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BACKGROUND: Since 2005, highly pathogenic avian influenza A H5N1 viruses have spread from Asia worldwide, infecting poultry, humans and wild birds. Subsequently, global interest in avian influenza (AI) surveillance increased. OBJECTIVES: Mongolia presents an opportunity to study viruses in wild birds because the country has very low densities of domestic poultry and supports large concentrations of migratory water birds. METHODS: We conducted AI surveillance in Mongolia over two time periods, 2009-2013 and 2016-2018, utilizing environmental fecal sampling. Fresh fecal samples were collected from water bird congregation sites. Hemagglutinin (HA) and neuraminidase (NA) subtypes of positive samples were identified through viral isolation or molecular assays, with pathogenicity determined by HA subtype or sequencing the HA cleavage site. RESULTS: A total of 10,222 samples were collected. Of these, 7,025 fecal samples were collected from 2009 to 2013, and 3,197 fecal samples were collected from 2016 to 2018. Testing revealed 175 (1.7%) positive samples for low-pathogenicity influenza A, including 118 samples from 2009 to 2013 (1.7%) and 57 samples from 2016 to 2018 (1.8%). HA and NA subtyping of all positives identified 11 subtypes of HA and nine subtypes of NA in 29 different combinations. Within periods, viruses were detected more frequently during the fall season than in the early summer. CONCLUSION: Mongolias critical wild bird habitat is positioned as a crossroad of multiple migratory flyways. Our work demonstrates the feasibility of using an affordable environmental fecal sampling approach for AI surveillance and contributes to understanding the prevalence and ecology of low-pathogenicity avian influenza viruses in this important location, where birds from multiple flyways mix.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10−7 ng/μL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian influenza (HPAI) viruses have potential to cross species barriers and cause pandemics. Since 2022, HPAI A(H5N1) belonging to the goose/Guangdong 2.3.4.4b hemagglutinin phylogenetic clade have infected poultry, wild birds, and mammals across North America. Continued circulation in birds and infection of multiple mammalian species with strains possessing adaptation mutations increase the risk for infection and subsequent reassortment with influenza A viruses endemic in swine. We assessed the susceptibility of swine to avian and mammalian HPAI H5N1 clade 2.3.4.4b strains using a pathogenesis and transmission model. All strains replicated in the lung of pigs and caused lesions consistent with influenza A infection. However, viral replication in the nasal cavity and transmission was only observed with mammalian isolates. Mammalian adaptation and reassortment may increase the risk for incursion and transmission of HPAI viruses in feral, backyard, or commercial swine.

Introduction: During the outbreak of avian influenza, A (H5N1) (IA) in wild and domestic birds recorded in January 2023, the epidemiological alert has been extended due to its potential contagion to humans, particularly in those exposed occupational groups. Objective: to identify the primary occupational risk groups, as well as the preventive, safety, and control measures against IA intended or implemented in these positions. Material and methods: A systematic search was conducted in Pubmed, Scopus, Web of science, Scielo and literature databases. Scientific articles, normative documents, and technical reports identifying vulnerable occupational groups and preventive measures against IA were included. Two authors conducted a full-text review, extracting information independently, and findings were summarized narratively. Results: A total of 5518 documents were identified, and 30 reports were included. 20% of the reports were published in 2023, 13/30 were affiliated to a university institution. Occupationally exposed groups were identified both directly and indirectly. 63.3% of reports identified breeders, poultry farmers and sellers as the most concerning occupational group, while 60% identified biosecurity practices (use of PPE, handwashing) as the primary measure against IA, followed by strategies such as education (training and capacity-building). Conclusion: Occupational groups of interest were identified, primarily those involved in sales, commerce, and the handling of bird waste with potential exposure to IA. Furthermore, the maintenance of biosecurity measures, cleaning-disinfection practices, and educational strategies in workplace settings are recommended.

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses (lineage 2.3.4.4b) are rapidly invading the Americas, threatening wildlife, poultry, and potentially evolving into the next global pandemic. In November 2022 HPAI arrived in Peru, triggering massive pelican and sea lion die-offs. We report genomic characterization of HPAI/H5N1 in five species of marine mammals and seabirds (dolphins, sea lions, sanderlings, pelicans and cormorants). Peruvian viruses belong to lineage 2.3.4.4b, but they are 4:4 reassortants where 4 genomic segments (PA, HA, NA and MP) position within the Eurasian lineage that initially entered North America from Eurasia, while the other 4 genomic segments (PB2, PB1, NP and NS) position within the American lineage (clade C) that circulated in North America. These viruses are rapidly accruing mutations, including mutations of concern, that warrant further examination and highlight an urgent need for active local surveillance to manage outbreaks and limit spillover into other species, including humans.

Avian influenza is caused by avian influenza virus infection; the H5N1 avian influenza virus is a highly pathogenic subtype, affecting poultry and human health. Since the discovery of the highly pathogenic subtype of the H5N1 avian influenza virus, it has caused enormous losses to the poultry farming industry. It was recently found that the H5N1 avian influenza virus tends to spread among mammals. Therefore, early rapid detection methods are highly significant for effectively preventing the spread of H5N1. This paper discusses the detection technologies used in the detection of the H5N1 avian influenza virus, including serological detection technology, immunological detection technology, molecular biology detection technology, genetic detection technology, and biosensors. Comparisons of these detection technologies were analyzed, aiming to provide some recommendations for the detection of the H5N1 avian influenza virus. [ABSTRACT FROM AUTHOR]

An infectious agent affecting both domestic and wild birds may cause avian influenza. All of them can be transmitted by coming into contact with tainted food, drink, or bird emissions, particularly feces. Numerous clades of H5N1 infections have been circulating since 2003, including one introduced to the United States in 2014 by wild birds, which persisted until 2016. There were 2,240 wild birds found in 45 states and 519 counties in the United States alone by September 14, 2022. According to the World Organization for Animal Health (WOAH), the predominant Highly Pathogenic Avian Influenza (HPAI) A (H5) virus subtype causing poultry outbreaks worldwide from late 2021 to early 2022 is A (H5N1). Most notifications from wild birds across multiple countries and regions suggest that the virus may have been introduced and spread via uncontrolled bird migration. The primary instance of a goose/Guangdong/1/96-lineage H5 HPAI infection inside the Americas since June 2015 was checked by the later disclosure of an H5N1 HPAI outbreak in Newfoundland, Canada. The avian flu Type A viruses, or bird flu viruses, rarely cause human infection; some bird flu viruses have done so in the past. The HPAI (H5) virus has been persistent in wild bird populations in Europe since the 2020-21 epidemic wave, according to the paper titled “Avian Influenza Overview: March-June 2022.” Even regions like Antarctica had avian influenza cases in 2023-24. Prevention and control can be done by monitoring and reporting outbreaks, preventing avian influenza at its source in animals, banning chicken farms, controlling methodologies, remuneration for ranchers, and vaccination.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10−7 ng/μL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

We found highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b associated with meningoencephalitis in a stranded harbor porpoise (Phocoena phocoena). The virus was closely related to strains responsible for a concurrent avian influenza outbreak in wild birds. This case highlights the potential risk for virus spillover to mammalian hosts.

Highly pathogenic avian influenza viruses (HPAIVs) in gallinaceous poultry are associated with viral infection of the endothelium, the induction of a 'cytokine storm, and severe disease. In contrast, in Pekin ducks, HPAIVs are rarely endothelial tropic, and a cytokine storm is not observed. To date, understanding these species-dependent differences in pathogenesis has been hampered by the absence of a pure culture of duck and chicken endothelial cells. Here, we use our recently established in vitro cultures of duck and chicken aortic endothelial cells to investigate species-dependent differences in the response of endothelial cells to HPAIV H5N1 infection. We demonstrate that chicken and duck endothelial cells display a different transcriptional response to HPAI H5N1 infection in vitro-with chickens displaying a more pro-inflammatory response to infection. As similar observations were recorded following in vitro stimulation with the viral mimetic polyI:C, these findings were not specific to an HPAIV H5N1 infection. However, similar species-dependent differences in the transcriptional response to polyI:C were not observed in avian fibroblasts. Taken together, these data demonstrate that chicken and duck endothelial cells display a different response to HPAIV H5N1 infection, and this may help account for the species-dependent differences observed in inflammation in vivo.

MotivationThe 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.ResultsWe 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 implementationOur new method allowing heterogeneous sampling priors for continuous phylogeographic inference is implemented in the open-source multi-platform software package BEAST 1.10.Supplementary informationSupplementary data are available at Bioinformatics online.

The live poultry trade is thought to play an important role in the spread and maintenance of highly pathogenic avian influenza A viruses (HP AIVs) in Asia. Despite an abundance of small-scale observational studies, the role of the poultry trade in disseminating AIV over large geographic areas is still unclear, especially for developing countries with complex poultry production systems. Here we combine virus genomes and reconstructed poultry transportation data to measure and compare the spatial spread in China of three key subtypes of AIV: H5N1, H7N9, and H5N6. Although it is difficult to disentangle the contribution of confounding factors, such as bird migration and spatial distance, we find evidence that the dissemination of these subtypes among domestic poultry is geographically continuous and likely associated with the intensity of the live poultry trade in China. Using two independent data sources and network analysis methods, we report a regional-scale community structure in China that might explain the spread of AIV subtypes in the country. The identification of this structure has the potential to inform more targeted strategies for the prevention and control of AIV in China.

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, as well as sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to the growth of human H1N1 and H3N2 viruses and low-pathogenic avian influenza (LPAI) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) H5N1 virus successfully propagated in HeLa cells to levels comparable to those in a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 virus growth, suggesting the absence of a host factor(s) required for the replication of H1N1, but not H5N1, viruses in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, as well as deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from an H5N1 virus was necessary and sufficient to enable H1N1 virus growth. Overall, this study suggests that the absence of one or more cellular factors in HeLa cells results in abortive replication of H1N1, H3N2, and LPAI viruses, which can be circumvented upon the introduction of H5N1 virus NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into the virus-host interactions that underlie IAV pathogenesis and tropism.IMPORTANCE Many zoonotic avian influenza A viruses have successfully crossed the species barrier and caused mild to life-threatening disease in humans. While human-to-human transmission is limited, there is a risk that these zoonotic viruses may acquire adaptive mutations enabling them to propagate efficiently and cause devastating human pandemics. Therefore, it is important to identify viral determinants that provide these viruses with a replicative advantage in human cells. Here, we tested the growth of influenza A virus in a subset of human cell lines and found that abortive replication of H1N1 viruses in HeLa cells can be circumvented upon the introduction of H5N1 virus HA and NP. Overall, this work leverages the genetic diversity of multiple human cell lines to highlight viral determinants that could contribute to H5N1 virus pathogenesis and tropism.

In August 2021, we detected highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses in poultry in southern Benin. The isolates were genetically similar to H5N1 viruses of clade 2.3.4.4b isolated during the same period in Africa and Europe. We also found evidence for 2 separate introductions of these viruses into Benin.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research.

Avian influenza is caused by avian influenza virus infection; the H5N1 avian influenza virus is a highly pathogenic subtype, affecting poultry and human health. Since the discovery of the highly pathogenic subtype of the H5N1 avian influenza virus, it has caused enormous losses to the poultry farming industry. It was recently found that the H5N1 avian influenza virus tends to spread among mammals. Therefore, early rapid detection methods are highly significant for effectively preventing the spread of H5N1. This paper discusses the detection technologies used in the detection of the H5N1 avian influenza virus, including serological detection technology, immunological detection technology, molecular biology detection technology, genetic detection technology, and biosensors. Comparisons of these detection technologies were analyzed, aiming to provide some recommendations for the detection of the H5N1 avian influenza virus.

The epidemiology of H5N1 and H7N9 avian viruses of humans infected in China differs despite both viruses being avian reassortants that have inherited six internal genes from a common ancestor, H9N2. The median age of infected populations is substantially younger for H5N1 virus (26 years) compared with H7N9 virus (63 years). Population susceptibility to infection with seasonal influenza is understood to be influenced by cross-reactive CD8+ T cells directed towards immunogenic peptides derived from internal viral proteins which may provide some level of protection against further influenza infection. Prior exposure to seasonal influenza peptides may influence the age-related infection patterns observed for H5N1 and H7N9 viruses. A comparison of relatedness of immunogenic peptides between historical human strains and the two avian emerged viruses was undertaken for a possible explanation in the differences in age incidence observed. There appeared to be some relationship between past exposure to related peptides and the lower number of H5N1 virus cases in older populations, however the relationship between prior exposure and older populations among H7N9 virus patients was less clear.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research. [ABSTRACT FROM AUTHOR]

In August 2021, we detected highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses in poultry in southern Benin. The isolates were genetically similar to H5N1 viruses of clade 2.3.4.4b isolated during the same period in Africa and Europe. We also found evidence for 2 separate introductions of these viruses into Benin. [ABSTRACT FROM AUTHOR]

How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.

The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV.

The spread of highly pathogenic avian influenza (HPAI) viruses worldwide has serious consequences for animal health and a major economic impact on the poultry production sector. Since 2014, Europe has been severely hit by several HPAI epidemics, with France being the most affected country. Most recently, France was again affected by two devastating HPAI epidemics in 2020–21 and 2021–22. We conducted a descriptive analysis of the 2020–21 and 2021–22 epidemics, as a first step towards identifying the poultry sector's remaining vulnerabilities regarding HPAI viruses in France. We examined the spatio‐temporal distribution of outbreaks that occurred in France in 2020–21 and 2021–22, and we assessed the outbreaks' spatial distribution in relation to the 2016–17 epidemic and to the two 'high‐risk zones' recently incorporated into French legislation to strengthen HPAI prevention and control. There were 468 reported outbreaks during the 2020–21 epidemic and 1375 outbreaks during the 2021–22 epidemic. In both epidemics, the outbreaks' distribution matched extremely well that of 2016–17, and most outbreaks (80.6% and 68.4%) were located in the two high‐risk zones. The southwestern high‐risk zone was affected in both epidemics, while the western high‐risk zone was affected for the first time in 2021–22, explaining the extremely high number of outbreaks reported. As soon as the virus reached the high‐risk zones, it started to spread between farms at very high rates, with each infected farm infecting between two and three other farms at the peaks of transmission. We showed that the spatial distribution model used to create the two high‐risk zones was able to predict the location of outbreaks for the 2020–21 and 2021–22 epidemics. These zones were characterized by high poultry farm densities; future efforts should, therefore, focus on reducing the density of susceptible poultry in highly dense areas. [ABSTRACT FROM AUTHOR]

We used surveillance data collected in California before, concurrent with, and subsequent to an outbreak of highly pathogenic (HP) clade 2.3.4.4 influenza A viruses (IAVs) in 2014-2015 to (i) evaluate IAV prevalence in waterfowl, (ii) assess the evidence for spill-over infections in marine mammals and (iii) genetically characterize low-pathogenic (LP) and HP IAVs to refine inference on the spatiotemporal extent of HP genome constellations and to evaluate possible evolutionary pathways. We screened samples from 1496 waterfowl and 1142 marine mammals collected from April 2014 to August 2015 and detected IAV RNA in 159 samples collected from birds (n=157) and pinnipeds (n=2). HP IAV RNA was identified in three samples originating from American wigeon (Anas americana). Genetic sequence data were generated for a clade 2.3.4.4 HP IAV-positive diagnostic sample and 57 LP IAV isolates. Phylogenetic analyses revealed that the HP IAV was a reassortant H5N8 virus with gene segments closely related to LP IAVs detected in mallards (Anas platyrhynchos) sampled in California and other IAVs detected in wild birds sampled within the Pacific Americas Flyway. In addition, our analysis provided support for common ancestry between LP IAVs recovered from waterfowl sampled in California and gene segments of reassortant HP H5N1 IAVs detected in British Columbia, Canada and Washington, USA. Our investigation provides evidence that waterfowl are likely to have played a role in the evolution of reassortant HP IAVs in the Pacific Americas Flyway during 2014-2015, whereas we did not find support for spill-over infections in potential pinniped hosts.

Since 1997, highly pathogenic avian influenza viruses of the H5N1 subtype have been transmitted from avian hosts to humans. The severity of H5N1 infection in humans, as well as the sporadic nature of H5N1 outbreaks, both geographically and temporally, make generation of an effective vaccine a global public health priority. An effective H5N1 vaccine must ultimately provide protection against viruses from diverse clades. Toll-like receptor (TLR) agonist adjuvant formulations have a demonstrated ability to broaden H5N1 vaccine responses in pre-clinical models. However, many of these agonist molecules have proven difficult to develop clinically. Here, we describe comprehensive adjuvant formulation development of the imidazoquinoline TLR-7/8 agonist 3M-052, in combination with H5N1 hemagglutinin (HA) based antigens. We find that 3M-052 in multiple formulations protects both mice and ferrets from lethal H5N1 homologous virus challenge. Furthermore, we conclusively demonstrate the ability of 3M-052 adjuvant formulations to broaden responses to H5N1 HA based antigens, and show that this broadening is functional using a heterologous lethal virus challenge in ferrets. Given the extensive clinical use of imidazoquinoline TLR agonists for other indications, these studies identify multiple adjuvant formulations which may be rapidly advanced into clinical trials in an H5N1 vaccine.

New influenza vaccines that provide effective and broad protection are desperately needed. Live attenuated viruses are attractive vaccine candidates because they can elicit both humoral and cellular immune responses. However, recent formulations of live attenuated influenza vaccines (LAIVs) have not been protective. We combined high-coverage transposon mutagenesis of influenza virus with a rapid high-throughput screening for attenuation to generate W7-791, a live attenuated mutant virus strain. W7-791 produced only a transient asymptomatic infection in adult and neonatal mice even at doses 100-fold higher than the LD50 of the parent strain. A single administration of W7-791 conferred full protection to mice against lethal challenge with H1N1, H3N2, and H5N1 strains, and improved viral clearance in ferrets. Adoptive transfer of T cells from W7-791-immunized mice conferred heterologous protection, indicating a role for T cell-mediated immunity. These studies present an LAIV development strategy to rapidly generate and screen entire libraries of viral clones.

Background: Small interfering RNA technology has been considered a prospective alternative antiviral treatment using gene silencing against influenza viruses with high mutations rates. On the other hand, there are no reports on its effectiveness against the highly pathogenic avian influenza H5N1 virus isolated from Indonesia. Objectives: The main objective of this study was to improve the siRNA design based on the nucleoprotein gene (siRNA-NP) for the Indonesian H5N1 virus. Methods: The effectiveness of these siRNA-NPs (NP672, NP1433, and NP1469) was analyzed in vitro in Marbin-Darby canine kidney cells. Results: The siRNA-NP672 caused the largest decrease in viral production and gene expression at 24, 48, and 72 h post-infection compared to the other siRNA-NPs. Moreover, three serial passages of the H5N1 virus in the presence of siRNA-NP672 did not induce any mutations within the nucleoprotein gene. Conclusions: These findings suggest that siRNA-NP672 can provide better protection against the Indonesian strain of the H5N1 virus. [ABSTRACT FROM AUTHOR]

We detected infection with highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b in 2 red fox (Vulpes vulpes) cubs found in the wild with neurologic signs in the Netherlands. The virus is related to avian influenza viruses found in wild birds in the same area.

Two zoonotic influenza A viruses (IAV) of global concern, H5N1 and H7N9, exhibit unexplained differences in age distribution of human cases. Using data from all known human cases of these viruses, we show that an individual's first IAV infection confers lifelong protection against severe disease from novel hemagglutinin (HA) subtypes in the same phylogenetic group. Statistical modeling shows that protective HA imprinting is the crucial explanatory factor, and it provides 75% protection against severe infection and 80% protection against death for both H5N1 and H7N9. Our results enable us to predict age distributions of severe disease for future pandemics and demonstrate that a novel strain's pandemic potential increases yearly when a group-mismatched HA subtype dominates seasonal influenza circulation. These findings open new frontiers for rational pandemic risk assessment.

Human outbreaks with avian influenza have been, so far, constrained by poor viral adaptation to non-avian hosts. This could be overcome via co-infection, whereby two strains share genetic material, allowing new hybrid strains to emerge. Identifying areas where co-infection is most likely can help target spaces for increased surveillance. Ecological niche modeling using remotely-sensed data can be used for this purpose. H5N1 and H9N2 influenza subtypes are endemic in Egyptian poultry. From 2006 to 2015, over 20,000 poultry and wild birds were tested at farms and live bird markets. Using ecological niche modeling we identified environmental, behavioral, and population characteristics of H5N1 and H9N2 niches within Egypt. Niches differed markedly by subtype. The subtype niches were combined to model co-infection potential with known occurrences used for validation. The distance to live bird markets was a strong predictor of co-infection. Using only single-subtype influenza outbreaks and publicly available ecological data, we identified areas of co-infection potential with high accuracy (area under the receiver operating characteristic (ROC) curve (AUC) 0.991).

Since 2005, H5N1 highly pathogenic avian influenza virus (HPAIV) has severely impacted the economy and public health in the Middle East (ME) with Egypt as the most affected country. Understanding the high-risk areas and spatiotemporal distribution of the H5N1 HPAIV in poultry is prerequisite for establishing risk-based surveillance activities at a regional level in the ME. Here, we aimed to predict the geographic range of H5N1 HPAIV outbreaks in poultry in the ME using a set of environmental variables and to investigate the spatiotemporal clustering of outbreaks in the region. Data from the ME for the period 2005-14 were analyzed using maximum entropy ecological niche modeling and the permutation model of the scan statistics. The predicted range of high-risk areas (P > 0.60) for H5N1 HPAIV in poultry included parts of the ME northeastern countries, whereas the Egyptian Nile delta and valley were estimated to be the most suitable locations for occurrence of H5N1 HPAIV outbreaks. The most important environmental predictor that contributed to risk for H5N1 HPAIV was the precipitation of the warmest quarter (47.2%), followed by the type of global livestock production system (18.1%). Most significant spatiotemporal clusters (P < 0.001) were detected in Egypt, Turkey, Kuwait, Saudi Arabia, and Sudan. Results suggest that more information related to poultry holding demographics is needed to further improve prediction of risk for H5N1 HPAIV in the ME, whereas the methodology presented here may be useful in guiding the design of surveillance programs and in identifying areas in which underreporting may have occurred.

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

IGHV polymorphism provides a rich source of humoral immune system diversity. One important example is the IGHV1-69 germline gene where the biased use of alleles that encode the critical CDR-H2 Phe54 (F-alleles) to make broadly neutralizing antibodies (HV1-69-sBnAb) to the influenza A hemagglutinin stem domain has been clearly established. However, whether IGHV1-69 polymorphism can also modulate B cell function and Ab repertoire expression through promoter and copy number (CN) variations has not been reported, nor has whether IGHV1-69 allelic distribution is impacted by ethnicity. Here we studied a cohort of NIH H5N1 vaccinees and demonstrate for the first time the influence of IGHV1-69 polymorphism on V-segment usage, somatic hypermutation and B cell expansion that elucidates the dominance of F-alleles in HV1-69-sBnAbs. We provide evidence that Phe54/Leu54 (F/L) polymorphism correlates with shifted repertoire usage of other IGHV germline genes. In addition, we analyzed ethnically diverse individuals within the 1000 genomes project and discovered marked variations in F- and L- genotypes and CN among the various ethnic groups that may impact HV1-69-sBnAb responses. These results have immediate implications for understanding HV1-69-sBnAb responses at the individual and population level and for the design and implementation of universal influenza vaccine.

Since its first isolation in 1996 in Guangdong, China, the highly pathogenic avian influenza virus (HPAIV) H5N1 has circulated in avian hosts for almost two decades and spread to more than 60 countries worldwide. The role of different avian hosts and the domestic-wild bird interface has been critical in shaping the complex HPAIV H5N1 disease ecology, but remains difficult to ascertain. To shed light on the large-scale H5N1 transmission patterns and disentangle the contributions of different avian hosts on the tempo and mode of HPAIV H5N1 dispersal, we apply Bayesian evolutionary inference techniques to comprehensive sets of hemagglutinin and neuraminidase gene sequences sampled between 1996 and 2011 throughout Asia and Russia. Our analyses demonstrate that the large-scale H5N1 transmission dynamics are structured according to different avian flyways, and that the incursion of the Central Asian flyway specifically was driven by Anatidae hosts coinciding with rapid rate of spread and an epidemic wavefront acceleration. This also resulted in long-distance dispersal that is likely to be explained by wild bird migration. We identify a significant degree of asymmetry in the large-scale transmission dynamics between Anatidae and Phasianidae, with the latter largely representing poultry as an evolutionary sink. A joint analysis of host dynamics and continuous spatial diffusion demonstrates that the rate of viral dispersal and host diffusivity is significantly higher for Anatidae compared with Phasianidae. These findings complement risk modeling studies and satellite tracking of wild birds in demonstrating a continental-scale structuring into areas of H5N1 persistence that are connected through migratory waterfowl.

Introduction: During the outbreak of avian influenza, A (H5N1) (IA) in wild and domestic birds recorded in January 2023, the epidemiological alert has been extended due to its potential contagion to humans, particularly in those exposed occupational groups., Objective: to identify the primary occupational risk groups, as well as the preventive, safety, and control measures against IA intended or implemented in these positions., Material and Methods: A systematic search was conducted in Pubmed, Scopus, Web of science, Scielo and literature databases. Scientific articles, normative documents, and technical reports identifying vulnerable occupational groups and preventive measures against IA were included. Two authors conducted a full-text review, extracting information independently, and findings were summarized narratively., Results: A total of 5518 documents were identified, and 30 reports were included. 20% of the reports were published in 2023, 13/30 were affiliated to a university institution. Occupationally exposed groups were identified both directly and indirectly. 63.3% of reports identified breeders, poultry farmers and sellers as the most concerning occupational group, while 60% identified biosecurity practices (use of PPE, handwashing) as the primary measure against IA, followed by strategies such as education (training and capacity-building)., Conclusion: Occupational groups of interest were identified, primarily those involved in sales, commerce, and the handling of bird waste with potential exposure to IA. Furthermore, the maintenance of biosecurity measures, cleaning-disinfection practices, and educational strategies in workplace settings are recommended., Competing Interests: The authors do not have conflict of interest., (© 2024 The Authors.)

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Previous Bayesian phylogeographic studies of H5N1 highly pathogenic avian influenza viruses (HPAIVs) explored the origin and spread of the epidemic from China into Russia, indicating that HPAIV circulated in Russia prior to its detection there in 2005. In this study, we extend this research to explore the evolution and spread of HPAIV within Europe during the 2005-2010 epidemic, using all available sequences of the hemagglutinin (HA) and neuraminidase (NA) gene regions that were collected in Europe and Russia during the outbreak. We use discrete-trait phylodynamic models within a Bayesian statistical framework to explore the evolution of HPAIV. Our results indicate that the genetic diversity and effective population size of HPAIV peaked between mid-2005 and early 2006, followed by drastic decline in 2007, which coincides with the end of the epidemic in Europe. Our results also suggest that domestic birds were the most likely source of the spread of the virus from Russia into Europe. Additionally, estimates of viral dispersal routes indicate that Russia, Romania, and Germany were key epicenters of these outbreaks. Our study quantifies the dynamics of a major European HPAIV pandemic and substantiates the ability of phylodynamic models to improve molecular surveillance of novel AIVs.

Emerging and re-emerging infectious diseases of zoonotic origin like highly pathogenic avian influenza pose a significant threat to human and animal health due to their elevated transmissibility. Identifying the drivers of such viruses is challenging, and estimation of spatial diffusion is complicated by the fact that the variability of viral spread from locations could be caused by a complex array of unknown factors. Several techniques exist to help identify these drivers, including bioinformatics, phylogeography, and spatial epidemiology, but these methods are generally evaluated separately and do not consider the complementary nature of each other. Here, we studied an approach that integrates these techniques and identifies the most important drivers of viral spread by focusing on H5N1 influenza A virus in Egypt because of its recent emergence as an epicenter for the disease. We used a Bayesian phylogeographic generalized linear model (GLM) to reconstruct spatiotemporal patterns of viral diffusion while simultaneously assessing the impact of factors contributing to transmission. We also calculated the cross-species transmission rates among hosts in order to identify the species driving transmission. The densities of both human and avian species were supported contributors, along with latitude, longitude, elevation, and several meteorological variables. Also supported was the presence of a genetic motif found near the hemagglutinin cleavage site. Various genetic, geographic, demographic, and environmental predictors each play a role in H1N1 diffusion. Further development and expansion of phylogeographic GLMs such as this will enable health agencies to identify variables that can curb virus diffusion and reduce morbidity and mortality.

Both H5N1 and H7N9 subtype avian influenza viruses cause enormous economic losses and pose considerable threats to public health. Bivalent vaccines against both two subtypes are more effective in control of H5N1 and H7N9 viruses in poultry and novel egg-independent vaccines are needed. Herein, H5 and H7 virus like particle (VLP) were generated in a baculovirus expression system and a bivalent H5+H7 VLP vaccine candidate was prepared by combining these two antigens. Single immunization of the bivalent VLP or commercial inactivated vaccines elicited effective antibody immune responses, including hemagglutination inhibition, virus neutralizing and HA-specific IgG antibodies. All vaccinated birds survived lethal challenge with highly pathogenic H5N1 and H7N9 viruses. Furthermore, the bivalent VLP significantly reduced viral shedding and virus replication in chickens, which was comparable to that observed for the commercial inactivated vaccine. However, the bivalent VLP was better than the commercial vaccine in terms of alleviating pulmonary lesions caused by H7N9 virus infection in chickens. Therefore, our study suggests that the bivalent H5+H7 VLP vaccine candidate can serve as a critical alternative for the traditional egg-based inactivated vaccines against H5N1 and H7N9 avian influenza virus infection in poultry.

UnlabelledModulating the host response is a promising approach to treating influenza, caused by a virus whose pathogenesis is determined in part by the reaction it elicits within the host. Though the pathogenicity of emerging H7N9 influenza virus in several animal models has been reported, these studies have not included a detailed characterization of the host response following infection. Therefore, we characterized the transcriptomic response of BALB/c mice infected with H7N9 (A/Anhui/01/2013) virus and compared it to the responses induced by H5N1 (A/Vietnam/1203/2004), H7N7 (A/Netherlands/219/2003), and pandemic 2009 H1N1 (A/Mexico/4482/2009) influenza viruses. We found that responses to the H7 subtype viruses were intermediate to those elicited by H5N1 and pdm09H1N1 early in infection but that they evolved to resemble the H5N1 response as infection progressed. H5N1, H7N7, and H7N9 viruses were pathogenic in mice, and this pathogenicity correlated with increased transcription of cytokine response genes and decreased transcription of lipid metabolism and coagulation signaling genes. This three-pronged transcriptomic signature was observed in mice infected with pathogenic H1N1 strains such as the 1918 virus, indicating that it may be predictive of pathogenicity across multiple influenza virus strains. Finally, we used host transcriptomic profiling to computationally predict drugs that reverse the host response to H7N9 infection, and we identified six FDA-approved drugs that could potentially be repurposed to treat H7N9 and other pathogenic influenza viruses.ImportanceEmerging avian influenza viruses are of global concern because the human population is immunologically naive to them. Current influenza drugs target viral molecules, but the high mutation rate of influenza viruses eventually leads to the development of antiviral resistance. As the host evolves far more slowly than the virus, and influenza pathogenesis is determined in part by the host response, targeting the host response is a promising approach to treating influenza. Here we characterize the host transcriptomic response to emerging H7N9 influenza virus and compare it with the responses to H7N7, H5N1, and pdm09H1N1. All three avian viruses were pathogenic in mice and elicited a transcriptomic signature that also occurs in response to the legendary 1918 influenza virus. Our work identifies host responses that could be targeted to treat severe H7N9 influenza and identifies six FDA-approved drugs that could potentially be repurposed as H7N9 influenza therapeutics.

Highly pathogenic influenza A virus subtype H5N1 causes significant poultry mortality in the six countries where it is endemic and can also infect humans. Egypt has reported the third highest number of poultry outbreaks (n=1084) globally. The objective of this cross-sectional study was to identify putative risk factors for H5N1 infections in backyard poultry in 16 villages in Damietta, El Gharbia, Fayoum, and Menofia governorates from 2010-2012. Cloacal and tracheal swabs and serum samples from domestic (n=1242) and wild birds (n=807) were tested for H5N1 via RT-PCR and hemagglutination inhibition, respectively. We measured poultry rearing practices with questionnaires (n=306 households) and contact rates among domestic and wild bird species with scan sampling. Domestic birds (chickens, ducks, and geese, n=51) in three governorates tested positive for H5N1 by PCR or serology. A regression model identified a significant correlation between H5N1 in poultry and the practice of disposing of dead poultry and poultry feces in the garbage (F=15.7, p

Anti-influenza drugs, amantadine and rimantadine, targeting the M2 channel from influenza A virus are no longer effective because of widespread drug resistance. S31N is the predominant and amantadine-resistant M2 mutant, present in almost all of the circulating influenza A strains as well as in the pandemic 2009 H1N1 and the highly pathogenic H5N1 flu strains. Thus, there is an urgent need to develop second-generation M2 inhibitors targeting the S31N mutant. However, the S31N mutant presents a huge challenge to drug discovery, and it has been considered undruggable for several decades. Using structural information, classical medicinal chemistry approaches, and M2-specific biological testing, we discovered benzyl-substituted amantadine derivatives with activity against both S31N and WT, among which 4-(adamantan-1-ylaminomethyl)-benzene-1,3-diol (44) is the most potent dual inhibitor. These inhibitors demonstrate that S31N is a druggable target and provide a new starting point to design novel M2 inhibitors that address the problem of drug-resistant influenza A infections.

Influenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PAR1 antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses.

Wild birds are suspected to have played a role in highly pathogenic avian influenza (HPAI) H5N1 outbreaks in West Bengal. Cluster analysis showed that H5N1 was introduced in West Bengal at least 3 times between 2008 and 2010. We simulated the introduction of H5N1 by wild birds and their contact with poultry through a stochastic continuous-time mathematical model. Results showed that reducing contact between wild birds and domestic poultry, and increasing the culling rate of infected domestic poultry communities will reduce the probability of outbreaks. Poultry communities that shared habitat with wild birds or those indistricts with previous outbreaks were more likely to suffer an outbreak. These results indicate that wild birds can introduce HPAI to domestic poultry and that limiting their contact at shared habitats together with swift culling of infected domestic poultry can greatly reduce the likelihood of HPAI outbreaks.

Despite strong commitments of the central and provincial veterinary authorities in Vietnam to control highly pathogenic avian influenza H5N1 (HPAI‐H5N1) in poultry and to diminish the risk of human infection, outbreaks continue to occur in poultry. This study describes the spatio‐temporal distribution of HPAI‐H5N1 outbreaks in Vietnam for the period December 2014–April 2018 using the space‐time K‐function and the space‐time scan statistic. The space‐time K‐function analyses showed statistically significant spatio‐temporal clustering of HPAI‐H5N1 outbreaks in poultry during the study period of up to 50 days and 60 kilometres. The space‐time scan statistic test identified three statistically significant space‐time clusters of HPAI‐H5N1 in the south of the country where the incidence of HPAI‐H5N1 outbreaks was greater than that expected if outbreaks were randomly distributed in space and time. The analyses indicated shortfalls in the effectiveness of control measures used to control HPAI‐H5N1 throughout the study period. Development of a better understanding of the relative impact of HPAI‐H5N1 control measures (depopulation of infected flocks, vaccination, movement restrictions) on space‐time interaction would allow animal health authorities to focus their efforts on control measures shown to have the greatest relative effect. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian influenza (HPAI) viruses have potential to cross species barriers and cause pandemics. Since 2022, HPAI A(H5N1) belonging to the goose/Guangdong 2.3.4.4b hemagglutinin phylogenetic clade have infected poultry, wild birds, and mammals across North America. Continued circulation in birds and infection of multiple mammalian species with strains possessing adaptation mutations increase the risk for infection and subsequent reassortment with influenza A viruses endemic in swine. We assessed the susceptibility of swine to avian and mammalian HPAI H5N1 clade 2.3.4.4b strains using a pathogenesis and transmission model. All strains replicated in the lung of pigs and caused lesions consistent with influenza A infection. However, viral replication in the nasal cavity and transmission was only observed with mammalian isolates. Mammalian adaptation and reassortment may increase the risk for incursion and transmission of HPAI viruses in feral, backyard, or commercial swine.

More than 60% of human infectious diseases are caused by pathogens shared with wild or domestic animals. Zoonotic disease organisms include those that are endemic in human populations or enzootic in animal populations with frequent cross-species transmission to people. Some of these diseases have only emerged recently. Together, these organisms are responsible for a substantial burden of disease, with endemic and enzootic zoonoses causing about a billion cases of illness in people and millions of deaths every year. Emerging zoonoses are a growing threat to global health and have caused hundreds of billions of US dollars of economic damage in the past 20 years. We aimed to review how zoonotic diseases result from natural pathogen ecology, and how other circumstances, such as animal production, extraction of natural resources, and antimicrobial application change the dynamics of disease exposure to human beings. In view of present anthropogenic trends, a more effective approach to zoonotic disease prevention and control will require a broad view of medicine that emphasises evidence-based decision making and integrates ecological and evolutionary principles of animal, human, and environmental factors. This broad view is essential for the successful development of policies and practices that reduce probability of future zoonotic emergence, targeted surveillance and strategic prevention, and engagement of partners outside the medical community to help improve health outcomes and reduce disease threats.

Influenza viruses exist in each host as a collection of genetically diverse variants, which might enhance their adaptive potential. To assess the genetic and functional diversity of highly pathogenic avian influenza A(H5N1) viruses within infected humans, we used deep-sequencing methods to characterize samples obtained from infected patients in northern Vietnam during 2004–2010 on different days after infection, from different anatomic sites, or both. We detected changes in virus genes that affected receptor binding, polymerase activity, or interferon antagonism, suggesting that these factors could play roles in influenza virus adaptation to humans. However, the frequency of most of these mutations remained low in the samples tested, implying that they were not efficiently selected within these hosts. Our data suggest that adaptation of influenza A(H5N1) viruses is probably stepwise and depends on accumulating combinations of mutations that alter function while maintaining fitness.

Abstract Background Efficient A(H5N1) control is unlikely to be based on epidemiological data alone. Such control depends on a thorough understanding and appreciation of the interconnectedness of epidemiological, social, and economic factors that contribute to A(H5N1) vulnerability. To date, the control of A(H5N1) in Egypt has been challenging. The disease has been endemic for more than 10 years with a dramatic increase in human cases between December 2014 and March 2015. Part of the problem has been a lack of understanding of the inter-play of drivers, conditions and motives that influence preventive behaviours at the household level. Methods To address this issue, the authors developed a Composite Risk Index (CRI) to inform decision-makers of critical epidemiological, livelihood, food security and risk perception factors that were found to contribute to A(H5N1) vulnerability at the community level. The CRI consists of seven constructs that were individually scored for each community. The seven constructs included poultry sales, previous flock exposure to A(H5N1), human risk probability, sense of control over the disease, preventative actions taken, level of household food insecurity and community norms toward certain handling and disposal practices. One hundred forty female poultry keepers across four governorates were interviewed in 2010 using a mix of random and purposive sampling techniques. A mixed method approach underpinned the analysis. The study used wealth ranking in order to help decision-makers in understanding the specific constraints of different wealth groups and aid better targeting of A(H5N1) control and prevention strategies. Results Poverty, widowhood and lack of education were among the factors associated with high risk scores. CRI scores in those villages where awareness raising had taken place were not significantly different compared to those villages where awareness raising had not taken place. Conclusions The aim of the tool is to enable targeting those communities that are likely to be highly vulnerable to A(H5N1) outbreaks and where control and awareness-raising efforts are expected to be most effective. In this manner, policy makers and practitioners will be able to better allocate limited resources to those communities most vulnerable to the negative impact of A(H5N1).