Evolution Of Avian Influenza Viruses Research Articles

Ecology and evolution of avian influenza viruses

Four types of influenza virus have been identified in nature: influenza A, B, and C viruses are capable of infecting humans, and influenzas A and B cause annual epidemics (seasonal flu) in humans; however, influenza D is currently known to infect only pigs and cattle. The influenza A viruses (IAVs) are of greatest importance to humans, causing widespread significant morbidity and mortality, and have been responsible for at least five pandemics documented since the beginning of the 20th century (Table 1). The H1N1 and H3N2 IAVs continue to circulate in humans as seasonal influenza. In addition to humans, IAVs have a wide range of host animal species in nature, especially wild aquatic birds, the reservoir hosts of IAVs. The IAVs isolated from or adapted to an avian host are named avian influenza viruses (AIVs), and are of great concern owing to their involvement in the genesis of pandemic and outbreak strains. Moreover, the majority of AIVs persist in wild birds and domestic poultry, and novel variants continue to emerge in birds and other hosts, posing non-negligible threats to host ecology and public health.

Evolution of H6N6 viruses in China between 2014 and 2019 involves multiple reassortment events

ABSTRACT H6N6 avian influenza viruses (AIVs) have been widely detected in wild birds, poultry, and even mammals. Recently, H6N6 viruses were reported to be involved in the generation of H5 and H7 subtype viruses. To investigate the emergence, evolutionary pattern, and potential for an epidemic of H6N6 viruses, the complete genomes of 198 H6N6 viruses were analyzed, including 168 H6N6 viruses deposited in the NCBI and GISAID databases from inception to January 2019 and 30 isolates collected from China between November 2014 and January 2019. Using phylogenetic analysis, the 198 strains of H6N6 viruses were identified as 98 genotypes. Molecular clock analysis indicated that the evolution of H6N6 viruses in China was constant and not interrupted by selective pressure. Notably, the laboratory isolates reassorted with six subtype viruses: H6N2, H5N6, H7N9, H5N2, H4N2, and H6N8, resulting in nine novel H6N6 reassortment events. These results suggested that H6N6 viruses can act as an intermediary in the evolution of H5N6, H6N6, and H7N9 viruses. Animal experiments demonstrated that the 10 representative H6N6 viruses showed low pathogenicity in chickens and were capable of infecting mice without prior adaptation. Our findings suggest that H6N6 viruses play an important role in the evolution of AIVs, and it is necessary to continuously monitor and evaluate the potential epidemic of the H6N6 subtype viruses.

Evolution of Avian Influenza Virus (H3) with Spillover into Humans, China.

The continuous evolution of avian influenza viruses (AIVs) of subtype H3 in China and the emergence of human infection with AIV subtype H3N8 highlight their threat to public health. Through surveillance in poultry-associated environments during 2009-2022, we isolated and sequenced 188 H3 AIVs across China. Performing large-scale sequence analysis with publicly available data, we identified 4 sublineages of H3 AIVs established in domestic ducks in China via multiple introductions from wild birds from Eurasia. Using full-genome analysis, we identified 126 distinct genotypes, of which the H3N2 G23 genotype predominated recently. H3N8 G25 viruses, which spilled over from birds to humans, might have been generated by reassortment between H3N2 G23, wild bird H3N8, and poultry H9N2 before February 2021. Mammal-adapted and drug-resistance substitutions occasionally occurred in H3 AIVs. Ongoing surveillance for H3 AIVs and risk assessment are imperative for potential pandemic preparedness.

Influenza A viruses in gulls in landfills and freshwater habitats in Minnesota, United States.

Introduction: The unpredictable evolution of avian influenza viruses (AIVs) presents an ongoing threat to agricultural production and public and wildlife health. Severe outbreaks of highly pathogenic H5N1 viruses in US poultry and wild birds since 2022 highlight the urgent need to understand the changing ecology of AIV. Surveillance of gulls in marine coastal environments has intensified in recent years to learn how their long-range pelagic movements potentially facilitate inter-hemispheric AIV movements. In contrast, little is known about inland gulls and their role in AIV spillover, maintenance, and long-range dissemination. Methods: To address this gap, we conducted active AIV surveillance in ring-billed gulls (Larus delawarensis) and Franklin's gulls (Leucophaeus pipixcan) in Minnesota's natural freshwater lakes during the summer breeding season and in landfills during fall migration (1,686 samples). Results: Whole-genome AIV sequences obtained from 40 individuals revealed three-lineage reassortants with a mix of genome segments from the avian Americas lineage, avian Eurasian lineage, and a global "Gull" lineage that diverged more than 50 years ago from the rest of the AIV global gene pool. No poultry viruses contained gull-adapted H13, NP, or NS genes, pointing to limited spillover. Geolocators traced gull migration routes across multiple North American flyways, explaining how inland gulls imported diverse AIV lineages from distant locations. Migration patterns were highly varied and deviated far from assumed "textbook" routes. Discussion: Viruses circulating in Minnesota gulls during the summer breeding season in freshwater environments reappeared in autumn landfills, evidence of AIV persistence in gulls between seasons and transmission between habitats. Going forward, wider adoption of technological advances in animal tracking devices and genetic sequencing is needed to expand AIV surveillance in understudied hosts and habitats.

Potential Impact of Environmental Pollution by Human Antivirals on Avian Influenza Virus Evolution

Antiviral (AV) drugs are the main line of defense against pandemic influenza. However, different administration policies are applied in countries with different stocks of AV drugs. These policies lead to different occurrences of drug metabolites in the aquatic environment, altering animal behavior with evolutionary consequences on viruses. The aim of this study was to investigate the potential impact of environmental pollution by human antivirals, such as oseltamivir carboxylate (OC), on the evolutionary rate of avian influenza. We used NA, HA, NP, and MP viral segments from two groups of neighboring countries sharing migratory routes of wild birds and characterized by different AV stockpiles. BEAST analyses were performed using the uncorrelated lognormal clock evolutionary model and the Bayesian skyline tree prior model. The ratios between the rate of evolution of the NA gene and the HA, NP, and MP segments were considered. The two groups of countries were compared by analyzing the differences in the ratio distributions. Our analyses highlighted a possible different behavior in the evolution of H5N1 2.3 clade viral strains when OC environmental pollution is present. In conclusion, the widespread consumption of antivirals and their presence in wastewater could influence the selective pressure on viruses.

Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets.

Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS.

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia.

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats.

Surveillance of avian influenza viruses in live bird markets of Shandong province from 2013 to 2019.

Avian influenza viruses (AIVs) seriously affect the poultry industry and pose a great threat to humans. Timely surveillance of AIVs is the basis for preparedness of the virus. This study reported the long-term surveillance of AIVs in the live bird market (LBM) of 16 cities in Shandong province from 2013 to 2019. A total of 29,895 samples were obtained and the overall positive rate of AIVs was 9.7%. The H9 was found to be the most predominant subtype in most of the time and contributed most to the monthly positve rate of AIVs as supported by the univariate and multivariate analysis, while H5 and H7 only circulated in some short periods. Then, the whole-genome sequences of 62 representative H9N2 viruses including one human isolate from a 7-year-old boy in were determined and they were genetically similar to each other with the median pairwise sequence identities ranging from 0.96 to 0.98 for all segments. The newly sequenced viruses were most similar to viruses isolated in chickens in mainland China, especially the provinces in Eastern China. Phylogenetic analysis showed that these newly sequenced H9N2 viruses belonged to the same clade for all segments except PB1. Nearly all of these viruses belonged to the G57 genotype which has dominated in China since 2010. Finally, several molecular markers associated with human adaptation, mammalian virulence, and drug resistance were identified in the newly sequenced H9N2 viruses. Overall, the study deepens our understanding of the epidemic and evolution of AIVs and provides a basis for effective control of AIVs in China.

Genetic subtyping and phylogenetic analysis of HA and NA from avian influenza virus in wild birds from Peru reveals unique features among circulating strains in America.

Avian influenza virus (AIV) represents a major concern with productive implications in poultry systems but it is also a zoonotic agent that possesses an intrinsic pandemic risk. AIV is an enveloped, negative-sense and single-stranded RNA virus with a segmented genome. The eight genomic segments, comprising the whole genome, encode for eleven proteins. Within these proteins, Hemagglutinin (HA) and Neuraminidase (NA) are the most relevant for studies of evolution and pathogenesis considering their role in viral replication, and have also been used for classification purposes. Migratory birds are the main hosts and play a pivotal role in viral evolution and dissemination due to their migratory routes that comprise large regions worldwide. Altogether, viral and reservoir factors contribute to the emergence of avian influenza viruses with novel features and pathogenic potentials. The study aimed to conduct surveillance of AIVs in wild birds from Peru. A multi-site screening of feces of migratory birds was performed to isolate viruses and to characterize the whole genome sequences, especially the genes coding for HA and NA proteins. Four-hundred-twenty-one (421) fecal samples, collected between March 2019 and March 2020 in Lima, were obtained from 21 species of wild birds. From these, we isolated five AIV from whimbrel, kelp gull, Franklin’s gulls and Mallard, which were of low pathogenicity, including four subtypes as H6N8, H13N6, H6N2 and H2N6. Genetic analysis of HA and NA genes revealed novel features in these viruses and phylogenetic analysis exhibited a close relationship with those identified in North America (US and Canada). Furthermore, H2N6 isolate presented a NA sequence with higher genetic relationship to Chilean isolates. These results highlight that the geographical factor is of major relevance in the evolution of AIV, suggesting that AIV circulating in Peru might represent a new site for the emergence of reassortant AIVs.

Virus Adaptation Following Experimental Infection of Chickens with a Domestic Duck Low Pathogenic Avian Influenza Isolate from the 2017 USA H7N9 Outbreak Identifies Polymorphic Mutations in Multiple Gene Segments.

In March 2017, highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) subtype H7N9 were detected from poultry farms and backyard birds in several states in the southeast United States. Because interspecies transmission is a known mechanism for evolution of AIVs, we sought to characterize infection and transmission of a domestic duck-origin H7N9 LPAIV in chickens and genetically compare the viruses replicating in the chickens to the original H7N9 clinical field samples used as inoculum. The results of the experimental infection demonstrated virus replication and transmission in chickens, with overt clinical signs of disease and shedding through both oral and cloacal routes. Unexpectedly, higher levels of virus shedding were observed in some cloacal swabs. Next generation sequencing (NGS) analysis identified numerous non-synonymous mutations at the consensus level in the polymerase genes (i.e., PA, PB1, and PB2) and the hemagglutinin (HA) receptor binding site in viruses recovered from chickens, indicating possible virus adaptation in the new host. For comparison, NGS analysis of clinical samples obtained from duck specimen collected during the outbreak indicated three polymorphic sides in the M1 segment and a minor population of viruses carrying the D139N (21.4%) substitution in the NS1 segment. Interestingly, at consensus level, A/duck/Alabama (H7N9) had isoleucine at position 105 in NP protein, similar to HPAIV (H7N9) but not to LPAIV (H7N9) isolated from the same 2017 influenza outbreak in the US. Taken together, this work demonstrates that the H7N9 viruses could readily jump between avian species, which may have contributed to the evolution of the virus and its spread in the region.

Avian Influenza in Wild Birds and Poultry: Dissemination Pathways, Monitoring Methods, and Virus Ecology.

Avian influenza is one of the largest known threats to domestic poultry. Influenza outbreaks on poultry farms typically lead to the complete slaughter of the entire domestic bird population, causing severe economic losses worldwide. Moreover, there are highly pathogenic avian influenza (HPAI) strains that are able to infect the swine or human population in addition to their primary avian host and, as such, have the potential of being a global zoonotic and pandemic threat. Migratory birds, especially waterfowl, are a natural reservoir of the avian influenza virus; they carry and exchange different virus strains along their migration routes, leading to antigenic drift and antigenic shift, which results in the emergence of novel HPAI viruses. This requires monitoring over time and in different locations to allow for the upkeep of relevant knowledge on avian influenza virus evolution and the prevention of novel epizootic and epidemic outbreaks. In this review, we assess the role of migratory birds in the spread and introduction of influenza strains on a global level, based on recent data. Our analysis sheds light on the details of viral dissemination linked to avian migration, the viral exchange between migratory waterfowl and domestic poultry, virus ecology in general, and viral evolution as a process tightly linked to bird migration. We also provide insight into methods used to detect and quantify avian influenza in the wild. This review may be beneficial for the influenza research community and may pave the way to novel strategies of avian influenza and HPAI zoonosis outbreak monitoring and prevention.

Parallel evolution in the emergence of highly pathogenic avian influenza A viruses

Parallel molecular evolution and adaptation are important phenomena commonly observed in viruses. Here, we exploit parallel molecular evolution to understand virulence evolution in avian influenza viruses (AIV). Highly-pathogenic AIVs evolve independently from low-pathogenic ancestors via acquisition of polybasic cleavage sites. Why some AIV lineages but not others evolve in this way is unknown. We hypothesise that the parallel emergence of highly-pathogenic AIV may be facilitated by permissive or compensatory mutations occurring across the viral genome. We combine phylogenetic, statistical and structural approaches to discover parallel mutations in AIV genomes associated with the highly-pathogenic phenotype. Parallel mutations were screened using a statistical test of mutation-phenotype association and further evaluated in the contexts of positive selection and protein structure. Our resulting mutational panel may help to reveal new links between virulence evolution and other traits, and raises the possibility of predicting aspects of AIV evolution.

Prevalence and Diversity of Avian Influenza Virus Hemagglutinin Sero-Subtypes in Poultry and Wild Birds in Bangladesh.

Highly pathogenic avian influenza H5 viruses have pandemic potential, cause significant economic losses and are of veterinary and public health concerns. This study aimed to investigate the distribution and diversity of hemagglutinin (HA) subtypes of avian influenza virus (AIV) in poultry and wild birds in Bangladesh. We conducted an avian influenza sero-surveillance in wild and domestic birds in wetlands of Chattogram and Sylhet in the winter seasons 2012–2014. We tested serum samples using a competitive enzyme-linked immunosorbent assay (c-ELISA), and randomly selected positive serum samples (170 of 942) were tested using hemagglutination inhibition (HI) to detect antibodies against the 16 different HA sero-subtypes. All AIV sero–subtypes except H7, H11, H14 and H15 were identified in the present study, with H5 and H9 dominating over other subtypes, regardless of the bird species. The diversity of HA sero-subtypes within groups ranged from 3 (in household chickens) to 10 (in migratory birds). The prevalence of the H5 sero-subtype was 76.3% (29/38) in nomadic ducks, 71.4% (5/7) in household chicken, 66.7% (24/36) in resident wild birds, 65.9% (27/41) in migratory birds and 61.7% (29/47) in household ducks. Moreover, the H9 sero-subtype was common in migratory birds (56%; 23/41), followed by 38.3% (18/47) in household ducks, 36.8% (14/38) in nomadic ducks, 30.6% (11/66) in resident wild birds and 28.5% (2/7) in household chickens. H1, H4 and H6 sero-subtypes were the most common sero-subtypes (80%; 8/10, 70%; 7/10 and 70%; 7/10, respectively) in migratory birds in 2012, H9 in resident wild birds (83.3%; 5/6) and H2 in nomadic ducks (73.9%; 17/23) in 2013, and the H5 sero-subtype in all types of birds (50% to 100%) in 2014. The present study demonstrates that a high diversity of HA subtypes circulated in diverse bird species in Bangladesh, and this broad range of AIV hosts may increase the probability of AIVs’ reassortment and may enhance the emergence of novel AIV strains. A continued surveillance for AIV at targeted domestic–wild bird interfaces is recommended to understand the ecology and evolution of AIVs.

Detection of avian influenza virus: a comparative study of the in silico and in vitro performances of current RT-qPCR assays

Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; hence, an accurate and rapid AIV diagnosis is essential. To date AIV detection relies on molecular methods, mainly RT-qPCR directed against AIV M gene segment. The evolution of AIV represents a relevant issue in diagnostic RT-qPCR due to possible mispriming and/or probe-binding failures resulting in false negative results. Consequently, RT-qPCR for AIV detection should be periodically re-assessed both in silico and in vitro. To this end, a specific workflow was developed to evaluate in silico the complementarity of primers and probes of four published RT-qPCR protocols to their target regions. The four assays and one commercially available kit for AIV detection were evaluated both for their analytical sensitivity using eight different viral dilution panels and for their diagnostic performances against clinical specimens of known infectious status. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances. This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions.

Infection of chicken H9N2 influenza viruses in different species of domestic ducks

Domestic ducks are considered as the interface between wild aquatic birds and terrestrial poultry and play an important role in the transmission and evolution of avian influenza viruses (AIVs). However, the infectivity of H9N2 AIVs in different domestic duck species has not been systematically evaluated. Here we investigated the infectivity of various genotypes of chicken H9N2 AIVs in Pekin duck (Anas Platyrhynchos), Mallard duck (Anas Platyrhynchos) and Muscovy duck (Cairina Moschata) through intranasal inoculation. We found that Pekin ducks and Mallard ducks were generally resistant to chicken H9N2 virus infection, while Muscovy ducks were relatively susceptible to H9N2 AIVs. All the tested viruses were isolated from oropharynx, trachea and lung tissues of Muscovy ducks. Additionally, genotype 57 (G57) H9N2 AIVs, which was predominant in chickens since 2010, showed increased virus replication in this duck species, indicating an improved interspecies transmission ability of recent H9N2 viruses from chickens to ducks. Our results demonstrated the role of Muscovy ducks in the ecology of H9N2 AIVs. More attentions should be paid to this host during viral surveillances. Additionally, inactivated H9N2 vaccine may be unnecessarily used in Pekin and Mallard ducks.

Active surveillance and genetic evolution of avian influenza viruses in Egypt, 2016–2018

ABSTRACT Egypt is a hotspot for avian influenza virus (AIV) due to the endemicity of H5N1 and H9N2 viruses. AIVs were isolated from 329 samples collected in 2016–2018; 48% were H9N2, 37.1% were H5N8, 7.6% were H5N1, and 7.3% were co-infections with 2 of the 3 subtypes. The 32 hemagglutinin (HA) sequences of the H5N1 viruses formed a well-defined lineage within clade 2.2.1.2. The 10 HA sequences of the H5N8 viruses belonged to a subclade within 2.3.4.4. The 11 HA of H9N2 isolates showed high sequence homology with other Egyptian G1-like H9N2 viruses. The prevalence of H5N8 viruses in ducks (2.4%) was higher than in chickens (0.94%). Genetic reassortment was detected in H9N2 viruses. Antigenic analysis showed that H9N2 viruses are homogenous, antigenic drift was detected among H5N1 viruses. AI H5N8 showed higher replication rate followed by H9N2 and H5N1, respectively. H5N8 was more common in Southern Egypt, H9N2 in the Nile Delta, and H5N1 in both areas. Ducks and chickens played a significant role in transmission of H5N1 viruses. The endemicity and co-circulation of H5N1, H5N8, and H9N2 AIV coupled with the lack of a clear control strategy continues to provide avenues for further virus evolution in Egypt.

Electrostatic Variation of Haemagglutinin as a Hallmark of the Evolution of Avian Influenza Viruses

Avian influenza virus is a zoonotic agent that significantly impacts public health and the poultry industry. Monitoring viral evolution and spread is crucial for surveillance and tracing programmes, which are currently based on serological or DNA sequencing-phylogenetics analysis. However, virus-host interactions, antigenic drift and spreading of viral clades strongly depend on variation in the surface features of capsid proteins. We report here that in silico comparative structural analysis of haemagglutinin can reveal relevant evolutionary fingerprints, particularly when integrated with sequence-based analyses. Phylogenetic analyses of H9 viral strains from wild birds and poultry, performed with different methods, reliably led to clustering of viruses into five main groups. Subsequent comparison of structural features showed congruence between such clustering and surface electrostatic fingerprints. These latter fingerprints relate group-specific variations in electrostatic charges and isocontours to well-known haemagglutinin sites involved in the modulation of immune escape and host specificity. This work suggests that the integration of structural and sequence comparisons may enhance investigations of trends and relevant mechanisms in viral evolution.

Genetic characterization of low-pathogenic avian influenza viruses isolated on the Izumi plain in Japan: possible association of dynamic movements of wild birds with AIV evolution.

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site of endangered cranes (hooded cranes and white-naped cranes) and of many other migratory birds (including wild ducks) that are considered carriers of avian influenza viruses (AIVs). To assess the risks of a highly pathogenic avian influenza outbreak in the crane populations, we tested various environmental samples for AIVs in this area. In the 2014-2015 winter season, we isolated one AIV of the H6N2 subtype from the cranes' roost water and two AIVs of the H11N9 subtype from a crane fecal sample and a cloacal swab of a dead spot-billed duck. Genetic analysis of these AIV isolates indicated that our H6N2 isolate is genetically close to AIVs isolated from wild birds in Southeast Asian countries, except that the PB1 and NS genes belong to the North American virus lineage. All genes of the two H11N9 isolates are related to AIVs belonging to the Eurasian virus lineage. Notably, in our phylogenetic trees, H11 HA and N9 NA genes showing high sequence similarity to the corresponding genes of isolates from wild birds in South Africa and Spain, respectively, did not cluster in the major groups with recent wild-bird isolates from East Asia. These results suggest that AIVs with viral gene segments derived from various locations and bird species have been brought to the Izumi plain. These findings imply a possible association of dynamic movements of wild birds with AIV evolution.

Identification of polymerase gene mutations that affect viral replication in H5N1 influenza viruses isolated from pigeons.

Highly pathogenic avian influenza virus H5N1 infects a wide range of host species, with a few cases of sporadic pigeon infections reported in the Middle East and Asia. However, the role of pigeons in the ecology and evolution of H5N1 viruses remains unclear. We previously reported two H5N1 virus strains, isolated from naturally infected pigeons in Egypt, that have several unique mutations in their viral polymerase genes. Here, we investigated the effect of these mutations on H5N1 polymerase activity and viral growth and identified three mutations that affected viral polymerase activity. The results showed that the PB1-V3D mutation significantly decreased polymerase activity and viral growth in both mammalian and avian cells. In contrast, the PB2-K627E and PA-K158R mutations had moderate effects: PB2-K627E decreased and PA-K158R increased polymerase activity. Structural homology modelling indicated that the PB1-V3D residue was located in the PB1 core region that interacts with PA, predicting that the PB1 mutation would produce a stronger interaction between PB1 and PA that results in decreased replication of pigeon-derived H5N1 viruses. Our results identified several unique mutations responsible for changes in polymerase activity in H5N1 virus strains isolated from infected pigeons, emphasizing the importance of avian influenza surveillance in pigeons and in studying the possible role of pigeon-derived H5N1 viruses in avian influenza virus evolution.

Avian Influenza A Viruses: Evolution and Zoonotic Infection.

Although efficient human-to-human transmission of avian influenza virus has yet to be seen, in the past two decades avian-to-human transmission of influenza A viruses has been reported. Influenza A/H5N1, in particular, has repeatedly caused human infections associated with high mortality, and since 1998 the virus has evolved into many clades of variants with significant antigenic diversity. In 2013, three (A/H7N9, A/H6N1, and A/H10N8) novel avian influenza viruses (AIVs) breached the animal-human host species barrier in Asia. In humans, roughly 35% of A/H7N9-infected patients succumbed to the zoonotic infection, and two of three A/H10N8 human infections were also lethal; however, neither of these viruses cause influenza-like symptoms in poultry. While most of these cases were associated with direct contact with infected poultry, some involved sustained human-to-human transmission. Thus, these events elicited concern regarding potential AIV pandemics. This article reviews the human incursions associated with AIV variants and the potential role of pigs as an intermediate host that may hasten AIV evolution. In addition, we discuss the known influenza A virus virulence and transmission factors and their evaluation in animal models. With the growing number of human AIV infections, constant vigilance for the emergence of novel viruses is of utmost importance. In addition, careful characterization and pathobiological assessment of these novel variants will help to identify strains of particular concern for future pandemics.