Emergence, Evolution, and Pathogenicity of Influenza A(H7N4) Virus in Shorebirds in China.

A 2-year surveillance study of influenza A viruses in migratory birds was conducted to understand the subsequent risk during the migratory seasons in Dandong Yalu River Estuary Coastal Wetland National Nature Reserve, Liaoning Province, China, a major stopover site on the East Asian-Australasian flyway. Overall, we isolated 27 influenza A viruses with multiple subtypes, including H3N8 (n = 2), H4N6 (n = 2), H4N7 (n = 2), H7N4 (n = 9), H7N7 (n = 1), H10N7 (n = 7), and H13N6 (n = 4). Particularly, a novel reassortant influenza A(H7N4) virus was first identified in a woman and her backyard poultry flock in Jiangsu Province, China, posing a serious threat to public health. Here, we describe the genetic characterization and pathogenicity of the nine influenza A(H7N4) isolates. Phylogenetic analysis indicated that complex viral gene flow occurred among Asian countries. We also demonstrated a similar evolutionary trajectory of the surface genes of the A(H7N4) isolates and Jiangsu human-related A(H7N4) viruses. Our A(H7N4) isolates exhibited differing degrees of virulence in mice, suggesting a potential risk to other mammalian species, including humans. We revealed multiple mutations that might affect viral virulence in mice. Our report highlights the importance and need for the long-term surveillance of avian influenza virus in migratory birds combined with domestic poultry surveillance along migratory routes and flyways and, thereby, the development of measures to manage potential health threats. IMPORTANCE The H7 subtype avian influenza viruses, such as H7N2, H7N3, H7N4, H7N7, and H7N9, were documented as being capable of infecting humans, and the H7 subtype low pathogenicity avian influenza viruses are capable of mutating into highly pathogenic avian influenza; therefore, they pose a serious threat to public health. Here, we investigated the evolutionary history, molecular characteristics, and pathogenicity of shorebird-origin influenza A(H7N4) viruses, showing a similar evolutionary trajectory with Jiangsu human A(H7N4) viruses in HA and NA genes. Moreover, our isolates exhibited variable virulence (including moderate virulence) in mice, suggesting a potential risk to other mammalian species, including humans.

H7N7 Avian Influenza Virus Mutation from Low to High Pathogenicity on a Layer Chicken Farm in the UK.

Avian influenza virus (AIV) subtypes H5 and H7 are capable of mutating from low to high pathogenicity strains, causing high mortality in poultry with significant economic losses globally. During 2015, two outbreaks of H7N7 low pathogenicity AIV (LPAIV) in Germany, and one each in the United Kingdom (UK) and The Netherlands occurred, as well as single outbreaks of H7N7 high pathogenicity AIV (HPAIV) in Germany and the UK. Both HPAIV outbreaks were linked to precursor H7N7 LPAIV outbreaks on the same or adjacent premises. Herein, we describe the clinical, epidemiological, and virological investigations for the H7N7 UK HPAIV outbreak on a farm with layer chickens in mixed free-range and caged units. H7N7 HPAIV was identified and isolated from clinical samples, as well as H7N7 LPAIV, which could not be isolated. Using serological and molecular evidence, we postulate how the viruses spread throughout the premises, indicating potential points of incursion and possible locations for the mutation event. Serological and mortality data suggested that the LPAIV infection preceded the HPAIV infection and afforded some clinical protection against the HPAIV. These results document the identification of a LPAIV to HPAIV mutation in nature, providing insights into factors that drive its manifestation during outbreaks.

Equine Influenza Virus in Asia: Phylogeographic Pattern and Molecular Features Reveal Circulation of an Autochthonous Lineage.

Equine influenza virus (EIV) causes severe acute respiratory disease in horses. Currently, the strains belonging to the H3N8 subtype are divided into two clades, Florida clade 1 (FC1) and Florida clade 2 (FC2), which emerged in 2002. Both FC1 and FC2 clades were reported in Asian and Middle East countries in the last decade. In this study, we described the evolution, epidemiology, and molecular characteristic of the EIV lineages, with focus on those detected in Asia from 2007 to 2017. The full genome phylogeny showed that FC1 and FC2 constituted separate and divergent lineages, without evidence of reassortment between the clades. While FC1 evolved as a single lineage, FC2 showed a divergent event around 2004 giving rise to two well-supported and coexisting sublineages, European and Asian. Furthermore, two different spread patterns of EIV in Asian countries were identified. The FC1 outbreaks were caused by independent introductions of EIV from the Americas, with the Asian isolates genetically similar to the contemporary American lineages. On the other hand, the FC2 strains detected in Asian mainland countries conformed to an autochthonous monophyletic group with a common ancestor dated in 2006 and showed evidence of an endemic circulation in a local host. Characteristic aminoacidic signature patterns were detected in all viral proteins in both Asian-FC1 and FC2 populations. Several changes were located at the top of the HA1 protein, inside or near antigenic sites. Further studies are needed to assess the potential impact of these antigenic changes in vaccination programs.IMPORTANCE The complex and continuous antigenic evolution of equine influenza viruses (EIVs) remains a major hurdle for vaccine development and the design of effective immunization programs. The present study provides a comprehensive analysis showing the EIV evolutionary dynamics, including the spread and circulation within the Asian continent and its relationship to global EIV populations over a 10-year period. Moreover, we provide a better understanding of EIV molecular evolution in Asian countries and its consequences on the antigenicity. The study underscores the association between the global horse movement and the circulation of EIV in this region. Understanding EIV evolution is imperative in order to mitigate the risk of outbreaks affecting the horse industry and to help with the selection of the viral strains to be included in the formulation of future vaccines.

H7N7 Highly Pathogenic Avian Influenza in Poultry Farms in Italy in 2016.

After the H7N7 highly pathogenic (HP) avian influenza (AI) outbreak in 2013, and a single case of H5N8 HPAI in 2014, in April 2016, a H7N7 HPAI virus was detected in northeastern Italy. The case occurred in an organic free-range laying hen farm located in proximity with one of the highest densely populated poultry areas (DPPAs) in Italy. Control measures provided by the Council of the European Union in directive 2005/94/CE were promptly applied, and enhanced surveillance activities were implemented in the DPPAs. On May 16, 2016, a second case was confirmed in a fattening turkey farm within the protection zone of the previous outbreak. Following an epidemiologic inquiry, another turkey farm was considered at risk of transmission and was subjected to preemptive culling. Epidemiologic data and phylogenetic analyses indicated that the virus was likely introduced from wild birds as a low pathogenicity AI strain, through direct contact. The rapid containment of the outbreak proves the level of preparedness of the veterinary public health sector in Italy. Nevertheless, the recurrent introductions from wild birds indicate the need of improving both the biosecurity levels in the DPPA and the surveillance activities in wild birds to quickly detect the presence of AI in the territory.

Genetic and phylogenetic characterizations of a novel genotype of highly pathogenic avian influenza (HPAI) H5N8 viruses in 2016/2017 in South Korea.

During the outbreaks of highly pathogenic avian influenza (HPAI) H5N6 viruses in 2016 in South Korea, novel H5N8 viruses were also isolated from migratory birds. Phylogenetic analysis revealed that the HA gene of these H5N8 viruses belonged to clade 2.3.4.4, similarly to recent H5Nx viruses, and originated from A/Brk/Korea/Gochang1/14(H5N8), a minor lineage of H5N8 that appeared in 2014 and then disappeared. At least four reassortment events occurred with different subtypes (H5N8, H7N7, H3N8 and H10N7) and a chicken challenge study revealed that they were classified as HPAI viruses according to OIE criteria.

New influenza A(H7N7) viruses detected in live poultry markets in China.

H7N7 avian influenza viruses have been widely detected in wild birds and domestic poultry since they were first detected in chickens in Italy in 1902. They can occasionally transmit to humans. Here, we isolated six H7N7 viruses in live poultry markets during routine surveillance from 2010 to 2013. Sequences analysis revealed that these viruses are reassortants bearing genes of H3N8, H7N3, H7N7, and H10N7 influenza viruses detected in wild birds and ducks, and can be categorized into three genotypes (A, B, and C). All six viruses bound to both human-type and avian-type receptors. The viruses in genotype B and C could replicate efficiently in the lungs and nasal turbinates of mice without prior adaptation, and the genotype C virus also replicated in the brain of two of three mice tested. It is important to continue to monitor the evolution of H7N7 viruses and to evaluate their potential to cause human infections.

Cross-reactivity between avian influenza A (H7N9) virus and divergent H7 subtypic- and heterosubtypic influenza A viruses.

The number of human avian H7N9 influenza infections has been increasing in China. Understanding their antigenic and serologic relationships is crucial for developing diagnostic tools and vaccines. Here, we evaluated the cross-reactivities and neutralizing activities among H7 subtype influenza viruses and between H7N9 and heterosubtype influenza A viruses. We found strong cross-reactivities between H7N9 and divergent H7 subtypic viruses, including H7N2, H7N3, and H7N7. Antisera against H7N2, H7N3, and H7N7 could also effectively neutralize two distinct H7N9 strains. Two-way cross-reactivities exist within group 2, including H3 and H4, whereas one-way cross-reactivities were found across other groups, including H1, H10, H9, and H13. Our data indicate that the hemaglutinins from divergent H7 subtypes may facilitate the development of vaccines for distinct H7N9 infections. Moreover, serologic diagnoses for H7N9 infections need to consider possible interference from the cross-reactivity of H7N9 with other subtype influenza viruses.

A synchronized global sweep of the internal genes of modern avian influenza virus.

Zoonotic infectious diseases such as influenza continue to pose a grave threat to human health. However, the factors that mediate the emergence of RNA viruses such as influenza A virus (IAV) are still incompletely understood. Phylogenetic inference is crucial to reconstructing the origins and tracing the flow of IAV within and between hosts. Here we show that explicitly allowing IAV host lineages to have independent rates of molecular evolution is necessary for reliable phylogenetic inference of IAV and that methods that do not do so, including 'relaxed' molecular clock models, can be positively misleading. A phylogenomic analysis using a host-specific local clock model recovers extremely consistent evolutionary histories across all genomic segments and demonstrates that the equine H7N7 lineage is a sister clade to strains from birds--as well as those from humans, swine and the equine H3N8 lineage--sharing an ancestor with them in the mid to late 1800s. Moreover, major western and eastern hemisphere avian influenza lineages inferred for each gene coalesce in the late 1800s. On the basis of these phylogenies and the synchrony of these key nodes, we infer that the internal genes of avian influenza virus (AIV) underwent a global selective sweep beginning in the late 1800s, a process that continued throughout the twentieth century and up to the present. The resulting western hemispheric AIV lineage subsequently contributed most of the genomic segments to the 1918 pandemic virus and, independently, the 1963 equine H3N8 panzootic lineage. This approach provides a clear resolution of evolutionary patterns and processes in IAV, including the flow of viral genes and genomes within and between host lineages.

The genetics of virus particle shape in equine influenza A virus.

BACKGROUND: Many human strains of influenza A virus produce highly pleomorphic virus particles that at the extremes can be approximated as either spheres of around 100 nm diameter or filaments of similar cross-section but elongated to lengths of many microns. The role filamentous virions play in the virus life cycle remains enigmatic. OBJECTIVES/METHODS: Here, we set out to define the morphology and genetics of virus particle shape in equine influenza A virus, using reverse genetics and microscopy of infected cells. RESULTS AND CONCLUSIONS: The majority of H3N8 strains tested were found to produce filamentous virions, as did the prototype H7N7 A/eq/Prague/56 strain. The exception was the prototype H3N8 isolate, A/eq/Miami/63. Reassortment of equine influenza virus M genes from filamentous and non-filamentous strains into the non-filamentous human virus A/PR/8/34 confirmed that segment 7 is a major determinant of particle shape. Sequence analysis identified three M1 amino acid polymorphisms plausibly associated with determining virion morphology, and the introduction of these changes into viruses confirmed the importance of two: S85N and N231D. However, while either change alone affected filament production, the greatest effect was seen when the polymorphisms were introduced in conjunction. Thus, influenza A viruses from equine hosts also produce filamentous virions, and the major genetic determinants are set by the M1 protein. However, the precise sequence determinants are different to those previously identified in human or porcine viruses.

The genesis and source of the H7N9 influenza viruses causing human infections in China.

A novel H7N9 influenza A virus first detected in March 2013 has since caused more than 130 human infections in China, resulting in 40 deaths. Preliminary analyses suggest that the virus is a reassortant of H7, N9 and H9N2 avian influenza viruses, and carries some amino acids associated with mammalian receptor binding, raising concerns of a new pandemic. However, neither the source populations of the H7N9 outbreak lineage nor the conditions for its genesis are fully known. Using a combination of active surveillance, screening of virus archives, and evolutionary analyses, here we show that H7 viruses probably transferred from domestic duck to chicken populations in China on at least two independent occasions. We show that the H7 viruses subsequently reassorted with enzootic H9N2 viruses to generate the H7N9 outbreak lineage, and a related previously unrecognized H7N7 lineage. The H7N9 outbreak lineage has spread over a large geographic region and is prevalent in chickens at live poultry markets, which are thought to be the immediate source of human infections. Whether the H7N9 outbreak lineage has, or will, become enzootic in China and neighbouring regions requires further investigation. The discovery here of a related H7N7 influenza virus in chickens that has the ability to infect mammals experimentally, suggests that H7 viruses may pose threats beyond the current outbreak. The continuing prevalence of H7 viruses in poultry could lead to the generation of highly pathogenic variants and further sporadic human infections, with a continued risk of the virus acquiring human-to-human transmissibility.

Comparative analysis of avian influenza virus diversity in poultry and humans during a highly pathogenic avian influenza A (H7N7) virus outbreak.

Although increasing data have become available that link human adaptation with specific molecular changes in nonhuman influenza viruses, the molecular changes of these viruses during a large highly pathogenic avian influenza virus (HPAI) outbreak in poultry along with avian-to-human transmission have never been documented. By comprehensive virologic analysis of combined veterinary and human samples obtained during a large HPAI A (H7N7) outbreak in the Netherlands in 2003, we mapped the acquisition of human adaptation markers to identify the public health risk associated with an HPAI outbreak in poultry. Full-length hemagglutinin (HA), neuraminidase (NA), and PB2 sequencing of A (H7N7) viruses obtained from 45 human cases showed amino acid variations at different codons in HA (n=20), NA (n=23), and PB2 (n=23). Identification of the avian sources of human virus infections based on 232 farm sequences demonstrated that for each gene about 50% of the variation was already present in poultry. Polygenic accumulation and farm-to-farm spread of known virulence and human adaptation markers in A (H7N7) virus-infected poultry occurred prior to farm-to-human transmission. These include the independent emergence of HA A143T mutants, accumulation of four NA mutations, and farm-to-farm spread of virus variants harboring mammalian host determinants D701N and S714I in PB2. This implies that HPAI viruses with pandemic potential can emerge directly from poultry. Since the public health risk of an avian influenza virus outbreak in poultry can rapidly change, we recommend virologic monitoring for human adaptation markers among poultry as well as among humans during the course of an outbreak in poultry.

Evidence of persistence and multiple genetic modifications of H7N7 low-pathogenic avian influenza virus in wild mallards in Poland provided by phylogenetic studies.

Genetic characterization of the whole genome of four avian influenza H7N7 viruses isolated in three successive winter seasons (2007 to 2009) from wild mallards in three cities in Poland was performed. All of the tested strains were of low pathogenicity and no molecular marker associated with an increased adaptation to poultry, mammals or resistance to antiviral drugs was found. The major outcome of the phylogenetic studies was that the isolate A/mallard/Poland/446/09 (detected in December 2009) shared a recent common ancestor with A/mallard/Poland/41/09 (isolated in February 2009) in relation to HA and PB1 genes, with A/mallard/Poland/16/09 (found in January 2009) regarding NA and NS genes, and with A/mallard/Poland/01/08 (recovered in December 2007) as regards the NS gene. Interestingly, A/mallard/Poland/16/09 and A/mallard/Poland/446/09 were isolated at the same sampling site almost exactly 1 year apart, which points to resident population of mallards (and other resident waterfowl) as responsible for the perpetuation of avian influenza virus (AIV) in the given area between successive winters. On the other hand, the ornithological data discussed in detail in the paper strongly suggest that the virus transmission between close sites but located in different urban areas is most probably achieved by migratory birds, a fact additionally supported by a close relatedness between different gene segments of Polish H7N7 and AIV detected in wild birds in Europe. A high heterogeneity of the gene pool found in the study is indicative of frequent reassortment events. Additionally, two H7N7 isolates were shown to possess selected genes closely related to AIV detected in domestic poultry in Italy and the Czech Republic. The present study corroborates the importance of active surveillance in wild birds as a valuable tool for early warning of avian influenza in poultry.

Development of real time RT-PCR assays for detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes.

Rapid detection and subtyping of H5 and H7 subtypes influenza A viruses are important for disease control in poultry and potential transmission to humans. Currently, virus isolation and subsequent HA and NA subtyping constitute the standard for avian influenza viruses detection and subtype identification. These methods are highly accurate and sensitive but are also laborious and time-consuming. Reverse transcription PCR and real time reverse transcription PCR assays, suitable tests for rapid detection, have previously been used for the specific diagnosis of H5 and H7 viruses, however, at present, no primer and probe sets are available for the identification of all H5 and H7 strains. Herein, we have developed specific and sensitive real time reverse transcription PCR assays for the detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes and we have also compared these molecular assays with viral isolation in terms of sensitivity. Our results demonstrate that the real time reverse transcription PCR assays are more sensitive, specific, less expensive compared to viral isolation. In conclusion, molecular assays could represent an useful tool for rapid detection and screening of H5 and H7 isolates during influenza A virus outbreaks alternatively to viral isolation.

Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus.

The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of >or=10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

Development of a pen-site test kit for the rapid diagnosis of H7 highly pathogenic avian influenza.

As well as H5 highly pathogenic avian influenza viruses (HPAIV), H7 HPAIV strains have caused serious damages in poultry industries worldwide. Cases of bird-to-human transmission of H7 HPAIV have also been reported [11]. On the outbreak of avian influenza, rapid diagnosis is critical not only for the control of HPAI but also for human health. In the present study, a rapid diagnosis kit based on immunochromatography for the detection of H7 hemagglutinin (HA) antigen of influenza A virus was developed using 2 monoclonal antibodies that recognize different epitopes on the H7 HAs. The kit detected each of the tested 15 H7 influenza virus strains and did not react with influenza A viruses of the other subtypes than H7 or other avian viral and bacterial pathogens. The kit detected H7 HA antigen in the swabs and tissue homogenates of the chickens experimentally infected with HPAIV strain A/chicken/Netherlands/2586/03 (H7N7). The results indicate that the present kit is specific and sensitive enough for the diagnosis of HPAI caused by H7 viruses, thus, recommended for the field application as a pen-site test kit.

Molecular analysis of avian H7 influenza viruses circulating in Eurasia in 1999-2005: detection of multiple reassortant virus genotypes.

Avian influenza infections by high and low pathogenicity H7 influenza viruses have caused several outbreaks in European poultry in recent years, also resulting in human infections. Although in some cases the source of H7 strains from domestic poultry was shown to be the viruses circulating in the wild bird reservoir, a thorough characterization of the entire genome of H7 viruses from both wild and domestic Eurasian birds, and their evolutionary relationships, has not been conducted. In our study, we have analysed low pathogenicity H7 influenza strains isolated from wild and domestic ducks in Italy and southern China and compared them with those from reared terrestrial poultry such as chicken and turkey. Phylogenetic analysis demonstrated that the H7 haemagglutinin genes were all closely related to each other, whereas the remaining genes could be divided into two or more phylogenetic groups. Almost each year different H7 reassortant viruses were identified and in at least two different years more than one H7 genotype co-circulated. A recent precursor in wild waterfowl was identified for most of the gene segments of terrestrial poultry viruses. Our data suggest that reassortment allows avian influenza viruses, in their natural reservoir, to increase their genetic diversity. In turn this might help avian influenza viruses colonize a wider range of hosts, including domestic poultry.

Evaluation of replication and pathogenicity of avian influenza a H7 subtype viruses in a mouse model.

Avian influenza A H7 subtype viruses pose a significant threat to human health because of their ability to transmit directly from domestic poultry to humans and to cause disease and, sometimes, death. Although it is important to develop vaccines against viruses of this subtype, very limited information is available on the immune response and pathogenesis of H7 viruses in animal models such as mice and ferrets. Ten H7 viruses were selected for possible vaccine development on the basis of their phylogenetic relationships and geographical locations. The virulence of the 10 viruses for mice and the immunogenicity of the viruses in mice and ferrets were evaluated to study the extent of antigenic relatedness and the level of cross-reactivity of antibodies. Most of the viruses showed similar patterns of cross-reactivity with mouse and ferret antisera. The Eurasian viruses elicited broadly cross-reactive antibodies that neutralized viruses from both Eurasian and North American lineages, but the converse was not true. A subset of the viruses was also evaluated for the ability to replicate and cause disease in BALB/c mice following intranasal administration. H7 subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. On the basis of phylogenetic data, induction of broadly cross-neutralizing antibodies in mouse and ferret antisera, and their ability to replicate in mice, we have selected A/Netherlands/219/03 (subtype H7N7) and A/chicken/BC/CN-7/04 (subtype H7N3) viruses for vaccine development. The mouse model can be used for the preclinical evaluation of these vaccines against H7 subtype viruses.

Properties of H7N7 influenza A virus strain SC35M lacking interferon antagonist NS1 in mice and chickens.

Non-structural protein NS1 of influenza A virus counteracts the host immune response by blocking the synthesis of type I interferon (IFN). As deletion of the complete NS1 gene has to date been reported only in the human H1N1 strain A/PR/8/34, it remained unclear whether NS1 is a non-essential virulence factor in other influenza A virus strains as well. In this report, the properties of NS1-deficient mutants derived from strain SC35M (H7N7) are described. A mutant of SC35M that completely lacks the NS1 gene was an excellent inducer of IFN in mammalian and avian cells in culture and, consequently, was able to multiply efficiently only in cell lines with defects in the type I IFN system. Virus mutants carrying C-terminally truncated versions of NS1 were less powerful inducers of IFN and were attenuated less strongly in human A549 cells. Although attenuated in wild-type mice, these mutants remained highly pathogenic for mice lacking the IFN-regulated antiviral factor Mx1. In contrast, the NS1-deficient SC35M mutant was completely non-pathogenic for wild-type mice, but remained pathogenic for mice lacking Mx1 and double-stranded RNA-activated protein kinase (PKR). Wild-type SC35M, but not the NS1-deficient mutant virus, was able to replicate in the upper respiratory tract of birds, but neither virus induced severe disease in adult chickens. Altogether, this study supports the view that NS1 represents a non-essential virulence factor of different influenza A viruses.

Genomic signatures of human versus avian influenza A viruses.

Position-specific entropy profiles created from scanning 306 human and 95 avian influenza A viral genomes showed that 228 of 4591 amino acid residues yielded significant differences between these 2 viruses. We subsequently used 15,785 protein sequences from the National Center for Biotechnology Information (NCBI) to assess the robustness of these signatures and obtained 52 "species-associated" positions. Specific mutations on those points may enable an avian influenza virus to become a human virus. Many of these signatures are found in NP, PA, and PB2 genes (viral ribonucleoproteins [RNPs]) and are mostly located in the functional domains related to RNP-RNP interactions that are important for viral replication. Upon inspecting 21 human-isolated avian influenza viral genomes from NCBI, we found 19 that exhibited > or =1 species-associated residue changes; 7 of them contained > or =2 substitutions. Histograms based on pairwise sequence comparison showed that NP disjointed most between human and avian influenza viruses, followed by PA and PB2.