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Pathobiological Origins and Evolutionary History of Highly Pathogenic Avian Influenza Viruses
Abstract
High-pathogenicity avian influenza (HPAI) viruses have arisen from low-pathogenicity avian influenza (LPAI) viruses via changes in the hemagglutinin proteolytic cleavage site, which include mutation of multiple nonbasic to basic amino acids, duplication of basic amino acids, or recombination with insertion of cellular or viral amino acids. Between 1959 and 2019, a total of 42 natural, independent H5 (n = 15) and H7 (n = 27) LPAI to HPAI virus conversion events have occurred in Europe (n = 16), North America (n = 9), Oceania (n = 7), Asia (n = 5), Africa (n = 4), and South America (n = 1). Thirty-eight of these HPAI outbreaks were limited in the number of poultry premises affected and were eradicated. However, poultry outbreaks caused by A/goose/Guangdong/1/1996 (H5Nx), Mexican H7N3, and Chinese H7N9 HPAI lineages have continued. Active surveillance and molecular detection and characterization efforts will provide the best opportunity for early detection and eradication from domestic birds.
... In addition, there is an adaptation, mainly referring to avian species: AIV adapted to chickens have an affinity to the SA α−2,3 Gal receptor with a β1,4 linkage to N-acetyl galactosamine (GalNac), and the particular case of AIV associated with waterfowl (ducks) has a preference for the SA α−2,3 Gal receptor with a β1,3 linkage to N-acetylglucosamine (GlcNac) [62]. In addition to its participation during cell binding, the amino acid sequence of the HA, in particular, the cleavage site (CS) sequence [48,63], is decisive for the pathotypes [64] classified as either low pathogenicity avian influenza virus (LPAIV) or high pathogenicity avian influenza virus (HPAIV) [48,63,64], based on the capacity of CS of furin-like proteases, increasing the virus's tissue tropism [65]. In the case of NA, its sialidase enzymatic activity promotes the degradation of the avian cellular receptor or mammalian cellular receptor [56,58,66]. ...
... According to the interspecies transmission model, it is important to mention the appearance of the spillover of subtypes H5 and H7 in the poultry industry [65,109] mainly due to the acquisition of multibasic amino acid sequences in the CS, which allowed the emergence of HPAIV [65,110]. However, this model becomes altered when examining the transmission of HPAIV in wildlife avian species [84,111], which occurs less frequently, as exemplified by the H5N3 virus in common terns (Sterna hirundo), reported in the Cape Province coast between Elizabeth Harbour and Lamberts Bay in 1961 [14,16,17,112]. ...
... According to the interspecies transmission model, it is important to mention the appearance of the spillover of subtypes H5 and H7 in the poultry industry [65,109] mainly due to the acquisition of multibasic amino acid sequences in the CS, which allowed the emergence of HPAIV [65,110]. However, this model becomes altered when examining the transmission of HPAIV in wildlife avian species [84,111], which occurs less frequently, as exemplified by the H5N3 virus in common terns (Sterna hirundo), reported in the Cape Province coast between Elizabeth Harbour and Lamberts Bay in 1961 [14,16,17,112]. ...
The high pathogenicity avian influenza virus H5N1, which first emerged in the winter of 2021, has resulted in multiple outbreaks across the American continent through the summer of 2023 and they continue based on early 2025 records, presenting significant challenges for global health and food security. The viruses causing the outbreaks belong to clade 2.3.4.4b, which are descendants of the lineage A/Goose/Guangdong/1/1996 (Gs/Gd) through genetic reassortments with several low pathogenicity avian influenza viruses present in populations of Anseriformes and Charadriiformes orders. This review addresses these issues by thoroughly analysing available epidemiological databases and specialized literature reviews. This project explores the mechanisms behind the resurgence of the H5N1 virus. It provides a comprehensive overview of the origin, timeline and factors contributing to its prevalence among wild bird populations on the American continent.
... Since their first emergence, the HA gene of H5 Gs/Gd HPAIV has genetically diversified into an overabundance of clades and subclades. Among these, clade 2.3.4.4b has undergone explosive expansion in wild birds and domestic poultry and almost entirely replaced other circulating clades in just a short period of time [19][20][21]. Strains from clade 2.3.4.4b have been reported to cause severe systemic infections and high mortality rates among waterfowl, as well as mass mortality events in sea birds, marine mammals, and others [22]. ...
... Due to their persistent circulation in poultry in Asia, Gs/Gd H5Nx HPAI viruses have found their way back to their natural reservoir, the wild aquatic bird species. Through waterfowl migrations, four waves of intercontinental transmission of Gs/Gd lineage H5Nx virus from Asia to other continents have been identified from 2003 onward [21]. This has led these viruses to undergo reassortment of the internal gene segments with other influenza viruses and genetic evolution of the hemagglutinin H5 gene. ...
... The first intercontinental spread of the H5 clade 2.3.4.4c (previously classified as 2.3.4.4a) to North America dates to 2014-2015, with H5N8 viruses introduced most likely via migratory birds through the Bering Strait to breeding grounds in Alaska [55]. After reassorting with LPAI gene segments from North American wild birds [56], an H5N2 reassortant virus of clade 2.3.4.4c became predominant and affected over 50 million birds in the U.S. in spring 2015 [19,21,57]. After sporadic detections in wild birds, the H5 clade 2.3.4.4c viruses in North America were last isolated in late 2016 [58]. ...
Simple Summary
Avian influenza viruses are highly contagious respiratory viruses that severely impact bird populations, causing significant morbidity, mortality, and economic losses in the poultry industry worldwide. Particularly concerning are the Asian-origin H5 subtype highly pathogenic avian influenza viruses of clade 2.3.4.4b, which emerged in 2013 and have since spread across Asia, Europe, Africa, and America, leading to outbreaks in various poultry and animal species. The unique epidemiological and pathobiological characteristics specific to clade 2.3.4.4b viruses are discussed, emphasizing their distinct nature compared to other clades. Wild waterfowl, acting as reservoirs, frequently carry these viruses, posing threats not only to their populations but also to other wild bird species, including endangered ones. Furthermore, an increasing number of clade 2.3.4.4b virus infections in wild or domestic mammalian species raises significant concerns about potential spillover events to humans. This review highlights the diverse outcomes of HPAI infections in different hosts, ranging from asymptomatic cases to fatal infection, influenced by host and virus-related factors. Understanding these complexities is vital for developing effective strategies to mitigate the impact of AIVs, safeguard poultry production, protect wildlife, and prevent potential public health crises.
Abstract
Avian influenza viruses (AIVs) are highly contagious respiratory viruses of birds, leading to significant morbidity and mortality globally and causing substantial economic losses to the poultry industry and agriculture. Since their first isolation in 2013–2014, the Asian-origin H5 highly pathogenic avian influenza viruses (HPAI) of clade 2.3.4.4b have undergone unprecedented evolution and reassortment of internal gene segments. In just a few years, it supplanted other AIV clades, and now it is widespread in the wild migratory waterfowl, spreading to Asia, Europe, Africa, and the Americas. Wild waterfowl, the natural reservoir of LPAIVs and generally more resistant to the disease, also manifested high morbidity and mortality with HPAIV clade 2.3.4.4b. This clade also caused overt clinical signs and mass mortality in a variety of avian and mammalian species never reported before, such as raptors, seabirds, sealions, foxes, and others. Most notably, the recent outbreaks in dairy cattle were associated with the emergence of a few critical mutations related to mammalian adaptation, raising concerns about the possibility of jumping species and acquisition of sustained human-to-human transmission. The main clinical signs and anatomopathological findings associated with clade 2.3.4.4b virus infection in birds and non-human mammals are hereby summarized.
... Highly pathogenic avian influenza (HPAI) is cause3d by influenza A viruses of the family Orthomyxoviridae. Since its identification in China in 1996, there have been multiple waves of intercontinental transmission of the H5Nx Gs/GD lineage virus [1][2][3]. HPAI has resulted in the death and mass slaughter of nearly 556 million poultry worldwide between January 2005 and December 2023, with peaks or more than 141 million losses in 2022, 83 million in 2023, 72 million in 2021, and 40 million in 2016 [3,4]. In addition, as of April 2024, humans have been infected with subtypes H5N1 (around 880 cases in the world), H7N9 (more than 1500 cases), H5N6 (around 90 cases), H9N2 (around 125 cases reported), and sporadic cases have been reported with subtypes H3N8, H6N1, H7N4, H7N7 and H10N3, H10N7, H10N8 [5][6][7][8][9][10]. ...
... In our study, sampling heterogeneity between countries is likely to result in bias in the phylogenetic trees. Despite the methodological limitations described, phylogeographyinformed GLM results of the inferred epidemics dynamics are consistent with previous studies [1,[103][104][105], which gives some confidence to the results of the GLM. ...
The multiple waves of intercontinental transmission of the highly pathogenic avian influenza (HPAI) H5Nx Gs/GD lineage since its identification in 1996 are testament to its resistance to control and prevention efforts. Knowledge of the predictors of HPAI international spread can help identify strengths as well as areas for improvement in surveillance and controlling HPAI. We used 10 years of data with quarterly granularity from the World Organization for Animal Health (WOAH), United Nations (UN), International Union for Conservation of Nature (IUCN), and genetic databases for 2.3.2.1c and 2.3.4.4b H5Nx clades, to determine the impact on international viral spread of (1) six categories of poultry commodities of legal international trade, (2) wild birds’ migration, (3) five types of preventive measures, (4) resources allocated to veterinary services, and (5) geographic distance between countries. Two analytical approaches were used: a generalized linear mixed model (GLMM) for all targeted countries, based on epidemiological, trade, and bird migration data. Then, phylogeography-informed generalized linear models (GLMs) with time-dependent predictors were specified for analyzing the HPAI spread between countries with available genetic data. The main conclusions of this study are that results suggested (1) a role of poultry trade in disease spread; (2) a role of migratory birds in disease spread; (3) a strong role of proximity between countries in disease spread; (4) a protective effect for resources allocated to veterinary services; and (5) a protective effect for precautions at borders in exposed countries (protective against informal trade). Our findings show the importance of proper implementation of preventive measures, as advocated in WOAH standards. In addition, our results show the complementarity of epidemiological, trade, biological, and genetic data to trace back international H5NX spread.
... H5 High Pathogenicity Avian Influenza viruses (HPAIVs) belong to the A/Goose/Guangdong/1/1996 (Gs/Gd) lineage consistently cause outbreaks in both domestic poultry and wild birds worldwide, causing huge economic losses to related industries and becoming a threat to public health [1][2][3][4][5]. Owing to diverse and extensive reassortment events in poultry and wild birds, Gs/Gd H5 HPAIVs have evolved into multiple hemagglutinin (HA) phylogenetic clades [6][7][8]. ...
... In 2020-2022, clade 2.3.4.4b H5N8 viruses caused outbreaks and subsequently H5N1 HPAIVs emerged as the most prevalent viruses worldwide, including in Europe, Asia, and America [3][4][5][8][9][10]. Reportedly, H5N1 HPAIVs caused one of the largest HPAIV epidemics in Europe, extensively affecting both poultry and wild birds in 2021-2022 [11][12][13]. ...
During the 2022–2023 winter season in South Korea, a novel clade 2.3.4.4b H5N1 HPAIV was first detected in wild birds, which then subsequently caused multiple outbreaks in poultry farms and wild birds. This study aimed to investigate the genetic characteristics of H5N1 HPAIVs isolated during the 2022–2023, along with their pathogenicity and transmissibility in chickens and ducks. The clade 2.3.4.4b H5N1 HPAIV viruses caused outbreaks in 75 poultry farms and detected in 174 wild bird cases. Phylogenetic analysis of hemagglutinin genes revealed that the South Korean H5N1 HPAIV isolates were closely related to Eurasian and American HPAIVs isolated between 2022 and 2023. In total, 21 diverse genotypes (22G0–22G20) were identified in virus isolates from poultry and wild birds, among which 22G7 was the dominant genotype. The 22G1 genotype (A/duck/Korea/H493/2022(H5N1)) caused high virulence and pathogenicity, with a 100 % mortality rate in specific-pathogen-free chickens. Ducks inoculated with genotypes 22G1 or 22G7 (A/duck/Korea/H537/2022(H5N1)) showed neurological signs, with 60 %–80 % mortality rate. In the contact groups of ducks, 100 % of transmissibility was observed. Notably, in the 22G7-inoculated group, viral shedding via the cloacal route was longer, and viral replication in the cecal tonsil was higher than that in the 22G1-inoculated group, which may have contributed to the dominancy of the 22G7 genotype. Therefore, better understanding of the genetic and pathogenic features of HPAI viruses is important for effective virus control in the field.
... There have been sporadic reports of zoonotic infections in which the virus is transmitted from birds to farm animals and from animals to humans, resulting in fatalities of farm animals. These zoonotic infections are associated with specific subtypes of avian influenza viruses, such as H1N1, H2N2, H5N1, H7N7, and H7N9 [1,3,[100][101][102]. The various subtypes of the avian influenza (AI) virus are identified by the antigens found on the surface of the influenza A virus. ...
... Co-infection involving the H9 subtype alongside other pathogens can exacerbate respiratory signs and mortality rates. It is crucial to quickly differentiate between the H5, H7, and H9 subtypes to implement appropriate vaccination programs that target the circulating subtype(s) [101,102]. ...
Accurate and timely molecular diagnosis of respiratory diseases in chickens is essential for implementing effective control measures, preventing the spread of diseases within poultry flocks, minimizing economic loss, and guarding food security. Traditional molecular diagnostic methods like polymerase chain reaction (PCR) require expensive equipment and trained personnel, limiting their use to centralized labs with a significant delay between sample collection and results. Loop-mediated isothermal amplification (LAMP) of nucleic acids offers an attractive alternative for detecting respiratory viruses in broiler chickens with sensitivity comparable to that of PCR. LAMP’s main advantages over PCR are its constant incubation temperature (∼65 °C), high amplification efficiency, and contaminant tolerance, which reduce equipment complexity, cost, and power consumption and enable instrument-free tests. This review highlights effective LAMP methods and variants that have been developed for detecting respiratory viruses in chickens at the point of need.
... Since the emergence of A/goose/Guangdong/1/1996 (gs/GD/96) H5N1 highly pathogenic avian influenza (HPAI) virus, the lineage diversified into multiple genetic groups (clade 0-9) and multiple subclades [1,2]. Diversification of the hemagglutinin (HA) gene lead to the emergence of clade 2.3.4.4b ...
... Diversification of the hemagglutinin (HA) gene lead to the emergence of clade 2.3.4.4b H5, which became predominant in Asia, Africa, Europe, and the Middle East, causing devastating losses to the poultry industry since it was first identified in 2014 [1][2][3]. In late 2021, clade 2.3.4.4b ...
The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.
... The 2022-2023 viruses belong to the clade 2.3.4.4b of the Goose/Guangdong lineage (Gs/Gd) (Leguía et al. 2023;Rimondi et al. 2024;Uhart et al. 2024;Ulloa et al. 2023). This lineage of viruses originated in 1996 in farmed geese in Southeast Asia and slowly developed mutations until it spread globally (Lee et al. 2021;Pohlmann et al. 2023). The global spread prompted an unprecedented wave of marine mammal mortality, particularly since the virus reached South America in late 2022. ...
The colony of southern elephant seals ( Mirounga leonina ) at Península Valdés (Argentina) grew by 0.9% from 2000 to 2022, reaching a population of 18,000 reproductive females. In 2023, an epidemic of the High Pathogenicity Avian Influenza H5N1 virus led to the death of almost all pups and an unknown number of adults. We tested five scenarios that included complete pup mortality along with varying levels of adult mortality and reduced fertility. Newborn mortality had the smallest impact on the future population due to high natural mortality. Consequences of pup deaths will not appear until 2027, when those lost pups would have first reproduced. Scenarios including mature female mortality had more severe and immediate consequences, with a reduction in the breeding population in 2024 predicted to match the flu death rate. It took about 10 years for the population to readjust to the 2022 age distribution. In scenarios including adult mortality, it will take decades for the population to return to the 2022 level. The 2023 epidemic may thus reverse the conservation status of a population previously having no threats to continued growth.
... In general, the clade 2.3.4.4b virus incursion has indicated major shifts in the ecology of HPAI viruses, because those newly emerged viruses have displayed properties quite distinct from the clade 2.2.1.2 viruses circulating formerly [15,16]. ...
The evolution and adaptation of highly pathogenic avian influenza (HPAI) viruses pose ongoing challenges for animal and public health. We investigated the pathogenic characteristics of the newly emerged H5N1/2022 and H5N8/2022 of clade 2.3.4.4b compared to the previously circulating H5N1/2016 of clade 2.2.1.2 in Egypt using both avian and murine models. All strains demonstrated a 100% mortality in chickens after intranasal inoculation (10⁶ EID50), while the H5N8/2022 strain showing significantly higher viral shedding (8.34 ± 0.55 log10 EID50). Contact transmission rates varied between strains (50% for the 2.3.4.4b clade and 100% for the 2.2.1.2 clade). In the mouse model, H5N1/2016 infection resulted in an 80% mortality rate with significant weight loss and virus replication in organs. In contrast, H5N8/2022 and H5N1/2022 had 60% and 40% mortality rates, respectively. An histopathological analysis revealed pronounced lesions in the tissues of the infected mice, with the most severe lesions found in the H5N1/2016 group. These findings suggest the decreased pathogenicity of the newer H5Nx strains in mammalian models, emphasizing the need for continued surveillance and adaptive control strategies.
... avian influenza virus subtype H5N1 was first identified in Hong Kong in 1997. Recent outbreaks of a pandemic in Asia, Europe, North America, Oceania, and Africa have prompted concerns about its rapid spread and the virus conversion LPAI to HPAI (6) The potential of H5N1 to vary through mutations and reassortment certainly raises the possibility of viral adaptation to the human species, even though the virus has not been able to sustain human-to-human transmission (7). Planning for a pandemic of influenza requires a high level of awareness that is required to contain the virus's initial outbreaks by using case recognition, sensitive and quick diagnostic techniques, suitable treatment , Antimicrobials may aid in the prevention of Secondary bacterial infection occurs in flocks infected with low-pathogenicity virus strains (8). ...
Avian influenza (AI) is Viral respiratory disease caused by RNA virus type A orthomyxoviruses. Avian influenza was a zoonotic disease, has caused several epidemics in domestic fowl , The different degrees of pathogenicity demonstrated by viruses in diverse avian species, as well as the diversity of viral properties such as antigens and genes, make it difficult to reliably diagnose viral infections in poultry or wild birds. This is because viruses can infection many bird specie without clinical signs ,The different degrees of pathogenicity demonstrated by viruses in diverse avian species, as well as the diversity of viral properties such as anti gens and genes, make it difficult to reliably diagnose viral infections in poultry or wild birds notably the latest H5,H7 highly virulent avian influenza viruses. Aims of the study are; Identification of AI infection in the broilers , layer, geese and Gallinula in Diwaniyah and Najaf governorates. Fourty suspected birds for each avian species were investigated which are taken from local market, ,veterinary clinics and poultry farms in the area, then rapid test were performed using the AIV Rapid kit. samples were taken randomly from (lung and trachea) , samples kept in Eppendorf tube contain 1.5 ml trizol , from which the RNA was extracted by RNA extraction kit and RNA converted into CDNA , this last product was entered into a real-time PCR with Universal Primer NP to detect the infection, Special primers were designed to identify possible subtypes (H5 primers,H7 primers.H9). Results of rapid test kit revealed that only 5 samples shown positive results between others. Twenty samples were positive in PCR test against NP primer. From these eight samples were positive against H5 and twelve were positive against H7 primers consequently and were negative to H9 primers , all above samples were taken from broilers. layer, geese and Gallinula birds were negative to all primers despite having upper respiratory signs .Aims of the study Identification of AI infection in the broilers and Gallinula in Diwaniyah and Najaf governorates. by np primer /PCR and Identify the genotype which is diagnosed in each avian species. By H5 H7 H9 primers /PCR.
... As a result, subtype is used to denote the particular virus genotypes with a variety of observable and measurable characteristics. ( [14,10,12] . ...
This narrative study seeks to examine pneumonia induced by severe acute respiratory syndrome coronavirus-2, influenza, and adenovirus regarding clinical, laboratory, and radiological features. In conclusion, whereas these viral pneumonia clinics exhibit same symptomatology and laboratory results, we assert that there are notable distinctions, particularly in radiological findings.
... High pathogenicity avian influenza viruses (HPAIVs) of subtype H5 derived from A/goose/Guangdong/1/1996 (Gs/Gd) have spread globally and circulated in wild and domestic birds for a quarter century [1][2][3]. The hemagglutinin (HA) genes of Gs/Gd lineage H5 HPAIVs have evolved into 10 genetically distinct clades (0-9) and multiple subclades [4][5][6]. ...
Migratory water birds are considered to be carriers of high pathogenicity avian influenza viruses (HPAIVs). In Japan, mallards are often observed during winter, and HPAIV-infected mallards often shed viruses asymptomatically. In this study, we focused on mallards as potential carriers of HPAIVs and investigated whether individual wild mallards are repeatedly infected with HPAIVs and act as HPAIV carriers multiple times within a season. Mallards were experimentally infected with H5N1 and H5N8 HPAIVs that were isolated recently in Japan and phylogenetically belong to different hemagglutinin groups (G2a, G2b, and G2d). All of these strains are more infectious to mallards than to chickens, and the infected mallards shed enough virus to infect others, regardless of whether they exhibited clinical signs. Serum antibodies to the homologous antigen, induced by a single infection with a low virus dose (10 times the 50% mallard infectious dose), were maintained at detectable levels for 84 days. Immunity at 84 days post-inoculation fully protected the mallards from a challenge with the homologous strain, as demonstrated by a lack of viral shedding, and antibody levels did not increase significantly in most of these birds. Protection against heterologous challenge was also observed despite undetectable levels of antibodies to the challenge strain. Our findings suggest that repeated infections with homologous and heterologous HPAIV strains do not occur frequently in individual wild mallards within a season, particularly at low viral doses, and the frequency with which they act as carriers may be limited.
... Since emerging in 1996, the goose/Guangdong (Gs/GD) lineage of the H5 highly pathogenic avian influenza virus (HPAIV) has evolved into multiple distinct groups of viruses [1,2]. The 2.3.4.4b clade has resulted in widespread mortality of domestic and wild avian species across the globe [3]. ...
The avian influenza virus is a global pathogen with significant health and economic implications. While primarily a pathogen of wild and domestic birds, recent outbreaks of the H5N1 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b have caused mortality in a wide variety of mammals, including members of the Canidae family, on multiple continents. Despite sporadic mortality events globally, the epidemiology and pathobiology of H5N1 HPAIV in wild canids remains poorly defined. During 2022–2024, 41 wild canid carcasses (diagnostic cases), including 23 red foxes and 18 gray foxes, were tested for the influenza A virus (IAV) via PCR, with five red fox kits testing positive (12%). Infected animals had variably severe encephalitis, pneumonia, and occasionally myocarditis associated with strong immunolabeling for IAV. Serum from 269 wild canids in Pennsylvania was tested for antibodies to IAV, including 133 samples collected prior to 2021 (pre-H5N1 HPAIV 2.3.4.4b introduction) and 136 collected after 2022 (post-H5N1 HPAIV 2.3.4.4b introduction). All samples collected prior to 2021 were seronegative for IAV. Two coyotes from 2024 were seropositive for IAV but were negative for antibodies to the H5 and N1 subtypes. Collectively, these data suggest that while sporadic H5N1 HPAIV infection and mortality can occur in wild canids, particularly juvenile red foxes, infection was limited in these outwardly healthy and opportunistically sampled animals. Future studies should utilize a risk-based approach to target sampling of wild canids at increased risk for H5N1 HPAIV infection, such as those around waterfowl habitats or spatially around wild bird or domestic animal outbreaks.
... The disease is caused by various subtypes of influenza viruses a member of the genus Influenzavirus A and family Orthomyxoviridae such as H5N1, H5N3, and H5N8, with evolving genetic characteristics [16,17,5]. Avian influenza viruses are classified into low and high pathogenicity forms, with HPAI A(H5N1) known to infect mammals, particularly those in environments with high virus concentrations [23,18,19,20], instances of dairy cattle (or goats) contracting the H5N1 virus are relatively uncommon [44]. The disease, has been a global concern since its identification in 1878 in Italy [9,6].Avian influenza virus type A (H5N1) has been confirmed in dairy cattle in 12 states: 25 herds in Michigan, 22 in Idaho, 18 in Texas, eight in New Mexico, five each in South Dakota and Colorado, four in Kansas, three in Minnesota, two in Iowa, and one each in North Carolina, Ohio, and Wyoming [42,45]. ...
Avian Influenza (AI) poses a critical threat to cattle production worldwide, resulting in significant yield losses and economic damages. Despite the severity of AI, comprehensive modeling studies on its transmission dynamics within cattle populations remain limited. In this study, we present a mathematical model to describe the spread of AI among cattle. The model is based on the Susceptible-Infectious-Recovered (SIR) framework, adapted to capture the unique characteristics of AI transmission. The disease-free equilibrium of the model was computed, and the basic reproduction number for AI was calculated using the next-generation matrix method. Sensitivity analysis was conducted using normalized forward sensitivity method to determine the impact of various parameters on the basic reproduction number (). Analytical and numerical analyses indicate that increased contact rates between susceptible cattle and infected virus significantly raise the transmission rate of AI, impacting cattle health and productivity. Sensitivity analysis highlights that the recruitment rates of cattle and infection rates are the most influential parameters affecting . Control measures such as introducing AI-resistant cattle breeds and improving farm management practices to reduce infection rates may be used to mitigate the disease spread. This study enhances the understanding of AI transmission dynamics, providing valuable insights for developing targeted control strategies to protect cattle health and improve production.
... Since the first isolation of H10 subtype AIV from chickens in Germany, H10Ny isolates have been frequently detected in poultry and wild waterfowl worldwide, and a number of novel reassortant human cases of H10 subtype AIVs have been reported in recent years (Arzey et al., 2012;Kim et al., 2012;Lee et al., 2021). Studies have shown that H10Ny strains have become more adapted to mammals (Lv et al., 2023). ...
On January 30, 2024, China announced the first human case of H10N5 influenza infection. Prior to this, human cases of H10N7 and H10N8 had been reported. It is now appropriate to re-examine the evolution and future epidemiological trends of the H10 and N5 subtypes of avian influenza viruses (AIVs). In this study, we analyzed the reassortment characteristics of the first human-derived H10N5 AIV (A/Zhejiang/ZJU01/2023), as well as the evolutionary dynamics of the wild bird-derived H10 and N5 subtypes of AIVs over the past decade. Our findings indicate that the human-derived H10N5 AIV exhibited low pathogenicity. A/bean_goose/Korea/KNU-10/2022(H10N7) and A/mallard/Novosibirsk_region/962k/2018(H12N5) were identified as the potential reassortment parents. The virus has existed since 2022 and several isolations have been reported in Bangladesh. Phylogenetic analysis showed that H10Ny and HxN5 AIVs in China are clustered differently based on the East Asian-Australian (eastern) and Central Asian-Indian (western) migratory flyways. The H10Ny and HxN5 AIV reassortant strains may cause human infections through accidental spillover. It is possible that another center of AIV evolution, mutation, and reassortment may be developing along the migratory flyways in northeastern Asia, distinct from Europe, the Americas, and China's Yangtze River Delta and Pearl River Delta, which should be closely monitored to ensure the safety of the public.
... O utbreaks of highly pathogenic avian influenza (HPAI) viruses may cause high mortality in specific domestic poultry species and significant economic loss. Outbreaks of HPAI are often rapidly eliminated or eradicated through intervention, with only few viral lineages that continue evolving and causing outbreaks (1). One of them is the A/goose/Guangdong/1/96-like (Gs/Gd) lineage of H5Nx HPAI virus, which has evolved and persisted through diverse clades and genotypes since it first emerged in Southern China in 1996. ...
H5 highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/96 (Gs/Gd) lineage continue to evolve and cause outbreaks in domestic poultry and wild birds, with sporadic spillover infections in mammals. The global spread of clade 2.3.4.4b viruses via migratory birds since 2020 has facilitated the introduction of novel reassortants to China, where avian influenza of various subtypes have been epizootic or enzootic among domestic birds. To determine the impact of clade 2.3.4.4b re-introduction on local HPAI dynamics, we analyzed the genetic diversity of H5N6 and H5N8 detected from monthly poultry market surveillance in Guangdong, China, between 2020 and 2022. Our findings reveal that H5N6 viruses clustered in clades 2.3.4.4b and 2.3.4.4h, while H5N8 viruses were exclusively clustered in clade 2.3.4.4b. After 2020, the re-introduced clade 2.3.4.4b viruses replaced the clade 2.3.4.4h viruses detected in 2020. The N6 genes were divided into two clusters, distinguished by an 11 amino acid deletion in the stalk region, while the N8 genes clustered with clade 2.3.4.4 H5N8 viruses circulating among wild birds. Genomic analysis identified 10 transient genotypes. H5N6, which was more prevalently detected, was also clustered into more genotypes than H5N8. Specifically, H5N6 isolates contained genes derived from HPAI H5Nx viruses and low pathogenic avian influenza in China, while the H5N8 isolates contained genes derived from HPAI A(H5N8) 2.3.4.4b and A(H5N1) 2.3.2.1c. No positive selection on amino acid residues associated with mammalian adaptation was found. Our results suggest expanded genetic diversity of H5Nx viruses in China since 2021 with increasing challenges for pandemic preparedness.
IMPORTANCE
Since 2016/2017, clade 2.3.4.4b H5Nx viruses have spread via migratory birds to all continents except Oceania. Here, we evaluated the impact of the re-introduction of clade of 2.3.4.4b on highly pathogenic avian influenza (HPAI) virus genetic diversity in China. Twenty-two H5N6 and H5N8 HPAI isolated from monthly surveillance in two poultry markets in Guangdong between 2020 and 2022 were characterized. Our findings showed that clade 2.3.4.4h, detected in 2020, was replaced by clade 2.3.4.4b in 2021–2022. H5N6 (n = 18) were clustered into more genotypes than H5N8 (n = 4), suggesting that H5N6 may possess better replication fitness in poultry. Conversely, the H5N8 genotypes are largely derived from the clade 2.3.4.4b wild bird isolates. As clade 2.3.4.4b continues to spread via migratory birds, it is anticipated that the genetic diversity of H5N6 viruses circulating in China may continue to expand in the coming years. Continuous efforts in surveillance, genetic analysis, and risk assessment are therefore crucial for pandemic preparedness.
... LPAI viruses can evolve into HPAI viruses through mutational changes in the proteolytic cleavage site. Several H5 and H7 subtypic conversion events have occurred in Europe, Asia, North America, South America, Africa, and Oceania [16]. The robust replication property allows HPAI viruses to replicate in multiple organs, causing significant fatalities and leading to economic loss to the poultry industry. ...
The rapid geographic spread of the highly pathogenic avian influenza (HPAI) A(H5N1) virus in poultry, wild birds, and other mammalian hosts, including humans, raises significant health concerns globally. The recent emergence of HPAI A(H5N1) in agricultural animals such as cattle and goats indicates the ability of the virus to breach unconventional host interfaces, further expanding the host range. Among the four influenza types—A, B, C, and D, cattle are most susceptible to influenza D infection and serve as a reservoir for this seven-segmented influenza virus. It is generally thought that bovines are not hosts for other types of influenza viruses, including type A. However, this long-standing viewpoint has been challenged by the recent outbreaks of HPAI A(H5N1) in dairy cows in the United States. To date, HPAI A(H5N1) has spread into fourteen states, affecting 299 dairy herds and causing clinical symptoms such as reduced appetite, fever, and a sudden drop in milk production. Infected cows can also transmit the disease through raw milk. This review article describes the current epidemiological landscape of HPAI A(H5N1) in US dairy cows and its interspecies transmission events in other mammalian hosts reported across the globe. The review also discusses the viral determinants of tropism, host range, adaptative mutations of HPAI A(H5N1) in various mammalian hosts with natural and experimental infections, and vaccination strategies. Finally, it summarizes some immediate questions that need to be addressed for a better understanding of the infection biology, transmission, and immune response of HPAI A(H5N1) in bovines.
... As a result, subtype is used to denote the particular virus genotypes with a variety of observable and measurable characteristics. ( [14,10,12] . ...
This narrative study seeks to examine pneumonia induced by severe acute respiratory syndrome coronavirus-2, influenza, and adenovirus regarding clinical, laboratory, and radiological features. In conclusion, whereas these viral pneumonia clinics exhibit same symptomatology and laboratory results, we assert that there are notable distinctions, particularly in radiological findings.
... Avian influenza, commonly known as "bird flu," primarily affects poultry, causing high mortality rates and economic losses [1,2]. It is caused by low-pathogenic avian influenza (LPAI) and highly pathogenic (HPAI) viruses [3,4]. HPAI strains, including H5N6 and H5N8, have caused outbreaks. ...
Background and Aim
Avian influenza is a global threat to avian species, particularly in developing countries. Recombinant vaccines, including virus-like particles (VLPs), are promising strategies for preventing the spread of the disease. VLPs produced through the self-assembly of viral structural proteins without genomic material mimic native virions and are promising platforms for new vaccines. VLPs have been shown to elicit protective antibodies and are effective and safe vaccines against influenza. This study aimed to optimize the protocol for the production and characterization of H9N2 VLPs and their evaluation as a vaccine in broiler birds.
Materials and Methods
Low-pathogenic influenza virus (LPAI) H9N2 was isolated and characterized through whole-genome sequencing, and a VLP-based vaccine for LPAI H9N2 was prepared using a baculovirus expression system. Codon-optimized hemagglutinin (HA), neuraminidase (NA), and M1 were successfully cloned in pFastbac1 and expressed in SF9 cells. Proteins were characterized using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy after purification. Semi-purified proteins were tested as a vaccine in broiler chickens challenged with LPAI H9N2.
Results
Recombinant Bacmid DNA from positive clones was extracted and confirmed using a polymerase chain reaction. The transfection showed cytopathic effects, and the proteins were confirmed through western blotting and SDS-PAGE, which showed the sizes of HA = 62–64 KD, NA = 52 KD, and M1 = 25 KD. The shape and morphology were confirmed through transmission electron microscopy which revealed 100–150 nm size particles. As a result, the semi-purified VLPs (HA assay: 256) were tested as a vaccine for specific-pathogen free broiler birds; administered through subcutaneous and intranasal routes. The birds were challenged on the 28th day after vaccination with the H9N2 strain, and the birds showed significant cross-reactivity with the H9N2 strain.
Conclusion
The semi-purified VLP-based vaccine induced a significant immune response in vivo. This vaccine formulation has the potential to control avian influenza outbreaks in Pakistan’s poultry industry.
... Highly pathogenic avian influenza viruses (HPAIVs) of the H5 subtype pose a significant threat to global poultry industries and public health. Since their initial emergence in poultry in 1996 as the goose/Guangdong lineage (gs/GD) [1], followed by the first human cases in 1997 [2], H5N1 viruses have undergone continuous evolution and have caused significant economic losses in the poultry industry [3]. Over decades, reassortment events between H5 HPAIVs and low pathogenicity avian influenza viruses have led to the emergence of clade 2.3.4.4 H5Nx, particularly novel H5N6 and H5N8 viruses in Eurasia [4,5]. ...
... Due to the close phylogenetic 40 relationship among the H1, H2, and H5 subtypes, shared conserved sequences underlie their comparable binding capacity for the #219-specific serum antibodies (Fig. 3d). Notably, H5 is the most frequently identified subtypes of highly pathogenic avian influenza (HPAI) 33 , implying that vaccinations with #219 can be expected to prevent H5 HPAI infections in humans. QIV features potent yet narrow neutralizing protection and was used as a control vaccine in immunization 45 experiments. ...
The conserved hemagglutinin (HA) stem region is widely regarded as a highly promising target for the development of a universal influenza vaccine. Through structure-based design and iterative mutagenesis screening, we successfully engineered a soluble trimeric H1 stem-only protein, designated #219, which does not contain heterologous trimerization motifs and thus prevents off-target immune responses. As confirmed by a 2.97 Å-resolution cryo-EM structure, the overall conformation of #219 remains intact. Vaccination of mice with #219 protected against lethal heterologous viral challenges. Specific serum antibodies strongly cross-reacted with HAs of diverse influenza A subtypes, cross-neutralized various H1N1 viruses, and exhibited multiple antibody-effector functions mediating infection-permissive protection. We have developed a promising H1 stem immunogen for broad cross-protection against influenza infection and also proposed an efficacious design strategy for soluble homo-oligomeric proteins.
... A total of 47 LPAIV to HPAIV conversion events were recorded between 1959 and 2024 worldwide [3,4]. On 43 occasions, the newly emerged HPAIV strain was first detected in terrestrial birds, and 41 HPAIV were first detected in Galliformes, more specifically 33 in chickens and 8 in turkeys. ...
... Among wild-bird reservoirs, the virus typically exhibits a highly host-adapted phenotype, replicating in the epithelial cells of the respiratory and gastrointestinal tracts without causing clinical signs [6]. This low pathogenic (LP) virus can be transmitted from wild to domestic Pathogens 2024, 13, 810 2 of 9 birds, resulting in subclinical or mild infections that may eventually progress to a highly pathogenic (HP) disease, which is responsible for massive losses in the poultry industry [7]. It has traditionally been assumed that HPAIV cannot be sustained in wild-bird populations [8]; however, this assumption has been reconsidered since the emergence of the H5 A/Goose/GuangDong/1/96 (H5 Gs/GD/96) lineage [9]. ...
Highly pathogenic avian influenza (HPAI) is a highly contagious viral disease that represents a significant threat to poultry production worldwide. Variants of the HPAI virus (HPAIV) H5A/Goose/GuangDong/1/96 (H5 Gs/GD/96) lineage have caused five intercontinental epizootic waves, with the most recent, clade 2.3.4.4b, reaching Argentina in February 2023. Initially detected in wild birds, the virus quickly spread to backyard and commercial poultry farms, leading to economic losses, including the loss of influenza-free status (IFS). By March/April 2023 the epidemic had peaked and vaccination was seriously considered. However, the success of strict stamping-out measures dissuaded the National Animal Health Authority (SENASA) from authorizing any vaccine. Suspected cases sharply declined by May, and the last detection in commercial poultry was reported in June. The effective control and potential eradication of HPAIV in Argentina were due to SENASA’s early detection and rapid response, supported by private companies, veterinarians, and other stakeholders. Stamping-out measures have been effective for virus elimination and reduced farm-to-farm transmission; however, as the virus of this clade may remain present in wild birds, the risk of reintroduction into poultry production is high. Therefore, maintaining continuous active surveillance will be crucial for promptly detecting any new HPAIV incursion and taking appropriate action to contain virus dissemination.
... Recent occurrences of a rare and deadly influenza pandemic in Asia, Europe, North America, Oceania, and Africa have raised fears about its rapid spread. years the virus turned LPAI into HPAI (6). Even though H5N1 has not been able to transmit human-to-human, its ability to mutate and reassort suggests viral adaptation to humans (7). ...
Abstract: Avian influenza was a zoonotic disease, has caused several epidemics in domestic fowl, Avian
influenza may be contracted by several species, such as birds, animals, including humans, starlings, cats,
and dogs. Poultry, such as chickens, turkeys, ducks, waders, beach birds, and ratites, are also vulnerable to
the illness. Avian influenza results in significant financial losses in chicken populations. Influenza
prevention The varying degrees of pathogenicity shown by viruses in various avian species, together with
the diversity in viral features such as antigens and genes, provide challenges in accurately diagnosing viral
infections in poultry or wild birds. This is because viruses may cause disease in several bird species. More
precisely, this applies to the very contagious avian influenza viruses H5 and H7, which are now spreading.
Aims of the study are; know the Inflammatory pathways activated during AIV infection by q-PCR for
INFG and IL-4 Proinflammatory Mediators
H5 and Positive H7 samples were sequenced for H5 and H7 genes and a phylogenetic tree was built.
RT- PCR INFG + IL-4 Proinflammatory Mediaor are carried out must comprehend how the host immune
system reacts to AIVs and ratio nally design new immunotherapies and vaccines. H5N1 the serotype isolate
in these study has high genetic similarity isolates genotypes H5 in nearby countries in chicken/Iran,
Sweden, Romania, Nigeria, swan Pakistan and in duck of Korea.
The serotype H7N1 isolate in these study smiler to genetic viruses H7 isolated in chicken in Italy, Sweden
and England.
q-PCR results show LI4 and INFG were upregulated from lung and treachea of positive samples The result
of this study reveal that H5N1 and H7N1 are the common serotypes in these area and development of
vaccine to control infection are strongly recommended. This should be done by isolation of the infected
virus of the above strains and according to the authority’s guidance.
... 110 Understanding the pathobiological origins and evolutionary history of HPAIVs, including H7N3, is crucial for effective control and prevention strategies. 111 Measures such as culling infected flocks, disinfection of materials, and treatment with antiviral medications like oseltamivir have been used to control outbreaks of H7N3 avian influenza in various countries. 112 Although HPAI H7N3 virus infections have been reported in a small number of people with conjunctivitis since 2004 in Canada and other countries, 113 with some cases resulting in mild symptoms in humans, such as conjunctivitis and mild influenza-like illness, no human infection cases were reported during the recent 5 years. ...
Avian influenza viruses (AIVs) have the potential to cause severe illness in wild birds, domestic poultry, and humans. The ongoing circulation of highly pathogenic avian influenza viruses (HPAIVs) has presented significant challenges to global poultry industry and public health in recent years. This study aimed to elucidate the circulation of HPAIVs during 2019 to 2023. Specifically, we assess the alarming global spread and continuous evolution of HPAIVs. Moreover, we discuss their transmission and prevention strategies to provide valuable references for future prevention and control measures against AIVs.
... According to pathogenicity, avian influenza viruses can be divided into highly pathogenic and low pathogenic avian influenza viruses [5]. Most low pathogenic avian influenza viruses can cause mild respiratory, intestinal or reproductive diseases [6], while highly pathogenic avian influenza viruses are characterized by high incidence rate and mortality [7]. Among various highly pathogenic strains, the H5N1 avian influenza virus not only infects poultry but also humans, with a very high mortality rate [8], and is considered the most pathogenic avian influenza virus [9]. ...
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.
... 1,4,5 Since 1955, most outbreaks have been caused by three lineages, with the so-called A/goose/Guangdong/1996 H5N1 lineage widespread in poultry and responsible for human infections. 4 The 2.3.4.4b clade evolved within this lineage and has spread throughout the world, including North America via wild birds in December 2021. 2,4,6-10 On March 25 th 2024, H5N1 2.3.4.4b was detected in Texas dairy cattle herds concomitantly with herds in Michigan and Kansas. ...
Avian influenza (serotype H5N1) is a highly pathogenic virus that emerged in domestic waterfowl in 1996. Over the past decade, zoonotic transmission to mammals, including humans, has been reported. Although human to human transmission is rare, infection has been fatal in nearly half of patients who have contracted the virus in past outbreaks. The increasing presence of the virus in domesticated animals raises substantial concerns that viral adaptation to immunologically naïve humans may result in the next flu pandemic. Wastewater-based epidemiology (WBE) to track viruses was historically used to track polio and has recently been implemented for SARS-CoV2 monitoring during the COVID-19 pandemic. Here, using an agnostic, hybrid-capture sequencing approach, we report the detection of H5N1 in wastewater in nine Texas cities, with a total catchment area population in the millions, over a two-month period from March 4 th to April 25 th , 2024. Sequencing reads uniquely aligning to H5N1 covered all eight genome segments, with best alignments to clade 2.3.4.4b. Notably, 19 of 23 monitored sites had at least one detection event, and the H5N1 serotype became dominant over seasonal influenza over time. A variant analysis suggests avian or bovine origin but other potential sources, especially humans, could not be excluded. We report the value of wastewater sequencing to track avian influenza.
... Upon the introduction of LPAIVs of the H5 and H7 subtypes in terrestrial poultry, highly pathogenic avian influenza viruses (HPAIVs) may emerge. HPAIVs cause severe disease in poultry with mortality rates as high as 100% 4 . Besides these disastrous consequences on animal welfare and the poultry industry, spillover events to humans pose a continuous pandemic threat 5 . ...
Highly pathogenic avian influenza viruses (HPAIVs) emerge from H5 and H7 low pathogenic avian influenza viruses (LPAIVs), most frequently upon insertions of nucleotides coding for basic amino acids at the cleavage site (CS) of the hemagglutinin (HA). The exact molecular mechanism(s) underlying this genetic change and reasons underlying the restriction to H5 and H7 viruses remain unknown. Here, we developed a novel experimental system based on frame repair through insertions or deletions (indels) of HAs with single nucleotide deletions. Indels were readily detected in a consensus H5 LPAIV CS at low frequency, which was increased upon the introduction of only one substitution leading to a longer stretch of adenines at the CS. In contrast, we only detected indels in H6 when multiple nucleotide substitutions were introduced. These data show that nucleotide sequence is a key determinant of insertions in the HA CS, and reveal novel insights about the subtype-specificity of HPAIV emergence.
... Conversion events from LAPI-to-HPAI viruses have occurred multiple times mainly in intensive poultry production systems, where they had been controlled and maintained geographically limited (8,9). However, this panorama changed in 1996 when the HA of the HPAI strain A/goose/Guangdong/1/1996 reached wild migratory birds, where it has been maintained and disseminated giving rise to the globally distributed goose/Guangdong (Gs/Gd) lineage (10,11). ...
High Pathogenicity Avian Influenza (HPAI) poses a significant threat to public and animal health. Clade 2.3.4.4b recently emerged from the Eastern hemisphere and disseminated globally, reaching the Latin American (LATAM) region in late 2022 for the first time. HPAI in LATAM has resulted in massive mortalities and culling of poultry and wild birds, causing infection in mammals and humans. Despite its meaningful impact in the region, only occasional evidence about the genetic and epitope characteristics of the introduced HPAI is reported. Hence, this study seeks to phylogenetically characterize the molecular features and the source of HPAI in LATAM by evaluating potential antigenic variations. For such a purpose, we analyzed 302 whole genome sequences. All Latin American viruses are descendants of the 2.3.4.4b clade of the European H5N1 subtype. According to genomic constellations deriving from European and American reassortments, the identification of three major subtypes and eight sub-genotypes was achievable. Based on the variation of antigenic motifs at the HA protein in LATAM, we detected three potential antigenic variants, indicating the HA-C group as the dominant variant. This study decidedly contributes to unraveling the origin of the 2.3.4.4b clade in LATAM, its geographic dissemination, and evolutionary dynamics within Latin American countries. These findings offer useful information for public health interventions and surveillance initiatives planned to prevent and manage the transmission of avian influenza viruses.
... Low-pathogenicity AIV (LPAIV) of the H5 and H7 subtypes may evolve toward highly pathogenic AIV (HPAIV) upon transmission into highly dense domestic bird populations (Lee, Criado, and Swayne 2021). HPAIV strains are primarily characterized by multiple basic amino acid residues at the HA cleavage site processed by ubiquitous proteases, resulting in a fast-spreading deadly disease with increased pathogenicity (de Bruin et al. 2022). ...
The highly pathogenic avian influenza viruses of the clade 2.3.4.4b have caused unprecedented deaths in South American wild birds, poultry, and marine mammals. In September 2023, pinnipeds and seabirds appeared dead on the Uruguayan Atlantic coast. Sixteen influenza virus strains were characterized by real-time reverse transcription PCR and genome sequencing in samples from sea lions (Otaria flavescens), fur seals (Arctocephalus australis), and terns (Sterna hirundinacea). Phylogenetic and ancestral reconstruction analysis showed that these strains have pinnipeds as the most likely ancestral host, representing a recent introduction of the clade 2.3.4.4b in Uruguay. The Uruguayan and closely related strains from Peru (sea lions) and Chile (sea lions and a human case) carry mammalian adaptative residues 591K and 701N in the viral polymerase basic protein 2 (PB2). Our findings suggest that the clade 2.3.4.4b strains in South America may have spread from mammals to mammals and seabirds, revealing a new transmission route.
... The major determinant of virulence is the HA gene, although other genes may contribute to increased pathogenicity [14,15]. For subtype H5 viruses, the HPAI phenotype correlates with the presence of multiple basic amino acids at the endoproteolytic cleavage site (CS) of the HA protein [16][17][18]. The IVPI experiments take long to perform, are associated with high costs, and are making use of animals. ...
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.
... LPAI has a low mortality rate and ability to infect, causing little to no disease in birds, because they can only replicate in tracheal tissues and the small intestine [24]. However, the H5/H7 subtypes of low pathogenicity (common in poultry and wild waterfowl) [37] can mutate by insertion and recombination processes in the proteolytic cleavage site of HA [6] until becoming HPAI viruses [38]. ...
Citation: Simancas-Racines, A.; Cadena-Ullauri, S.; Guevara-Ramírez, P.; Zambrano, A.K.; Simancas-Racines, D. Avian Influenza: Strategies to Manage an Outbreak. Pathogens 2023, 12, 610. Abstract: Avian influenza (AI) is a contagious disease among the poultry population with high avian mortality, which generates significant economic losses and elevated costs for disease control and outbreak eradication. AI is caused by an RNA virus part of the Orthomyxoviridae family; however, only Influenzavirus A is capable of infecting birds. AI pathogenicity is based on the lethality, signs, and molecular characteristics of the virus. Low pathogenic avian influenza (LPAI) virus has a low mortality rate and ability to infect, whereas the highly pathogenic avian influenza (HPAI) virus can cross respiratory and intestinal barriers, diffuse to the blood, damage all tissues of the bird, and has a high mortality rate. Nowadays, avian influenza is a global public health concern due to its zoonotic potential. Wild waterfowl is the natural reservoir of AI viruses, and the oral-fecal path is the main transmission route between birds. Similarly, transmission to other species generally occurs after virus circulation in densely populated infected avian species, indicating that AI viruses can adapt to promote the spread. Moreover, HPAI is a notifiable animal disease; therefore, all countries must report infections to the health authorities. Regarding laboratory diagnoses, the presence of influenza virus type A can be identified by agar gel immunodiffusion (AGID), enzyme immunoassay (EIA), immunofluorescence assays, and enzyme-linked immunoadsorption assay (ELISAs). Furthermore, reverse transcription polymerase chain reaction is used for viral RNA detection and is considered the gold standard for the management of suspect and confirmed cases of AI. If there is suspicion of a case, epidemiological surveillance protocols must be initiated until a definitive diagnosis is obtained. Moreover, if there is a confirmed case, containment actions should be prompt and strict precautions must be taken when handling infected poultry cases or infected materials. The containment measures for confirmed cases include the sanitary slaughter of infected poultry using methods such as environment saturation with CO 2 , carbon dioxide foam, and cervical dislocation. For disposal, burial, and incineration, protocols should be followed. Lastly, disinfection of affected poultry farms must be carried out. The present review aims to provide an overview of the avian influenza virus, strategies for its management, the challenges an outbreak can generate, and recommendations for informed decision making.
... The worldwide spread of H5 high-pathogenicity avian influenza (HPAI) viruses (HPAIVs) has caused huge outbreaks and economic losses in poultry over the past few years (World Organization for Animal Health: WOAH). Almost all recent H5 HPAIVs arose from A/goose/Guangdong/1/1996_(H5N1) (Gs/Gd lineage), which was first detected in China in 1996 [1,2] and evolved into multiple clades and subclades defined by hemagglutinin (HA) genes [3]. H5 HPAIVs in clade 2.3.4.4 emerged in 2013-2014 and became dominant among outbreaks worldwide [4]. ...
In winter 2021–2022, H5N1 and H5N8 high-pathogenicity avian influenza (HPAI) viruses (HPAIVs) caused serious outbreaks in Japan: 25 outbreaks of HPAI at poultry farms and 107 cases in wild birds or in the environment. Phylogenetic analyses divided H5 HPAIVs isolated in Japan in the winter of 2021–2022 into three groups—G2a, G2b, and G2d—which were disseminated at different locations and times. Full-genome sequencing analyses of these HPAIVs revealed a strong relationship of multiple genes between Japan and Siberia, suggesting that they arose from reassortment events with avian influenza viruses (AIVs) in Siberia. The results emphasize the complex of dissemination and reassortment events with the movement of migratory birds, and the importance of continual monitoring of AIVs in Japan and Siberia for early alerts to the intrusion of HPAIVs.
Since their emergence in Guangdong, China, in 1996, Gs/GD H5 highly pathogenic avian influenza viruses (HPAIVs) have diversified into multiple clades, spreading globally through wild bird migrations and causing substantial losses in poultry and wildlife. In South Korea, HPAIVs, including H5N1, H5N8, and H5N6 subtypes, have been repeatedly introduced since 2003. This review examines the epidemiology, genetic characteristics, and pathobiological features of these viruses in South Korea. Outbreaks typically occur between October and December, aligning with the arrival of wintering migratory birds. While outbreaks in poultry farms dominated before 2018, wild bird cases became more prevalent in subsequent years. Seasonal outbreaks in poultry have declined, but large-scale mortality events in wild birds emerged biennially from 2020. Genotypic diversity has increased since 2014 due to reassortment with low pathogenic viruses, with novel genomic traits detected in recent seasons. Infection studies show consistently fatal outcomes in chickens, while high mortality in domestic ducks was observed only with two of the studied strains, despite efficient transmission. Wild bird studies reveal species-specific roles in viral shedding and transmission. This review underscores the dynamic nature of HPAI outbreaks, highlighting the importance of surveillance, biosecurity, and genetic and pathogenicity analyses to mitigate future risks.
Background: Avian influenza (AI), caused by orthomyxoviruses, is a globally significant disease affecting avian and non-avian species. It manifests in two variants, according to the two biovariants of the virus differentiated as highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) strains, both of which compromise animal welfare, reduce productivity, and cause substantial economic loss. The zoonotic potential of HPAI strains, particularly the currently dominant clade 2.3.4.4b, raises concerns about public health and epidemic risks. This review assesses the results of current vaccine trials targeting HPAI clade 2.3.4.4b, emphasizing these studies because most outbreak strains in domestic poultry currently belong to this dominant clade. Methods: Multiple scientific databases comprised reports of research trials on vaccine efficacy against HPAI clade 2.3.4.4b. The Boolean term “Clade 2.3.4.4b AND vaccine” was entered into the following databases: PubMed, PubAg, Scopus, Cochrane Library, and ScienceDirect. Results: The resulting papers were analyzed. Studies revealed that antigenic similarity between vaccine and field strains enhances protective efficacy (PE), reduces viral shedding, and improves hemagglutination inhibition titers. While multivalent vaccines showed potential, results were inconsistent and varied depending on strain compatibility. Single-dose vaccines may provide sufficient PE for poultry, though ducks and geese often require multiple doses, and long-term PE is yet unknown. It was discovered that vector vaccines can provide appropriate PE against clade 2.3.4.4.b. Conclusions: Further analysis is needed as their effects may be short-lived, and subsequent doses may be required. Limited research exists on the long-term efficacy of these vaccines and their effectiveness in many avian species. Addressing these gaps is crucial for optimizing vaccination strategies. A re-evaluation of vaccination strategies is recommended but essential to implement adequate biosecurity measures on in poultry farms. This review synthesizes current evidence and may assist veterinarians and authorities in deciding whether to apply or license vaccines to reduce economic losses caused by AI.
The global dissemination of H5 avian influenza viruses represents a significant threat to both human and animal health. In this study, we conducted a genome‐wide siRNA library screening against the highly pathogenic H5N1 influenza virus, leading us to the identification of 457 cellular cofactors (441 proviral factors and 16 antiviral factors) involved in the virus replication cycle. Gene Ontology term enrichment analysis revealed that the candidate gene data sets were enriched in gene categories associated with mRNA splicing via spliceosome in the biological process, integral component of membrane in the cellular component, and protein binding in the molecular function. Reactome pathway analysis showed that the immune system (up to 63 genes) was the highest enriched pathway. Subsequent comparisons with four previous siRNA library screenings revealed that the overlapping rates of the involved pathways were 8.53%–62.61%, which were significantly higher than those of the common genes (1.85%–6.24%). Together, our genome‐wide siRNA library screening unveiled a panorama of host cellular networks engaged in the regulation of highly pathogenic H5N1 influenza virus replication, which may provide potential targets and strategies for developing novel antiviral countermeasures.
Influenza, a major “One Health” threat, has gained heightened attention following recent reports of
highly pathogenic avian influenza in dairy cattle and cow-to-human transmission in the USA. This
review explores general aspects of influenza A virus (IAV) biology, its interactions with mammalian
hosts, and discusses the key considerations for developing vaccines to prevent or curtail IAV infection
in the bovine mammary gland and its spread through milk
Background. Subtypes H5, H7, H9 and H10 of the influenza A virus have a high pandemic potential, due to their ability to adapt to new hosts by acquiring mutations and genetic reassortment. Sporadic cases of human infection are often characterized by severe course of disease and high mortality. The development of a PCR reagent kit for in vitro diagnostics of potentially pandemic influenza A viruses subtypes is important. Objective. Development and implementation of a diagnostic reagent kit for the detection of influenza A virus RNA and typing of subtypes H5, H7, H9, H10 in human biological material using RT-PCR with hybridization-fluorescence detection. Material and methods. Specific primers and hybridization-fluorescent probes have been developed for the amplification of influenza A virus RNA and H5, H7, H9, and H10 virus subtypes. Optimal RT-PCR conditions were selected and the analytical and diagnostic characteristics of test kit based on selected specific oligonucleotides were evaluated. Clinical and laboratory tests of a set of reagents were carried out to confirm its effectiveness and safety. Results. The analytical sensitivity of the PCR reagent kit for in vitro diagnostics was assessed on model samples of human biomaterial. The value is 1·103 GE of the target viral RNA per ml of nasopharyngeal and oropharyngeal mucosa swabs or sputum. The absence of cross-reactions with the genetic material of influenza A virus of other subtypes, influenza viruses B, C, other viruses and bacteria was shown. To assess the diagnostic characteristics, clinical material from patients with typical clinical symptoms of acute respiratory viral infections, as well as model samples simulating biological material from patients with influenza subtypes H5, H7, H9 and H10 were used. To obtain these model samples, clinical material was contaminated with strains of H5N1, H7N3, H9N2 and H10N5 subtypes or with biological material from birds affected by influenza. As a result, 343 samples containing RNA of influenza A virus of various subtypes, including H5, H7, H9, H10, and not containing RNA of influenza A virus, were studied. Complete agreement of the results with the results of the comparison system was shown. Conclusion. Based on the results of clinical laboratory tests, it was confirmed that the developed test kit has high analytical and diagnostic characteristics, is effective and safe when used as intended, and can be recommended for registration as a medical device for in vitro diagnostics.
Population immunity is a determining factor in relation to the spread of various variants of the influenza virus, and therefore is of great importance for predicting epidemics, characterizing the epidemic process and assessing the effectiveness of vaccination campaigns. The aim of the work was to monitor markers of seasonal influenza viruses and avian influenza viruses in the blood serum of residents of the Russian Federation in 2023–2024. Materials and methods . Blood serum samples from healthy donors were collected in the Siberian Federal District of the Russian Federation in October-November 2023. In addition, blood sera from people who had had contact with sick and/or dead birds and from residents of regions located on migration routes of wild waterfowl were studied in HI-test (hemagglutination inhibition) and virus neutralization. Results and discussion . It is shown that ahead of the epidemic season of 2023/2024, population immunity to influenza in the Siberian Federal District was at the level recommended by the World Health Organization (WHO) – at least 50 % of the immune population. However, among individuals who had had contact with sick and/or dead birds, humoral immunity to seasonal influenza was significantly lower – from 5 % to 30 % seropositive, depending on the region. HI-test on avian influenza viruses A/H5Nx and A/H9N2 has revealed 0 and 3.7 % of positive samples, respectively. The risk of a pandemic influenza virus emergence can be reduced by 75–100 % vaccination against seasonal influenza and monitoring antibody levels in poultry farm workers and employees of other organizations directly involved in poultry breeding and processing.
The emergence and evolution of avian influenza A viruses (AIVs) pose significant challenges to both public health and animal husbandry worldwide. Here, we characterized a novel reassortant highly pathogenic avian influenza virus (HPAIV), clade 2.3.4.4b H5N6, that was isolated from a mandarin duck in South Korea in December 2023. Phylogenetic and molecular analyses show that the hemagglutinin (HA) gene of the 23-JBN-F12-36/H5N6 virus clustered with HPAIV clade 2.3.4.4b H5N1 viruses, which were circulating in South Korea and Japan in 2022–2023. The M and polymerase acidic (PA) genes also revealed a close association with the HPAIV clade 2.3.4.4b H5N1 AIV that was identified previously in South Korea during November 2022. Notably, the neuraminidase (NA) gene of the 23-JBN-F12-36/H5N6 virus was estimated to have its origins in the HPAIV clade 2.3.4.4h H5N6 prevalent in poultry in China, and it is clustered with the AIVs that are associated with human infection cases. Taken together, these results show that the virus has been produced by reassortment with H5N1 HPAIV, which is prevalent in wild birds; H5N6 HPAIV, which is circulated in poultry in China; and the internal genes of low pathogenic avian influenza viruses (LPAIVs). In light of the reassortment of HPAIVs circulating in existing wild birds and HPAIVs circulating in poultry in China within the 2.3.4.4b H5Nx clade, it is imperative to strengthen active surveillance across wild bird populations, poultry farms, and live poultry markets, and to inform for the effective design of improved prevention and control strategies.
The prevalence of highly pathogenic avian influenza (HPAI) A(H5N1) viruses has increased in wild birds and poultry worldwide, and concomitant outbreaks in mammals have occurred. During 2023, outbreaks of HPAI H5N1 virus infections were reported in cats in South Korea. The H5N1 clade 2.3.4.4b viruses isolated from 2 cats harbored mutations in the polymerase basic protein 2 gene encoding single amino acid substitutions E627K or D701N, which are associated with virus adaptation in mammals. Hence, we analyzed the pathogenicity and transmission of the cat-derived H5N1 viruses in other mammals. Both isolates caused fatal infections in mice and ferrets. We observed contact infections between ferrets, confirming the viruses had high pathogenicity and transmission in mammals. Most HPAI H5N1 virus infections in humans have occurred through direct contact with poultry or a contaminated environment. Therefore, One Health surveillance of mammals, wild birds, and poultry is needed to prevent potential zoonotic threats.
Highly pathogenic avian influenza (HPAI) viruses have spread at an unprecedented scale, leading to mass mortalities in birds and mammals. In 2023, a transatlantic incursion of HPAI A(H5N5) viruses into North America was detected, followed shortly thereafter by a mammalian detection. As these A(H5N5) viruses were similar to contemporary viruses described in Eurasia, the transatlantic spread of A(H5N5) viruses was most likely facilitated by pelagic seabirds. Some of the Canadian A(H5N5) viruses from birds and mammals possessed the PB2-E627K substitution known to facilitate adaptation to mammals. Ferrets inoculated with A(H5N5) viruses showed rapid, severe disease onset, with some evidence of direct contact transmission. However, these viruses have maintained receptor binding traits of avian influenza viruses and were susceptible to oseltamivir and zanamivir. Understanding the factors influencing the virulence and transmission of A(H5N5) in migratory birds and mammals is critical to minimize impacts on wildlife and public health.
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.
Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.
IMPORTANCE
Influenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.
Since 2020, clade 2.3.4.4b highly pathogenic avian influenza H5N8 and H5N1 viruses have swept through continents, posing serious threats to the world. Through comprehensive analyses of epidemiological, genetic, and bird migration data, we found that the dominant genotype replacement of the H5N8 viruses in 2020 contributed to the H5N1 outbreak in the 2021/2022 wave. The 2020 outbreak of the H5N8 G1 genotype instead of the G0 genotype produced reassortment opportunities and led to the emergence of a new H5N1 virus with G1’s HA and MP genes. Despite extensive reassortments in the 2021/2022 wave, the H5N1 virus retained the HA and MP genes, causing a significant outbreak in Europe and North America. Furtherly, through the wild bird migration flyways investigation, we found that the temporal–spatial coincidence between the outbreak of the H5N8 G1 virus and the bird autumn migration may have expanded the H5 viral spread, which may be one of the main drivers of the emergence of the 2020–2022 H5 panzootic.
IMPORTANCE
Since 2020, highly pathogenic avian influenza (HPAI) H5 subtype variants of clade 2.3.4.4b have spread across continents, posing unprecedented threats globally. However, the factors promoting the genesis and spread of H5 HPAI viruses remain unclear. Here, we found that the spatiotemporal genotype replacement of H5N8 HPAI viruses contributed to the emergence of the H5N1 variant that caused the 2021/2022 panzootic, and the viral evolution in poultry of Egypt and surrounding area and autumn bird migration from the Russia–Kazakhstan region to Europe are important drivers of the emergence of the 2020–2022 H5 panzootic. These findings provide important targets for early warning and could help control the current and future HPAI epidemics.
Based on the pathogenicity in chickens, most H1-H16 avian influenza viruses (AIV) cause mild diseases, whereas some of the H5 and H7 AI viruses cause severe, systemic disease. The number of basic amino acids in the haemagglutinin (HA) cleavage site of AIV plays a critical role in pathogenicity. As we gain a greater understanding of the molecular mechanisms of pathogenicity, genome sequencing of the HA0 cleavage site has assumed a greater role in assessment of the potential pathogenicity of H5 and H7 viruses. We validated the use of HA cleavage site motif analysis by comparing molecular pathotyping data against experimental in vivo (intravenous pathogenicity index [IVPI] and lethality) data for determination of both low pathogenicity and high pathogenicity AI virus declaration with the goal of expediting pathotype confirmation and further reducing the reliance on in vivo testing. Our data provide statistical support to the continued use of molecular determination of pathotype for AI viruses based on the HA cleavage site sequence in the absence of an in vivo study determination. This approach not only expedites the declaration process of highly pathogenic AIV (HPAIV) but also reduces the need for experimental in vivo testing of H5 and H7 viruses.
Clade 2.3.4.4b highly pathogenic avian influenza A (HPAI) viruses have been detected in wild birds worldwide, causing recurrent outbreaks since 2016. During the winter of 2021–2022, we detected 1 H5N8 and 43 H5N1 clade 2.3.4.4b HPAI viruses from wild birds in South Korea. Phylogenetic analysis revealed that HA gene of H5N1 viruses was divided into two genetically distinct groups (N1.G1 and N1.G2). Bayesian phylodynamic analysis demonstrated that wild birds play a vital role in viral transmission and long-term maintenance. We identified five genotypes (N1.G1.1, N1.G2, N1.G2.1, N1.G2.2, and N1.G2.2.1) having distinct gene segment constellations most probably produced by reassortments with low-pathogenic avian influenza viruses. Our results suggest that clade 2.3.4.4b persists in wild birds for a long time, causing continuous outbreaks, compared with previous clades of H5 HPAI viruses. Our study emphasizes the need for enhancing control measures in response to the changing viral epidemiology.
The acquisition of a multibasic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein is the main determinant of the conversion of low pathogenic avian influenza viruses into highly pathogenic strains, facilitating HA cleavage and virus replication in a broader range of host cells. In nature, substitutions or insertions in HA RNA genomic segments that code for multiple basic amino acids have been observed only in the HA genes of two out of sixteen subtypes circulating in birds, H5 and H7. Given the compatibility of MBCS motifs with HA proteins of numerous subtypes, this selectivity was hypothesized to be determined by the existence of specific motifs in HA RNA, in particular structured domains. In H5 and H7 HA RNAs, predictions of such domains have yielded alternative conserved stem-loop structures with the cleavage site codons in the hairpin loops. Here, potential RNA secondary structures were analyzed in the cleavage site regions of HA segments of influenza viruses of different types and subtypes. H5- and H7-like stem-loop structures were found in all known influenza A virus subtypes and in influenza B and C viruses with homology modeling. Nucleotide covariations supported this conservation to be determined by RNA structural constraints that are stronger in the domain-closing bottom stems as compared to apical parts. The structured character of this region in (sub-)types other than H5 and H7 indicates its functional importance beyond the ability to evolve toward an MBCS responsible for a highly pathogenic phenotype.
Low pathogenicity avian influenza viruses (LPAIVs) are generally asymptomatic in their natural avian hosts. LPAIVs can evolve into highly pathogenic forms, which can affect avian and human populations with devastating consequences. The switch to highly pathogenic avian influenza virus (HPAIV) from LPAIV precursors requires the acquisition of multiple basic amino acids in the haemagglutinin cleavage site (HACS) motif. Through reverse genetics of an H5N1 HPAIV, and experimental infection of chickens, we determined that viruses containing five or more basic amino acids in the HACS motif were preferentially selected over those with three to four basic amino acids, leading to rapid replacement with virus types containing extended HACS motifs. Conversely, viruses harbouring low pathogenicity motifs containing two basic amino acids did not readily evolve to extended forms, suggesting that a single insertion of a basic amino acid into the cleavage site motif of low-pathogenic viruses may lead to escalating selection for extended motifs. Our results may explain why mid-length forms are rarely detected in nature. The stability of the short motif suggests that pathogenicity switching may require specific conditions of intense selection pressure (such as with high host density) to boost selection of the initial mid-length HACS forms.
Over the years, the emergence of novel H5 and H7 highly pathogenic avian influenza viruses (HPAI) has been taking place through two main mechanisms: first, the conversion of a low pathogenic into a highly pathogenic virus, and second, the reassortment between different genetic segments of low and highly pathogenic viruses already in circulation. We investigated and summarized the literature on emerging HPAI H5 and H7 viruses with the aim of building a spatio-temporal database of all these recorded conversions and reassortments events. We subsequently mapped the spatio-temporal distribution of known emergence events, as well as the species and production systems that they were associated with, the aim being to establish their main characteristics. From 1959 onwards, we identified a total of 39 independent H7 and H5 LPAI to HPAI conversion events. All but two of these events were reported in commercial poultry production systems, and a majority of these events took place in high-income countries. In contrast, a total of 127 reassortments have been reported from 1983 to 2015, which predominantly took place in countries with poultry production systems transitioning from backyard to intensive production systems. Those systems are characterized by several co-circulating viruses, multiple host species, regular contact points in live bird markets, limited biosecurity within value chains, and frequent vaccination campaigns that impose selection pressures for emergence of novel reassortants. We conclude that novel HPAI emergences by these two mechanisms occur in different ecological niches, with different viral, environmental and host associated factors, which has implications in early detection and management and mitigation of the risk of emergence of novel HPAI viruses.
In March 2017, highly pathogenic avian influenza A(H7N9) was detected at 2 poultry farms in Tennessee, USA. Surveillance data and genetic analyses indicated multiple introductions of low pathogenicity avian influenza virus before mutation to high pathogenicity and interfarm transmission. Poultry surveillance should continue because low pathogenicity viruses circulate and spill over into commercial poultry. © 2017, Centers for Disease Control and Prevention (CDC). All rights reserved.
Since its emergence in 2013, the H7N9 low-pathogenic avian influenza virus (LPAIV) has been circulating in domestic poultry in China, causing five waves of human infections. A novel H7N9 highly pathogenic avian influenza virus (HPAIV) variant possessing multiple basic amino acids at the cleavage site of the hemagglutinin (HA) protein was first reported in two cases of human infection in January 2017. More seriously, those novel H7N9 HPAIV variants have been transmitted and caused outbreaks on poultry farms in eight provinces in China. Herein, we demonstrate the presence of three different amino acid motifs at the cleavage sites of these HPAIV variants which were isolated from chickens and humans and likely evolved from the preexisting LPAIVs. Animal experiments showed that these novel H7N9 HPAIV variants are both highly pathogenic in chickens and lethal to mice. Notably, humanorigin viruses were more pathogenic in mice than avian viruses, and the mutations in the PB2 gene associated with adaptation to mammals (E627K, A588V, and D701N) were identified by next-generation sequencing (NGS) and Sanger sequencing of the isolates from infected mice. No polymorphisms in the key amino acid substitutions of PB2 and HA in isolates from infected chicken lungs were detected by NGS. In sum, these results highlight the high degree of pathogenicity and the valid transmissibility of this new H7N9 variant in chickens and the quick adaptation of this new H7N9 variant to mammals, so the risk should be evaluated and more attention should be paid to this variant.
The novel low-pathogenic avian influenza A H7N9 viruses (LPAI H7N9 viruses) have been a threat to public health since their emergence in 2013 because of the high rates of mortality and morbidity that they cause. Recently, highly pathogenic variants of these avian influenza A H7N9 viruses (HPAI H7N9 viruses) have emerged and caused human infections and outbreaks among poultry in mainland China. However, it is still unclear how the HPAI H7N9 virus was generated and how it evolved and spread in China. Here, we show that the ancestor virus of the HPAI H7N9 viruses originated in the Yangtze River Delta region and spread southward to the Pearl River Delta region, possibly through live poultry trade. After introduction into the Pearl River Delta region, the origin LPAI H7N9 virus acquired four amino acid insertions in the hemagglutinin (HA) protein cleavage site and mutated into the HPAI H7N9 virus in late May 2016. Afterward, the HPAI H7N9 viruses further reassorted with LPAI H7N9 or H9N2 viruses locally and generated multiple different genotypes. As of 14 July 2017, the HPAI H7N9 viruses had spread from Guangdong Province to at least 12 other provinces. The rapid geographical expansion and genetic evolution of the HPAI H7N9 viruses pose a great challenge not only to public health but also to poultry production. Effective control measures, including enhanced surveillance, are therefore urgently needed.
IMPORTANCE
The LPAI H7N9 virus has caused five outbreak waves in humans and was recently reported to have mutated into highly pathogenic variants. It is unknown how the HPAI H7N9 virus originated, evolved, and disseminated in China. In this study, we comprehensively analyzed the sequences of HPAI H7N9 viruses from 28 human and 21 environmental samples covering eight provinces in China that were taken from November 2016 to June 2017. The results show that the ancestor virus of the HPAI H7N9 viruses originated in the Yangtze River Delta region. However, the insertion of four amino acids into the HA protein cleavage site of an LPAI H7N9 virus occurred in late May 2016 in the Pearl River Delta region. The mutated HPAI H7N9 virus further reassorted with LPAI H7N9 or H9N2 viruses that were cocirculating in poultry. Considering the rapid geographical expansion of the HPAI H7N9 viruses, effective control measures are urgently needed.
In mid-January 2016, an outbreak of H7N8 high-pathogenicity avian influenza virus (HPAIV) in commercial turkeys occurred in Indiana. Surveillance within the 10km control zone identified H7N8 low-pathogenicity avian influenza virus (LPAIV) in nine surrounding turkey flocks but no other HPAIV-affected premises. We sequenced four of the H7N8 HPAIV isolated from the single farm and nine LPAIV identified during control zone surveillance. Evaluation included phylogenetic network analysis indicating close relatedness across the HPAIV and LPAIV, and that the progenitor H7N8 LPAIV spread among the affected turkey farms in Indiana, followed by spontaneous mutation to HPAIV on a single premise through acquisition of three basic amino acids at the hemagglutinin cleavage site. Deep sequencing of the available viruses failed to identify subpopulations in either the HPAIV or LPAIV suggesting mutation to HPAIV likely occurred on a single farm and the HPAIV did not spread to epidemiologically linked LPAIV-affected farms.
The genetic composition of an H5 subtype hemagglutinin gene quasispecies, obtained from ostrich tissues that had been infected with H5 subtype influenza virus was analysed using a next generation sequencing approach. The first evidence for the reiterative copying of a poly (U) stretch in the connecting peptide region in the haemagglutinin cleavage site (HACS) by the viral RNA-dependent RNA polymerase (RdRp) is provided. Multiple non-consensus species of RNA were detected in the infected host, corresponding to likely intermediate sequences between the putative low pathogenic precursor nucleotide sequence of the H5 influenza strain and the highly pathogenic avian influenza virus gene sequence. In silico analysis of the identified RNA sequences predicted that the intermediary H5 sequence PQREKRGLF plays an important role in subsequent mutational events that relocate the HACS coding region from stable base-paired RNA regions to a single-stranded bulge, thereby priming the connecting peptide coding region for RdRp slippage.
Several new highly pathogenic (HP) H5 avian influenza virus (AIV) have been detected in poultry farms from south-western France since November 2015, among which an HP H5N1. The zoonotic potential and origin of these AIVs immediately became matters of concern. One virus of each subtype H5N1 (150169a), H5N2 (150233) and H5N9 (150236) was characterised. All proved highly pathogenic for poultry as demonstrated molecularly by the presence of a polybasic cleavage site in their HA protein ? with a sequence (HQRRKR/GLF) previously unknown among avian H5 HPAI viruses ? or experimentally by the in vivo demonstration of an intravenous pathogenicity index of 2.9 for the H5N1 HP isolate. Phylogenetic analyses based on the full genomes obtained by NGS confirmed that the eight viral segments of the three isolates were all part of avian Eurasian phylogenetic lineage but differed from the Gs/Gd/1/96-like lineage. The study of the genetic characteristics at specific amino acid positions relevant for modulating the adaptation to and the virulence for mammals showed that presently, these viruses possess most molecular features characteristic of AIV and lack some major characteristics required for efficient respiratory transmission to or between humans. The three isolates are therefore predicted to have no significant pandemic potential.
Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes.
Highly pathogenic H5N1 avian influenza virus (A/H5N1) devastated the poultry industry and continues to pose a pandemic threat. Studying the progressive genetic changes in A/H5N1 after long term circulation in poultry may help to better understand A/H5N1 biology in birds. A/H5N1 clade 2.2.1.1 antigenic drift viruses have been isolated from vaccinated commercial poultry in Egypt. They exhibit a peculiar stepwise accumulation of glycosylation sites (GS) in the hemagglutinin (HA) with viruses carrying, beyond the conserved 5 GS, additional GS at amino acid residues 72, 154, 236 and 273 resulting in 6, 7, 8 or 9 GS in the HA. Available information about the impact of glycosylation on virus fitness and pathobiology is mostly derived from mammalian models. Here, we generated recombinant viruses imitating the progressive acquisition of GS in HA and investigated their biological relevance in vitro and in vivo. Our in vitro results indicated that the accumulation of GS correlated with increased glycosylation, increased virus replication, neuraminidase activity, cell-to-cell spread and thermostability, however, strikingly, without significant impact on virus escape from neutralizing antibodies. In vivo, glycosylation modulated virus virulence, tissue tropism, replication and chicken-to-chicken transmission. Predominance in the field was toward viruses with hyperglycosylated HA. Together, progressive glycosylation of the HA may foster persistence of A/H5N1 by increasing replication, stability and bird-to-bird transmission without significant impact on antigenic drift.
There are three conserved N-linked glycosites, namely, Asn10, Asn23, and Asn286, at the stem region of hemagglutinin (HA) in H5N1 avian influenza viruses (AIVs). To understand the effect of glycosylation in the stem domain of HA on the biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to generate different patterns of stem glycans on the HA of A/Mallard/Huadong/S/2005. The results indicated that these three N-glycans were dispensable for the generation of replication-competent influenza viruses. However, when N-glycans at Asn10 plus either Asn23 or Asn268 were removed, the cleavability of HA was almost completely blocked, leading to a significant decrease of the growth rates of the mutant viruses in MDCK and CEF in comparison with that of the wild-type (WT) virus. Moreover, the mutant viruses lacking these oligosaccharides, particularly the N-glycan at Asn10, revealed a significant decrease in thermostability and pH stability compared with the WT virus. Interestingly, the mutant viruses induced a lower level of neutralizing antibodies against the WT virus, and most of the mutant viruses were more sensitive to neutralizing antibodies than the WT virus. Taken together, these data strongly suggest that the HA stem glycans play a critical role on HA cleavage, replication, thermostability, pH stability, and antigenicity of H5N1 AIVs.
Influenza A viruses infect a remarkably diverse number of hosts. Two completely new influenza A virus subtypes were recently discovered in bats, dramatically expanding the host range of the virus. These bat viruses are extremely divergent from all other known strains and likely have unique replication cycles. Phylogenetic analysis indicates long-term, isolated evolution in bats. This is supported by a high seroprevalence in sampled bat populations. As bats represent ~20% of all classified mammals, these findings suggests the presence of a massive cryptic reservoir of poorly characterized influenza A viruses. Here, we review the exciting progress made on understanding these newly discovered viruses, and discuss their zoonotic potential.
Unlabelled:
The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pKas match the pHs of late endosomes (pK(a) of ∼ 6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines with different amino acids and analyzing the effect of these mutations on conformational change and fusion, we found that His184, but not His110, plays an essential role in the pH dependence of the conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central in a conserved interaction network possibly regulating the pH dependence of conformational change via its pKa. As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in the vicinity of histidine may affect its pK(a). The HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly pathogenic as well as the low pathogenic H5 HA, we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pK(a)-modulating effect of neighboring residues.
Importance:
The main pathogenic determinant of influenza viruses, the hemagglutinin (HA) protein, triggers a key step of the infection process: the fusion of the virus envelope with the endosomal membrane releasing the viral genome. Whereas essential aspects of the fusion-inducing mechanism of HA at low pH are well understood, the molecular trigger of the pH-dependent conformational change inducing fusion has been unclear. We provide evidence that His184 regulates the pH dependence of the HA conformational change via its pK(a). Mutations of neighboring residues which may affect the pK(a) of His184 could play an important role in virus adaptation to a specific host. We suggest that mutation of neighboring residue 216, which is present in all highly pathogenic phenotypes of H5N1 influenza virus strains, contributed to the adaptation of these viruses to the human host via its effect on the pKa of His184.
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Sporadic activity by H5N2 influenza viruses has been observed in chickens in Taiwan from 2003 to 2012. The available information suggests that these viruses were generated by reassortment between a Mexican-like H5N2 virus and a local enzootic H6N1 virus. Yet the origin, prevalence, and pathogenicity of these H5N2 viruses have not been fully defined. Following the 2012 highly pathogenic avian influenza (HPAI) outbreaks, surveillance was conducted from December 2012 to July 2013 at a live-poultry wholesale market in Taipei. Our findings showed that H5N2 and H6N1 viruses cocirculated at low levels in chickens in Taiwan. Phylogenetic analyses revealed that all H5N2 viruses had hemagglutinin (HA) and neuraminidase (NA) genes derived from a 1994 Mexican-like virus, while their internal gene complexes were incorporated from the enzootic H6N1 virus lineage by multiple reassortment events. Pathogenicity studies demonstrated heterogeneous results even though all tested viruses had motifs (R-X-K/R-R) supportive of high pathogenicity. Serological surveys for common subtypes of avian viruses confirmed the prevalence of the H5N2 and H6N1 viruses in chickens and revealed an extraordinarily high seroconversion rate to an H9N2 virus, a subtype that is not found in Taiwan but is prevalent in mainland China. These findings suggest that reassortant H5N2 viruses, together with H6N1 viruses, have become established and enzootic in chickens throughout Taiwan and that a large-scale vaccination program might have been conducted locally that likely led to the introduction of the 1994 Mexican-like virus to Taiwan in 2003.
Importance:
H5N2 avian influenza viruses first appeared in chickens in Taiwan in 2003 and caused a series of outbreaks afterwards. Phylogenetic analyses show that the chicken H5N2 viruses have H5 and N2 genes that are closely related to those of a vaccine strain originating from Mexico in 1994, while the contemporary duck H5N2 viruses in Taiwan belong to the Eurasian gene pool. The unusually high similarity of the chicken H5N2 viruses to the Mexican vaccine strain suggests that these viruses might have been introduced to Taiwan by using inadequately inactivated or attenuated vaccines. These chicken H5N2 viruses are developing varying levels of pathogenicity that could lead to significant consequences for the local poultry industry. These findings emphasize the need for strict quality control and competent oversight in the manufacture and usage of avian influenza virus vaccines and indicate that alternatives to widespread vaccination may be desirable.
The divergence of the hemagglutinin gene of A/goose/Guangdong/1/1996-lineage H5N1 viruses during 2011 and 2012 (807 new sequences collected through December 31, 2012) was analyzed by phylogenetic and p-distance methods to define new clades using the pre-established nomenclature system. Eight new clade designations were recommended based on division of clade 1.1 (Mekong River Delta), 2.1.3.2 (Indonesia), 2.2.2 (India/Bangladesh), 2.2.1.1 (Egypt/Israel), and 2.3.2.1 (Asia). A simplification to the previously defined criteria, which adds a letter rather than number to the right-most digit of fifth-order clades, was proposed to facilitate this and future updates.
Influenza virus entry is mediated by the acidic pH-induced activation of hemagglutinin (HA) protein. Here, we investigated how a decrease in HA activation pH (an increase in acid stability) influences the properties of highly pathogenic H5N1 influenza virus in mammalian hosts. We generated isogenic A/Vietnam/1203/2004 (H5N1) viruses containing either wild-type HA protein (activation pH 6.0) or an HA2-K58I point mutation (activation pH 5.5). The VN1203-HA2-K58I virus had similar replication kinetics compared to VN1203-wild-type in MDCK and normal human bronchial epithelial cells yet reduced growth in human alveolar A549 cells, which were found to have a higher endosomal pH than MDCK cells. Wild-type and HA2-K58I viruses promoted similar morbidity and mortality in C57BL/6J mice and ferrets, and neither virus transmitted efficiently to naïve contact cage-mate ferrets. The acid-stabilizing HA2-K58I mutation, which diminishes H5N1 replication and transmission in ducks, increased the virus load in the ferret nasal cavity early during infection while simultaneously reducing the virus load in the lungs. Overall, a single, acid-stabilizing mutation was found to enhance the growth of an H5N1 influenza virus in the mammalian upper respiratory tract yet was insufficient to enable contact transmission in ferrets in the absence of additional mutations that confer α (2, 6)-receptor binding specificity and remove a critical N-linked glycosylation site. Information provided here on the contribution of HA acid stability to H5N1 influenza virus fitness and transmissibility in mammals, in the background of a non-laboratory-adapted virus, provides essential information for surveillance and assessment of the pandemic potential of currently circulating H5N1 viruses.
Background
A characteristic difference between highly and non-highly pathogenic avian influenza strains is the presence of an extended, often multibasic, cleavage motif insertion in the hemagglutinin protein. Such motif is found in H7N3 strains from chicken farm outbreaks in 2012 in Mexico.
Methods
Through phylogenetic, sequence and structural analysis, we try to shed light on the role, prevalence, likelihood of appearance and origin of the inserted cleavage motifs in these H7N3 avian influenza strains.
Results
The H7N3 avian influenza strain which caused outbreaks in chicken farms in June/July 2012 in Mexico has a new extended cleavage site which is the likely reason for its high pathogenicity in these birds. This cleavage site appears to have been naturally acquired and was not present in the closest low pathogenic precursors. Structural modeling shows that insertion of a productive cleavage site is quite flexible to accept insertions of different length and with sequences from different possible origins. Different from recent cleavage site insertions, the origin of the insert here is not from the viral genome but from host 28S ribosomal RNA (rRNA) instead. This is a novelty for a natural acquisition as a similar insertion has so far only been observed in a laboratory strain before. Given the abundance of viral and host RNA in infected cells, the acquisition of a pathogenicity-enhancing extended cleavage site through a similar route by other low-pathogenic avian strains in future does not seem unlikely. Important for surveillance of these H7N3 strains, the structural sites known to enhance mammalian airborne transmission are dominated by the characteristic avian residues and the risk of human to human transmission should currently be low but should be monitored for future changes accordingly.
Conclusions
This highly pathogenic H7N3 avian influenza strain acquired a novel extended cleavage site which likely originated from recombination with 28S rRNA from the avian host. Notably, this new virus can infect humans but currently lacks critical host receptor adaptations that would facilitate human to human transmission.
After receptor binding and internalization during influenza virus entry, the hemagglutinin (HA) protein is triggered by low
pH to undergo irreversible conformational changes that mediate membrane fusion. To investigate how mutations that alter the
activation pH of the HA protein influence the fitness of an avian H5N1 influenza virus in a mammalian model, we infected C57BL/6J
or DBA/2J mice and compared the replication and virulence of recombinant A/chicken/Vietnam/C58/04 (H5N1) HA-Y231H mutant, wild-type, and HA-H241Q and HA-K582I mutant viruses that have HA activation pH values of 6.3, 5.9, 5.6, and 5.4, respectively. The HA-Y231H mutant virus was highly susceptible to acid inactivation in vitro and was attenuated for growth and virulence in mice, suggesting that an H5N1 HA protein triggered at pH 6.3 is too unstable
for the virus to remain fit. Wild-type and HA-H241Q viruses were similar in pathogenicity and grew to similar levels in mice, ducks, and cell cultures derived from both avian
and mammalian tissues, suggesting that H5N1 HA proteins triggered at pH values in the range of 5.9 to 5.6 broadly support
replication. The HA-K582I mutant virus had greater growth and virulence in DBA/2J mice than the wild type did, although the mutant virus was highly
attenuated in ducks. The data suggest that adaptation of avian H5N1 influenza virus for infection in mammals is supported
by a decrease in the HA activation pH to 5.4. Identification of the HA activation pH as a host-specific infectivity factor
is expected to aid in the surveillance and risk assessment of currently circulating H5N1 influenza viruses.
In early 2013, human infections caused by a novel H7N9 avian influenza virus (AIV) were first reported in China; these infections caused severe disease and death. The virus was initially low pathogenic to poultry, enabling it to spread widely in different provinces, especially in live poultry markets. Importantly, the H7N9 low pathogenic AIVs (LPAIVs) evolved into highly pathogenic AIVs (HPAIVs) in the beginning of 2017, causing a greater threat to human health and devastating losses to the poultry industry. Fortunately, nationwide vaccination of chickens with an H5/H7 bivalent inactivated avian influenza vaccine since September 2017 has successfully controlled H7N9 avian influenza infections in poultry and, importantly, has also prevented human infections. In this review, we summarize the biological properties of the H7N9 viruses, specifically their genetic evolution, adaptation, pathogenesis, receptor binding, transmission, drug resistance, and antigenic variation, as well as the prevention and control measures. The information obtained from investigating and managing the H7N9 viruses could improve our ability to understand other novel AIVs and formulate effective measures to control their threat to humans and animals.
The main historical features of avian influenza (AI) include: initial high pathogenicity avian influenza (HPAI) cases reported in Northern Italy and clinical differentiation of HPAI from fowl cholera in 1880 and etiology demonstrated as a filterable agent in 1901. Economic losses from AI have varied depending on the strain of virus, species of bird infected, number of farms involved, control methods used, and the speed of implementation of control or eradication strategies. HPAI and H5/H7 low pathogenicity AI are of interest to the World Organization for Animal Health. Based on morphologic and biochemical evidence, AI viruses exert pathological effect on avian cells by two mechanisms: necrosis or apoptosis. Because of the broad spectrum of signs and lesions reported with infections by AI viruses in several species, a definitive diagnosis must be made by virologic and serologic methods. Interventions for AI include measures designed to: prevent introduction of virus and eliminate/eradicate the virus.
Influenza A viruses are important veterinary and human health pathogens that all share many genetic characteristics. Wild birds are thought to be the primordial reservoirs of influenza viruses. The viruses, when given the opportunity to infect new species, can adapt to the new host and can cause disease that ranges from mild to severe. The hemagglutinin cleavage site is a critical virulence determinant in poultry, and certain sequence motifs can result in systemic replication, allowing increased virulence, and resulting in the highly pathogenic avian influenza phenotype. However all eight gene segments contribute to host adaptation and can influence viral replication and transmission. Influenza viruses can rapidly change through their ability to reassort gene segments, and with a high mutation rate can accumulate amino acids which result in antigenic changes that allow the virus to evade the host immune response. These processes are often referred to as antigenic shift and drift, respectively.
Avian influenza virus (AIV) is a global virus that knows no geographic boundaries, has no political agenda, and can infect poultry irrespective of their occupying ecosystem, agricultural production system, or other anthropocentric niches. AIVs or evidence of their infection have been detected in poultry and wild birds on all seven continents, but the largest numbers of cases detected have been in North America and Europe, where intensive surveillance is conducted in poultry and wild birds. Since 1959, there have been 37 epizootics of highly pathogenic avian influenza in poultry and wild birds, the largest one being the H5N1 HPAI epizootic, which began in China in 1996 and continues today. This epizootic has involved 67 countries in Asia, Africa, Europe, and North America, and has affected more than 400 million birds.
Since 2008, seven countries from five continents have experienced HPAI outbreaks in poultry due to viruses unrelated to H5 Goose/Guangdong-lineage viruses. These have covered a range of virus subtypes and affected different production species, from chickens to ostriches. Each outbreak section in this chapter describes in detail the disease presentation, diagnosis, control, and relevant epidemiology. The level of spread, economic impacts, and implications for human infection are also described.
The H5 highly pathogenic avian influenza viruses that emerged in China in 1996 have circulated and evolved for 18 years. They have caused severe losses for poultry producers across four continents, resulting in the death and destruction of millions of poultry. Some strains of H5 virus also cause severe zoonotic disease, and may have the potential to produce a human influenza pandemic. This chapter describes the major events that have occurred in this panzootic and the evolution of the viruses, including information on the various clades and genotypes that have emerged. It discusses the main modes of spread and the factors that have allowed these viruses to persist, including the importance of the development and structure of the poultry sector, especially in places where these viruses remain entrenched. It provides information on control and preventive measures that have been adopted in selected countries, and offers an assessment of expected developments, including the limited prospects for global eradication in the foreseeable future.
The 2014-15 H5Nx high pathogenicity avian influenza (HPAI) outbreak affected 211 commercial premises, 21 backyard flocks, 75 individual wild birds and four captive-reared raptors in 21 Western and upper Midwestern states, resulting in death or culling of over 50.4 million poultry in the stamping-out program that cost USA government 3.3 billion impact on the economy. Seventeen trading partners suspended imports of all U.S.-origin poultry and poultry products while 38 trading partners regionalized the United States, allowed trade in poultry and poultry products to continue from areas of the U.S. not affected by HPAI. Disease response and control activities in addition to the use of comprehensive surveillance and regionalization (zoning) as prescribed by the OIE Terrestrial Animal Health Code is a scientifically valid and effective means to maintain safe trade in poultry and poultry products. This was further realized during the 2016 H7N8 HPAI outbreak in Dubois county Indiana with greater acceptance of regionalization and continuity in trade with a more limited cost of $30 million for eradication.
Avian influenza (Al) viruses comprise the vast majority of the type A Orthomyxoviridae. Evolution has produced an enormous array of viral antigenic subtypes and variants based upon the structure of the two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). These viruses appear to be perpetuated in nature in a select few wild avian species, but some strains are capable of sporadic and unpredictable entry into other animal populations, including humans. The fate of these occasional entries is likewise unpredictable, and investigators are left only with retrospective analysis. It is clear, however, that Al viruses (or some of their genes) have fixed themselves into circulating lineages in some mammalian hosts. In birds, particularly commercial poultry, Al can undergo a dramatic shift and take the unique form of a highly lethal and systemic disease. This has happened at least eight times in this decade on four different continents. In this review we explore these outbreaks and what we have learned from them regarding virulence acquisition and interspecies transmission. We further attempt to explore the implications of these outbreaks for the future of both avian and non-avian species and discuss current methods of diagnosis and control of Al.
Introduction Great Britain South Africa Germany 1979 (H7N7) Ireland 1983 (H5N8) Italy The Netherlands, Belgium, and Germany 2003 (H7N7) Democratic People's Republic of Korea (North Korea) 2005 (H7N7) Conclusions References
Introduction General History Regulatory Aspects Low Pathogenicity Avian Influenza in Poultry and Captive Birds High Pathogenicity Avian Influenza (1959 to 2007) Conclusions References
Membrane fusion is not spontaneous. Therefore, enveloped viruses have evolved membrane-fusion mediating glycoproteins that, once activated, refold, and release energy that fuses viral and cellular membranes. The influenza A virus hemagglutinin (HA) protein is a prototypic structural class I viral fusion glycoprotein that, once primed by proteolytic cleavage, is activated by endosomal low pH to form a fusogenic "leash-in-grooves" hairpin structure. Low-pH induced HA protein refolding is an irreversible process, so acid exposure in the absence of a target membrane leads to virus inactivation. The HA proteins of diverse influenza virus subtypes isolated from a variety of species differ in their acid stabilities, or pH values at which irreversible HA protein conformational changes are triggered. Recently, efficient replication of highly pathogenic avian influenza (HPAI) viruses such as H5N1 in avian species has been associated with a relatively high HA activation pH. In contrast, a decrease in H5N1 HA activation pH has been shown to enhance replication and airborne transmission in mammals. Mutations that alter the acid stabilities of H1 and H3 HA proteins have also been discovered that influence the amantadine susceptibilities, replication rates, and pathogenicities of human influenza viruses. An understanding of the role of HA acid stability in influenza virus biology is expected to aid in identifying emerging viruses with increased pandemic potential and assist in developing live attenuated virus vaccines. Acid-induced HA protein activation, which has provided a paradigm for protein-mediated membrane fusion, is now identified as a novel determinant of influenza virus biology.
The aetiological agent of an epizootic among Common Terns ( Sterna hirundo ) in South Africa in 1961 was isolated from several sick birds and named Tern virus. It was classified on the basis of antigenic and morphological properties as a strain of avian influenza virus, Myxovirus influenzae A/Tern/South Africa/1961. The strain-specific antigen of Tern virus was unrelated to all known influenza strains with the single exception of Chicken/Scotland/1959 virus and the two viruses may be regarded as variants of the same strain. This relationship raised the interesting epidemiological possibility of the spread of infection between sea-birds and domestic poultry because the Common Tern migrates between Europe and South Africa.
Highly pathogenic avian influenza (HPAI) virus (H5N1) has appeared in >60 countries and continues to evolve and diversify at a concerning rate. Because different names have been used to describe emerging lineages of the virus, this study describes a unified nomenclature system to facilitate discussion and comparison of subtype H5N1 lineages.
Between the month of October 1997 and January 1998, eight outbreaks of highly pathogenic avian influenza were diagnosed in the Veneto and Friuli-Venezia Giulia regions in north-eastern Italy. For each of the eight outbreaks, influenza A virus of subtype H5N2 was isolated and the inoculation of susceptible chickens confirmed these viruses to be extremely virulent with intravenous pathogenicity indices in 6-week-old chickens of 2.98 to 3.00. Although it was not possible to trace the origin of infection, the epidemiological investigation revealed connections between several outbreaks and emphasized the well-known risk factors for avian influenza such as bird movement, rearing of mixed populations and contact with migratory waterfowl. Control measures listed in European Union directive 92/40/EEC were implemented promptly and spread of the infection to intensively-reared domestic poultry was avoided.
Influenza A viruses that infect poultry can be divided into two groups. Very virulent viruses cause highly pathogenic avian influenza (HPAI), with flock mortality as high as 100%. These viruses have been restricted to subtypes H5 and H7, although not all H5 and H7 viruses cause HPAI. All other viruses cause a milder, primarily respiratory, disease (LPAI), unless exacerbated. Until recently, HPAI viruses were rarely isolated from wild birds, but for LPAI viruses extremely high isolation rates have been recorded in surveillance studies. Influenza viruses may infect all types of domestic or captive birds in all areas of the world. The frequency with which primary infections occur in any type of bird usually depends on the degree of contact there is with feral birds. Secondary spread is typically associated with human involvement, either by birds or bird product movement or by transferring infective faeces from infected to susceptible birds, but potentially wild birds can be involved. In recent years the frequency of HPAI outbreaks appears to have increased and there have been particularly costly outbreaks of HPAI in densely populated poultry areas in Italy, The Netherlands and Canada. In each outbreak millions of birds were slaughtered to bring the outbreaks under control. Since the 1990s, AI infections due to two subtypes have been widespread in poultry across a large area of the World. LPAI H9N2 appears to have spread across the whole of Asia in that time and has become endemic in poultry in many of the affected countries. However, these outbreaks have been overshadowed by the H5N1 HPAI virus, initially isolated in China that has now spread in poultry and/or wild birds throughout Asia and into Europe and Africa, resulting in the death or culling of hundreds of millions of poultry and posing a significant zoonosis threat. To date control methods seem to have been unsuccessful on the larger scale and HPAI H5N1 outbreaks continue to be reported.
Introduction Avian Influenza in Birds other than Domestic Poultry in Australia Outbreaks of HPAI in Australian Poultry Lessons Learned Likely Approach to Control of HPAI Today Public Health Aspects Risks Posed by H5N1 HPAI in Asia References
Introduction Genetic Studies and Nomenclature of the H5N1 History of the H5N1 Panzootic Sources of Infection and Reasons for Spread Pathology of H5N1 HPAI Diagnostic Aspects Disease Control The Future Conclusions Acknowledgments References
Introduction Synonyms and Access to “Old” Literature Selected Historial Aspects Detailed Descriptions of Fowl Plague Differential Diagnoses Search for the Causative Agent of Fowl Plague General Modes of Spread of Fowl Plague Virus Spread of Fowl Plague to other European Countries The First “Mild” Case of Fowl Plague, the N Virus Spread of Fowl Plague across Asia Prevention and Control Conclusions Acknowledgments References
Recent isolations of H5N2 subtype avian influenza (AI) viruses in North America have raised questions concerning their origin, transmission to commercial poultry, and potential for virulence. One ratite-origin isolate of low pathogenicity, A/emu/TX/39924/93 (H5N2), was subjected to a procedure that rapidly selects and/or amplifies highly pathogenic (HP) strains. The resulting highly virulent derivative had an altered hemagglutinin (HA) gene containing an additional six nucleotides at position 970–975 in the HA1 coding region. This resulted in an arg-lys insertion near the proteolytic cleavage site of the HA protein. The remainder of the HA sequence differed by an additional seven amino acids from the parent. The HA precursor of the derivative, but not the parent, was readily cleaved during replication in cell culture without addition of trypsin. In experimentally infected chickens, the derivative produced lesions typical of highly pathogenic avian influenza. A reverse transcriptase-polymerase chain reaction (RT-PCR) primer set was designed to amplify exclusively from molecules with the inserted six nucleotides. The set yielded product only from the selected derivative samples and not the parent. Thus, the levels of the HP variants in the parent stock were undetectable, or the insertion occurred rapidly during the selection process.
In October 2009, highly pathogenic avian influenza virus (HPAIV) was isolated for the first time in poultry in Spain. Sequencing analysis revealed that it was an H7N7 HPAIV. The progenitors of H7 HPAIV strains involved in recent European poultry outbreaks were simultaneously circulating in wild birds. The infected Spanish farm is located close to a reservoir abundant in wild birds. Epidemiological investigation found no links to other poultry holdings and those located in the control area were negative for AIV. Previous spatial risk analyses had identified the area where the infected holding is located to beat high relative risk for the introduction and presence of H5N1 HPAIV by wild birds. We suggest a risk-based surveillance scheme that targets smaller geographical units but maintains the number of wild birds being sampled, as early detection of potentially pathogenic AIV is crucial in preventing spread to poultry.
Clinical signs, death, virus excretion and immune response were measured in 2-week-old chickens, turkeys, quail and ducks infected by intramuscular, intranasal and contact routes with eight influenza viruses of H5 subtype. Six of the viruses: A/chicken/Scotland/59 (H5N1), ck/Scot; A/tern/South Africa/61 (H5N3), tern/SA; A/turkey/Ontario/ 7732/66 (H5N9); ty/Ont; A/chicken/Pennsylvania/1370/83 (H5N2); Pa/1370; A/turkey/Ireland/83 (H5N8); ty/Ireland, and A/duck/Ireland/ 113/84 (HSN8); dk/Ireland, were highly pathogenic for chickens and turkeys. Two viruses, A/chicken/Pennsylvania/1/83 (H5N2), Pa/1 and A/turkey/Italy/ZA/80 (H5N2), ty/Italy, were of low pathogenicity. Ck/Scot was more pathogenic for chickens than turkeys while ty/Ont was more pathogenic for turkeys than chickens. Other viruses showed little difference in their pathogenicity for these two hosts. No clinical signs or deaths were seen in any of the infected ducks. Only two viruses, dk/Ireland and ty/Ireland, produced consistent serological responses in ducks, although intramuscular infection with tern/SA and ty/Italy resulted in some ducks with positive HI titres. These four were the only viruses reisolated from ducks. Quail showed some resistance to viruses which were highly pathogenic for chickens and turkeys, most notably to ck/Scot and ty/Ont and to a lesser extent tern/SA and Pa/1370. Transmission of virus from intranasally infected birds to birds placed in contact varied considerably with both host and infecting virus and the various combinations of these.