for its role as a protective immunogen. The neuraminidase is expressed at a lower abundance than the haemagglutinin on the virus surface but does elicit a robust antibody response. This review aims to compile the current information on haemagglutinin and neuraminidase epitopes and immune escape mutants of H5 and H7 highly pathogenic avian influenza viruses. Understanding the evolution of immune escape mutants and the location of epitopes is critical for identification of vaccine strains and development of broadly reactive vaccines that can be utilized in humans and animals.

Outbreak of Highly Pathogenic Avian Influenza A(H5N1) Virus in Seals, St. Lawrence Estuary, Quebec, Canada1.

We describe an unusual mortality event caused by a highly pathogenic avian influenza (HPAI) A(H5N1) virus clade 2.3.4.4b involving harbor (Phoca vitulina) and gray (Halichoerus grypus) seals in the St. Lawrence Estuary, Quebec, Canada, in 2022. Fifteen (56%) of the seals submitted for necropsy were considered to be fatally infected by HPAI H5N1 containing fully Eurasian or Eurasian/North American genome constellations. Concurrently, presence of large numbers of bird carcasses infected with HPAI H5N1 at seal haul-out sites most likely contributed to the spillover of infection to the seals. Histologic changes included meningoencephalitis (100%), fibrinosuppurative alveolitis, and multiorgan acute necrotizing inflammation. This report of fatal HPAI H5N1 infection in pinnipeds in Canada raises concerns about the expanding host of this virus, the potential for the establishment of a marine mammal reservoir, and the public health risks associated with spillover to mammals.Nous décrivons un événement de mortalité inhabituelle causé par un virus de l'influenza aviaire hautement pathogène A(H5N1) clade 2.3.4.4b chez des phoques communs (Phoca vitulina) et gris (Halichoerus grypus) dans l'estuaire du Saint-Laurent au Québec, Canada, en 2022. Quinze (56%) des phoques soumis pour nécropsie ont été considérés comme étant fatalement infectés par le virus H5N1 de lignées eurasiennes ou de réassortiment eurasiennes/nord-américaines. Un grand nombre simultané de carcasses d'oiseaux infectés par le H5N1 sur les sites d'échouement a probablement contribué à la contamination de ces phoques. Les changements histologiques associés à cette infection incluaient : méningo-encéphalite (100%), alvéolite fibrinosuppurée et inflammation nécrosante aiguë multi-organique. Cette documentation soulève des préoccupations quant à l'émergence de virus mortels, à la possibilité d'établissement de réservoirs chez les mammifères marins, et aux risques pour la santé publique associés aux propagations du virus chez les mammifères.

Development of a nucleoside-modified mRNA vaccine against clade 2.3.4.4b H5 highly pathogenic avian influenza virus.

mRNA lipid nanoparticle (LNP) vaccines would be useful during an influenza virus pandemic since they can be produced rapidly and do not require the generation of egg-adapted vaccine seed stocks. Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. Here, we generate an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. The H5 mRNA-LNP vaccine elicits strong T cell and antibody responses in female mice, including neutralizing antibodies and broadly-reactive anti-HA stalk antibodies. The H5 mRNA-LNP vaccine elicits antibodies at similar levels compared to whole inactivated vaccines in female mice with and without prior H1N1 exposures. Finally, we find that the H5 mRNA-LNP vaccine is immunogenic in male ferrets and prevents morbidity and mortality of animals following 2.3.4.4b H5N1 challenge. Together, our data demonstrate that a monovalent mRNA-LNP vaccine expressing 2.3.4.4b H5 is immunogenic and protective in pre-clinical animal models.

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.

Efficacy of live and inactivated recombinant Newcastle disease virus vaccines expressing clade 2.3.4.4b H5 hemagglutinin against H5N1 highly pathogenic avian influenza in SPF chickens, Broilers, and domestic ducks.

A Newcastle disease virus (NDV)-vectored vaccine expressing clade 2.3.4.4b H5 Hemagglutinin was developed and assessed for efficacy against H5N1 highly pathogenic avian influenza (HPAI) in specific pathogen-free (SPF) chickens, broilers, and domestic ducks. In SPF chickens, the live recombinant NDV-vectored vaccine, rK148/22-H5, achieved complete survival against HPAI and NDV challenges and significantly reduced viral shedding. Notably, the live rK148/22-H5 vaccine conferred good clinical protection in broilers despite the presence of maternally derived antibodies. Good clinical protection was observed in domestic ducks, with decreased viral shedding. It demonstrated complete survival and reduced cloacal viral shedding when used as an inactivated vaccine from SPF chickens. The rK148/22-H5 vaccine is potentially a viable and supportive option for biosecurity measure, effectively protecting in chickens against the deadly clade 2.3.4.4b H5 HPAI and NDV infections. Furthermore, it aligns with the strategy of Differentiating Infected from Vaccinated Animals (DIVA).

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.

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

ABSTRACTRapid 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 mammalian 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 adaptations such as PB2-E627 K 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.

Cross-protective efficacy and safety of an adenovirus-based universal influenza vaccine expressing nucleoprotein, hemagglutinin, and the ectodomain of matrix protein 2.

It is necessary to develop universal vaccines that act broadly and continuously to combat regular seasonal epidemics of influenza and rare pandemics. The aim of this study was to find the optimal dose regimen for the efficacy and safety of a mixture of previously developed recombinant adenovirus-based vaccines that expressed influenza nucleoprotein, hemagglutinin, and ectodomain of matrix protein 2 (rAd/NP and rAd/HA-M2e). The vaccine efficacy and safety were measured in the immunized mice with the mixture of rAd/NP and rAd/HA-M2e intranasally or intramuscularly. The minimum dose that would be efficacious in a single intranasal administration of the vaccine mixture and cross-protective efficacy against various influenza strains were examined. In addition, the immune responses that may affect the cross-protective efficacy were measured. We found that intranasal administration is an optimal route for 107 pfu of vaccine mixture, which is effective against pre-existing immunity against adenovirus. In a study to find the minimum dose with vaccine efficacy, the 106 pfu of vaccine mixture showed higher antibody titers to the nucleoprotein than did the same dose of rAd/NP alone in the serum of immunized mice. The 106 pfu of vaccine mixture overcame the morbidity and mortality of mice against the lethal dose of pH1N1, H3N2, and H5N1 influenza infections. No noticeable side effects were observed in single and repeated toxicity studies. We found that the mucosal administration of adenovirus-based universal influenza vaccine has both efficacy and safety, and can provide cross-protection against various influenza infections even at doses lower than those previously known to be effective.

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.

Iceland: an underestimated hub for the spread of high-pathogenicity avian influenza viruses in the North Atlantic.

High-pathogenicity avian influenza viruses (HPAIVs) of the goose/Guangdong lineage are enzootically circulating in wild bird populations worldwide. This increases the risk of entry into poultry production and spill-over to mammalian species, including humans. Better understanding of the ecological and epizootiological networks of these viruses is essential to optimize mitigation measures. Based on full genome sequences of 26 HPAIV samples from Iceland, which were collected between spring and autumn 2022, as well as 1 sample from the 2023 summer period, we show that 3 different genotypes of HPAIV H5N1 clade 2.3.4.4b were circulating within the wild bird population in Iceland in 2022. Furthermore, in 2023 we observed a novel introduction of HPAIV H5N5 of the same clade to Iceland. The data support the role of Iceland as an utmost northwestern distribution area in Europe that might act also as a potential bridging point for intercontinental spread of HPAIV across the North Atlantic.

A combination influenza mRNA vaccine candidate provided broad protection against diverse influenza virus challenge.

Influenza viruses present a significant threat to global health. The production of a universal vaccine is considered essential due to the ineffectiveness of current seasonal influenza vaccines against mutant strains. mRNA technology offers new prospects in vaccinology, with various candidates for different infectious diseases currently in development and testing phases. In this study, we encapsulated a universal influenza mRNA vaccine. The vaccine encoded influenza hemagglutinin (HA), nucleoprotein (NP), and three tandem repeats of matrix protein 2 (3M2e). Twice-vaccinated mice exhibited strong humoral and cell-mediated immune responses in vivo. Notably, these immune responses led to a significant reduction in viral load of the lungs in challenged mice, and also conferred protection against future wild-type H1N1, H3N2, or H5N1 influenza virus challenges. Our findings suggest that this mRNA-universal vaccine strategy for influenza virus may be instrumental in mitigating the impact of future influenza pandemics.

Structures of H5N1 influenza polymerase with ANP32B reveal mechanisms of genome replication and host adaptation.

Avian influenza A viruses (IAVs) pose a public health threat, as they are capable of triggering pandemics by crossing species barriers. Replication of avian IAVs in mammalian cells is hindered by species-specific variation in acidic nuclear phosphoprotein 32 (ANP32) proteins, which are essential for viral RNA genome replication. Adaptive mutations enable the IAV RNA polymerase (FluPolA) to surmount this barrier. Here, we present cryo-electron microscopy structures of monomeric and dimeric avian H5N1 FluPolA with human ANP32B. ANP32B interacts with the PA subunit of FluPolA in the monomeric form, at the site used for its docking onto the C-terminal domain of host RNA polymerase II during viral transcription. ANP32B acts as a chaperone, guiding FluPolA towards a ribonucleoprotein-associated FluPolA to form an asymmetric dimer-the replication platform for the viral genome. These findings offer insights into the molecular mechanisms governing IAV genome replication, while enhancing our understanding of the molecular processes underpinning mammalian adaptations in avian-origin FluPolA.

Risk assessment of a highly pathogenic H5N1 influenza virus from mink.

Outbreaks of highly pathogenic H5N1 clade 2.3.4.4b viruses in farmed mink and seals combined with isolated human infections suggest these viruses pose a pandemic threat. To assess this threat, using the ferret model, we show an H5N1 isolate derived from mink transmits by direct contact to 75% of exposed ferrets and, in airborne transmission studies, the virus transmits to 37.5% of contacts. Sequence analyses show no mutations were associated with transmission. The H5N1 virus also has a low infectious dose and remains virulent at low doses. This isolate carries the adaptive mutation, PB2 T271A, and reversing this mutation reduces mortality and airborne transmission. This is the first report of a H5N1 clade 2.3.4.4b virus exhibiting direct contact and airborne transmissibility in ferrets. These data indicate heightened pandemic potential of the panzootic H5N1 viruses and emphasize the need for continued efforts to control outbreaks and monitor viral evolution.

Blowflies are potential vector for avian influenza virus at enzootic area in Japan.

High pathogenicity avian influenza (HPAI) poses a significant threat to both domestic and wild birds globally. The avian influenza virus, known for environmental contamination and subsequent oral infection in birds, necessitates careful consideration of alternative introduction routes during HPAI outbreaks. This study focuses on blowflies (genus Calliphora), in particular Calliphora nigribarbis, attracted to decaying animals and feces, which migrate to lowland areas of Japan from northern or mountainous regions in early winter, coinciding with HPAI season. Our investigation aims to delineate the role of blowflies as HPAI vectors by conducting a virus prevalence survey in a wild bird HPAI-enzootic area. In December 2022, 648 Calliphora nigribarbis were collected. Influenza virus RT-PCR testing identified 14 virus-positive samples (2.2% prevalence), with the highest occurrence observed near the crane colony (14.9%). Subtyping revealed the presence of H5N1 and HxN1 in some samples. Subsequent collections in December 2023 identified one HPAI virus-positive specimen from 608 collected flies in total, underscoring the potential involvement of blowflies in HPAI transmission. Our observations suggest C. nigribarbis may acquire the HPAI virus from deceased wild birds directly or from fecal materials from infected birds, highlighting the need to add blowflies as a target of HPAI vector control.

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.

Highly pathogenic avian influenza A(H5N1) virus in a common bottlenose dolphin (Tursiops truncatus) in Florida.

Since late 2021, highly pathogenic avian influenza (HPAI) viruses of A/goose/Guangdong/1/1996 (H5N1) lineage have caused widespread mortality in wild birds and poultry in the United States. Concomitant with the spread of HPAI viruses in birds are increasing numbers of mammalian infections, including wild and captive mesocarnivores and carnivores with central nervous system involvement. Here we report HPAI, A(H5N1) of clade 2.3.4.4b, in a common bottlenose dolphin (Tursiops truncatus) from Florida, United States. Pathological findings include neuronal necrosis and inflammation of the brain and meninges, and quantitative real time RT-PCR reveal the brain carried the highest viral load. Virus isolated from the brain contains a S246N neuraminidase substitution which leads to reduced inhibition by neuraminidase inhibitor oseltamivir. The increased prevalence of A(H5N1) viruses in atypical avian hosts and its cross-species transmission into mammalian species highlights the public health importance of continued disease surveillance and biosecurity protocols.

In Ovo Models to Predict Virulence of Highly Pathogenic Avian Influenza H5-Viruses for Chickens and Ducks.

Highly pathogenic avian influenza (HPAI) H5-viruses are circulating in wild birds and are repeatedly introduced to poultry causing outbreaks in the Netherlands since 2014. The largest epizootic ever recorded in Europe was caused by HPAI H5N1 clade 2.3.4.4b viruses in the period 2021-2022. The recent H5-clade 2.3.4.4 viruses were found to differ in their virulence for chickens and ducks. Viruses causing only mild disease may remain undetected, increasing the risk of virus spread to other farms, wild birds and mammals. We developed in ovo models to determine the virulence of HPAI viruses for chickens and ducks, which are fast and have low costs. The virulence of five contemporary H5-viruses was compared studying replication rate, average time to death and virus spread in the embryo. Remarkable differences in virulence were observed between H5-viruses and between poultry species. The H5N1-2021 virus was found to have a fast replication rate in both the chicken and duck in ovo models, but a slower systemic virus dissemination compared to three other H5-clade 2.3.4.4b viruses. The results show the potential of in ovo models to quickly determine the virulence of novel HPAI viruses, and study potential virulence factors which can help to better guide the surveillance in poultry.