BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Highly pathogenic avian influenza A(H5N1) virus infections on fur farms connected to mass mortalities of black-headed gulls, Finland, July to October 2023.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe 2020-2023. In July 2023, HPAI A(H5N1) was detected on 27 fur farms in Finland. In total, infections in silver and blue foxes, American minks and raccoon dogs were confirmed by RT-PCR. The pathological findings in the animals include widespread inflammatory lesions in the lungs, brain and liver, indicating efficient systemic dissemination of the virus. Phylogenetic analysis of Finnish A(H5N1) strains from fur animals and wild birds has identified three clusters (Finland I-III), and molecular analyses revealed emergence of mutations known to facilitate viral adaptation to mammals in the PB2 and NA proteins. Findings of avian influenza in fur animals were spatially and temporally connected with mass mortalities in wild birds. The mechanisms of virus transmission within and between farms have not been conclusively identified, but several different routes relating to limited biosecurity on the farms are implicated. The outbreak was managed in close collaboration between animal and human health authorities to mitigate and monitor the impact for both animal and human health.

Evidence for Different Virulence Determinants and Host Response after Infection of Turkeys and Chickens with Highly Pathogenic H7N1 Avian Influenza Virus.

Wild birds are the reservoir for all avian influenza viruses (AIV). In poultry, the transition from low pathogenic (LP) AIV of H5 and H7 subtypes to highly pathogenic (HP) AIV is accompanied mainly by changing the hemagglutinin (HA) monobasic cleavage site (CS) to a polybasic motif (pCS). Galliformes, including turkeys and chickens, succumb with high morbidity and mortality to HPAIV infections, although turkeys appear more vulnerable than chickens. Surprisingly, the genetic determinants for virulence and pathogenesis of HPAIV in turkeys are largely unknown. Here, we determined the genetic markers for virulence and transmission of HPAIV H7N1 in turkeys, and we explored the host responses in this species compared to those of chickens. We found that recombinant LPAIV H7N1 carrying pCS was avirulent in chickens but exhibited high virulence in turkeys, indicating that virulence determinants vary in these two galliform species. A transcriptome analysis indicated that turkeys mount a different host response than do chickens, particularly from genes involved in RNA metabolism and the immune response. Furthermore, we found that the HA glycosylation at residue 123, acquired by LP viruses shortly after transmission from wild birds and preceding the transition from LP to HP, had a role in virus fitness and virulence in chickens, though it was not a prerequisite for high virulence in turkeys. Together, these findings indicate variable virulence determinants and host responses in two closely related galliformes, turkeys and chickens, after infection with HPAIV H7N1. These results could explain the higher vulnerability to HPAIV of turkeys compared to chickens. IMPORTANCE Infection with HPAIV in chickens and turkeys, two closely related galliform species, results in severe disease and death. Although the presence of a polybasic cleavage site (pCS) in the hemagglutinin of AIV is a major virulence determinant for the transition of LPAIV to HPAIV, there are knowledge gaps on the genetic determinants (including pCS) and the host responses in turkeys compared to chickens. Here, we found that the pCS alone was sufficient for the transformation of a LP H7N1 into a HPAIV in turkeys but not in chickens. We also noticed that turkeys exhibited a different host response to an HPAIV infection, namely, a widespread downregulation of host gene expression associated with protein synthesis and the immune response. These results are important for a better understanding of the evolution of HPAIV from LPAIV and of the different outcomes and the pathomechanisms of HPAIV infections in chickens and turkeys.

Highly pathogenic avian influenza A(H5N1) virus infection in farmed minks, Spain, October 2022.

In October 2022, an outbreak in Europe of highly pathogenic avian influenza (HPAI) A(H5N1) in intensively farmed minks occurred in northwest Spain. A single mink farm hosting more than 50,000 minks was involved. The identified viruses belong to clade 2.3.4.4b, which is responsible of the ongoing epizootic in Europe. An uncommon mutation (T271A) in the PB2 gene with potential public health implications was found. Our investigations indicate onward mink transmission of the virus may have occurred in the affected farm.

Outbreak of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in cats, Poland, June to July 2023.

BackgroundOver a 3-week period in late June/early July 2023, Poland experienced an outbreak caused by highly pathogenic avian influenza (HPAI) A(H5N1) virus in cats.AimThis study aimed to characterise the identified virus and investigate possible sources of infection.MethodsWe performed next generation sequencing and phylogenetic analysis of detected viruses in cats.ResultsWe sampled 46 cats, and 25 tested positive for avian influenza virus. The identified viruses belong to clade 2.3.4.4b, genotype CH (H5N1 A/Eurasian wigeon/Netherlands/3/2022-like). In Poland, this genotype was responsible for several poultry outbreaks between December 2022 and January 2023 and has been identified only sporadically since February 2023. Viruses from cats were very similar to each other, indicating one common source of infection. In addition, the most closely related virus was detected in a dead white stork in early June. Influenza A(H5N1) viruses from cats possessed two amino acid substitutions in the PB2 protein (526R and 627K) which are two molecular markers of virus adaptation in mammals. The virus detected in the white stork presented one of those mutations (627K), which suggests that the virus that had spilled over to cats was already partially adapted to mammalian species.ConclusionThe scale of HPAI H5N1 virus infection in cats in Poland is worrying. One of the possible sources seems to be poultry meat, but to date no such meat has been identified with certainty. Surveillance should be stepped up on poultry, but also on certain species of farmed mammals kept close to infected poultry farms.

Intercontinental Spread of Eurasian Highly Pathogenic Avian Influenza A(H5N1) to Senegal.

In January 2021, Senegal reported the emergence of highly pathogenic avian influenza virus A(H5N1), which was detected on a poultry farm in Thies, Senegal, and in great white pelicans in the Djoudj National Bird Sanctuary. We report evidence of new transcontinental spread of H5N1 from Europe toward Africa.

Early start of seasonal transmission and co-circulation of West Nile virus lineage 2 and a newly introduced lineage 1 strain, northern Italy, June 2022.

In spring 2022, Europe faced an unprecedented heatwave, increasing the risk of West Nile virus (WNV) outbreaks. As early as 7 June 2022, WNV was detected in Culex mosquitoes in northern Italy, and - in the following days - in two blood donors, a patient with encephalitis, wild birds and additional mosquito pools. Genome sequencing demonstrated co-circulation of WNV lineage 2 and a newly introduced WNV lineage 1, which was discovered in the region in 2021.

Incorporating heterogeneous sampling probabilities in continuous phylogeographic inference - Application to H5N1 spread in the Mekong region.

MOTIVATION: The potentially low precision associated with the geographic origin of sampled sequences represents an important limitation for spatially explicit (i.e. continuous) phylogeographic inference of fast-evolving pathogens such as RNA viruses. A substantial proportion of publicly available sequences is geo-referenced at broad spatial scale such as the administrative unit of origin, rather than more precise locations (e.g. geographic coordinates). Most frequently, such sequences are either discarded prior to continuous phylogeographic inference or arbitrarily assigned to the geographic coordinates of the centroid of their administrative area of origin for lack of a better alternative. RESULTS: We here implement and describe a new approach that allows to incorporate heterogeneous prior sampling probabilities over a geographic area. External data, such as outbreak locations, are used to specify these prior sampling probabilities over a collection of sub-polygons. We apply this new method to the analysis of highly pathogenic avian influenza H5N1 clade data in the Mekong region. Our method allows to properly include, in continuous phylogeographic analyses, H5N1 sequences that are only associated with large administrative areas of origin and assign them with more accurate locations. Finally, we use continuous phylogeographic reconstructions to analyse the dispersal dynamics of different H5N1 clades and investigate the impact of environmental factors on lineage dispersal velocities. AVAILABILITY AND IMPLEMENTATION: Our new method allowing heterogeneous sampling priors for continuous phylogeographic inference is implemented in the open-source multi-platform software package BEAST 1.10. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular, antigenic, and pathogenic characterization of H5N8 highly pathogenic avian influenza viruses isolated in the Democratic Republic of Congo in 2017.

In May 2017, high mortality of chickens and Muscovy ducks due to the H5N8 highly pathogenic avian influenza virus (HPAIV) was reported in the Democratic Republic of Congo (DR Congo). In this study, we assessed the molecular, antigenic, and pathogenic features in poultry of the H5N8 HPAIV from the 2017 Congolese outbreaks. Phylogenetic analysis of the eight viral gene segments revealed that all 12 DR Congo isolates clustered in clade 2.3.4.4B together with other H5N8 HPAIVs isolated in Africa and Eurasia, suggesting a possible common origin of these viruses. Antigenically, a slight difference was observed between the Congolese isolates and a representative virus from group C in the same clade. After intranasal inoculation with a representative DR Congo virus, high pathogenicity was observed in chickens and Muscovy ducks but not in Pekin ducks. Viral replication was higher in chickens than in Muscovy duck and Pekin duck organs; however, neurotropism was pronounced in Muscovy ducks. Our data confirmed the high pathogenicity of the DR Congo virus in chickens and Muscovy ducks, as observed in the field. National awareness and strengthening surveillance in the region are needed to better control HPAIVs.

Avian influenza H9N2 subtype in Ghana: virus characterization and evidence of co-infection.

Between November 2017 and February 2018, Ghanaian poultry producers reported to animal health authorities a dramatic increase in mortality rate and a relevant drop in egg production in several layer hen farms. Laboratory investigations revealed that the farms had been infected by the H9N2 influenza subtype. Virological and molecular characterization of the viruses identified in Ghana is described here for the first time. Whole genome analysis showed that the viruses belong to the G1-lineage and cluster with viruses identified in North and West Africa. The low pathogenicity of the virus was confirmed by the intravenous pathogenicity index assay. Further investigations revealed co-infection with infectious bronchitis virus of the GI-19 lineage, which very likely explained the severity of the disease observed during the outbreaks. The H9N2 outbreaks in Ghana highlight the importance of performing a differential diagnosis and an in-depth characterization of emerging viruses. In addition, the detection of a potentially zoonotic subtype, such as the H9N2, in a region where highly pathogenic avian influenza H5Nx is currently circulating highlights the urgency of implementing enhanced monitoring strategies and supporting improved investments in regional diagnostic technologies. RESEARCH HIGHLIGHTS Influenza A H9N2 subtype was detected in layer hens in Ghana in 2017-2018 Whole genome characterization of seven H9N2 viruses was performed Phylogenetic trees revealed that the H9N2 viruses belong to the G1 lineage The HA protein possesses the amino acid mutations 226L and 155T Co-infection with infectious bronchitis virus of the GI-19 lineage was identified.

Highly Pathogenic Avian Influenza A(H5N8) Virus, Democratic Republic of the Congo, 2017.

In 2017, highly pathogenic avian influenza A(H5N8) virus was detected in poultry in the Democratic Republic of the Congo. Whole-genome phylogeny showed the virus clustered with H5N8 clade 2.3.4.4B strains from birds in central and southern Asia. Emergence of this virus in central Africa represents a threat for animal health and food security.

Highly Pathogenic Avian Influenza A(H5N8) Virus, Cameroon, 2017.

Highly pathogenic avian influenza A(H5N8) viruses of clade 2.3.4.4 spread into West Africa in late 2016 during the autumn bird migration. Genetic characterization of the complete genome of these viruses detected in wild and domestic birds in Cameroon in January 2017 demonstrated the occurrence of multiple virus introductions.

Comparison of 2016-17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe.

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016-17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016-17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005-06 and 2016-17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016-17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control.

Highly pathogenic avian influenza A/H5N1 Clade 2.3.2.1c virus in poultry in Cameroon, 2016-2017.

In May 2016, highly pathogenic avian influenza virus of the subtype A/H5N1 was detected in Cameroon in an industrial poultry farm at Mvog-Betsi, Yaoundé (Centre region), with a recorded sudden increase of deaths among chickens, and an overall mortality rate of 75%. The virus spread further and caused new outbreaks in some parts of the country. In total, 21 outbreaks were confirmed from May 2016 to March 2017 (six in the Centre, six in the West, eight in the South and one in the Adamaoua regions). This resulted in an estimated total loss of 138,252 birds (44,451 deaths due to infection and 93,801 stamped out). Only domestic birds (chickens, ducks and geese) were affected in farms as well as in poultry markets. The outbreaks occurred in three waves, the first from May to June 2016, the second in September 2016 and the last wave in March 2017. The topology of the phylogeny based on the haemagglutinin gene segment indicated that the causative H5N1 viruses fall within the genetic clade 2.3.2.1c, within the same group as the A/H5N1 viruses collected in Niger in 2015 and 2016. More importantly, the gene constellation of four representative viruses showed evidence of H5N1/H9N2 intra-clade reassortment. Additional epidemiological and genetic data from affected countries in West Africa are needed to better trace the origin, spread and evolution of A/H5N1 in Cameroon. RESEARCH HIGHLIGHTS HPAI A/H5N1 was detected in May 2016 in domestic chickens in Yaoundé-Cameroon. Twenty-one outbreaks in total were confirmed from May 2016 to March 2017. The causative H5N1 viruses fall within the genetic clade 2.3.2.1c. The viral gene constellation showed evidence of H5N1/H9N2 intra-clade reassortment.

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody.

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.

Influenza A(H9N2) Virus, Burkina Faso.

We identified influenza A(H9N2) virus G1 lineage in poultry in Burkina Faso. Urgent actions are needed to raise awareness about the risk associated with spread of this zoonotic virus subtype in the area and to construct a strategy for effective prevention and control of influenza caused by this virus.

Genetic Diversity of Highly Pathogenic Avian Influenza A(H5N8/H5N5) Viruses in Italy, 2016-17.

In winter 2016-17, highly pathogenic avian influenza A(H5N8) and A(H5N5) viruses of clade 2.3.4.4 were identified in wild and domestic birds in Italy. We report the occurrence of multiple introductions and describe the identification in Europe of 2 novel genotypes, generated through multiple reassortment events.

Unexpected Interfarm Transmission Dynamics during a Highly Pathogenic Avian Influenza Epidemic.

UNLABELLED: Next-generation sequencing technology is now being increasingly applied to study the within- and between-host population dynamics of viruses. However, information on avian influenza virus evolution and transmission during a naturally occurring epidemic is still limited. Here, we use deep-sequencing data obtained from clinical samples collected from five industrial holdings and a backyard farm infected during the 2013 highly pathogenic avian influenza (HPAI) H7N7 epidemic in Italy to unravel (i) the epidemic virus population diversity, (ii) the evolution of virus pathogenicity, and (iii) the pathways of viral transmission between different holdings and sheds. We show a high level of genetic diversity of the HPAI H7N7 viruses within a single farm as a consequence of separate bottlenecks and founder effects. In particular, we identified the cocirculation in the index case of two viral strains showing a different insertion at the hemagglutinin cleavage site, as well as nine nucleotide differences at the consensus level and 92 minority variants. To assess interfarm transmission, we combined epidemiological and genetic data and identified the index case as the major source of the virus, suggesting the spread of different viral haplotypes from the index farm to the other industrial holdings, probably at different time points. Our results revealed interfarm transmission dynamics that the epidemiological data alone could not unravel and demonstrated that delay in the disease detection and stamping out was the major cause of the emergence and the spread of the HPAI strain. IMPORTANCE: The within- and between-host evolutionary dynamics of a highly pathogenic avian influenza (HPAI) strain during a naturally occurring epidemic is currently poorly understood. Here, we perform for the first time an in-depth sequence analysis of all the samples collected during a HPAI epidemic and demonstrate the importance to complement outbreak investigations with genetic data to reconstruct the transmission dynamics of the viruses and to evaluate the within- and between-farm genetic diversity of the viral population. We show that the evolutionary transition from the low pathogenic form to the highly pathogenic form occurred within the first infected flock, where we identified haplotypes with hemagglutinin cleavage site of different lengths. We also identify the index case as the major source of virus, indicating that prompt application of depopulation measures is essential to limit virus spread to other farms.

Genetically Different Highly Pathogenic Avian Influenza A(H5N1) Viruses in West Africa, 2015.

To trace the evolution of highly pathogenic influenza A(H5N1) virus in West Africa, we sequenced genomes of 43 viruses collected during 2015 from poultry and wild birds in 5 countries. We found 2 co-circulating genetic groups within clade 2.3.2.1c. Mutations that may increase adaptation to mammals raise concern over possible risk for humans.