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Highly pathogenic avian influenza viruses of the H5N1 clade 2.3.4.4b were detected in North America in the winter of 2021/2022. These viruses have spread across the Americas, causing morbidity and mortality in both wild and domestic birds as well as some mammalian species, including cattle. Many surveillance programs for wildlife as well as commercial poultry operations have detected these viruses. In this study, we conducted surveillance of avian species in the urban environment in New York City. We detected highly pathogenic H5N1 viruses in six samples from four different bird species and performed whole-genome sequencing. Sequencing analysis showed the presence of multiple different genotypes. Our work highlights that the interface between animals and humans that may give rise to zoonotic infections or even pandemics is not limited to rural environments and commercial poultry operations but extends into the heart of our urban centers.IMPORTANCEWhile surveillance programs for avian influenza viruses are often focused on migratory routes and their associated stop-over locations or commercial poultry operations, many bird species-including migratory birds-frequent or live in urban green spaces and wetlands. This brings them into contact with a highly dense population of humans and pets, providing an extensive urban animal-human interface in which the general public may have little awareness of circulating infectious diseases. This study focuses on virus surveillance of this interface, combined with culturally responsive science education and community outreach., Competing Interests: The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, influenza virus vaccines, and influenza virus therapeutics, which list Florian Krammer as the co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, Castlevax, to develop SARS-CoV-2 vaccines. Florian Krammer is co-founder and scientific advisory board member of Castlevax. Florian Krammer has consulted for Merck, Curevac, Seqirus, and Pfizer and is currently consulting for 3rd Rock Ventures, GSK, Gritstone, and Avimex. The Krammer laboratory is also collaborating with Dynavax on influenza vaccine development. All other authors declare no conflicts.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals., Competing Interests: The authors declare no conflict of interest.

Adaptive mutations and/or reassortments in avian influenza virus polymerase subunits PA, PB1, and PB2 are one of the major factors enabling the virus to overcome the species barrier to infect humans. The majority of human adaptation polymerase mutations have been identified in PB2; fewer adaptation mutations have been characterized in PA and PB1. Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and generally carry the human adaptation PB2-E627K substitution during their dissemination in nature. In this study, we identified other human adaptation polymerase mutations by analyzing phylogeny-associated PA mutations that H5N1 clade 2.2.1 viruses have accumulated during their evolution in the field. This analysis identified several PA mutations that produced increased replication by contemporary clade 2.2.1.2 viruses in vitro in human cells and in vivo in mice compared to ancestral clade 2.2.1 viruses. The PA mutations acted cooperatively to increase viral polymerase activity and replication in both avian and human cells, with the effect being more prominent in human cells at 33°C than at 37°C. These results indicated that PA mutations have a role in establishing contemporary clade 2.2.1.2 virus infections in poultry and in adaptation to infect mammals. Our study provided data on the mechanism for PA mutations to accumulate during avian influenza virus evolution and extend the viral host range. IMPORTANCE Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and have caused the highest number of human H5N1 influenza cases worldwide, presenting a serious global public health threat. These viruses may have the greatest evolutionary potential for adaptation from avian hosts to human hosts. Using a comprehensive phylogenetic approach, we identified several novel clade 2.2.1 virus polymerase mutations that increased viral replication in vitro in human cells and in vivo in mice. These mutations were in the polymerase PA subunit and acted cooperatively with the E627K mutation in the PB2 polymerase subunit to provide higher replication in contemporary clade 2.2.1.2 viruses than in ancestral clade 2.2.1 viruses. These data indicated that ongoing clade 2.2.1 dissemination in the field has driven PA mutations to modify viral replication to enable host range expansion, with a higher public health risk for humans., (Copyright © 2020 Arai et al.)

The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-β) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans. IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells., (Copyright © 2019 Monteagudo et al.)

Transmission of avian influenza (AI) viruses to mammals involves phylogenetic bottlenecks that select small numbers of variants for transmission to new host species. However, little is known about the AI virus quasispecies diversity that produces variants for virus adaptation to humans. Here, we analyzed the hemagglutinin (HA) genetic diversity produced during AI H5N1 single-virus infection of primary human airway cells and characterized the phenotypes of these variants. During single-virus infection, HA variants emerged with increased fitness to infect human cells. These variants generally had decreased HA thermostability, an indicator of decreased transmissibility, that appeared to compensate for their increase in α2,6-linked sialic acid (α2,6 Sia) binding specificity and/or in the membrane fusion pH threshold, each of which is an advantageous mutational change for viral infection of human airway epithelia. An HA variant with increased HA thermostability also emerged but could not outcompete variants with less HA thermostability. These results provided data on HA quasispecies diversity in human airway cells. IMPORTANCE The diversity of the influenza virus quasispecies that emerges from a single infection is the starting point for viral adaptation to new hosts. A few studies have investigated AI virus quasispecies diversity during human adaptation using clinical samples. However, those studies could be appreciably affected by individual variability and multifactorial respiratory factors, which complicate identification of quasispecies diversity produced by selective pressure for increased adaptation to infect human airway cells. Here, we found that detectable HA genetic diversity was produced by H5N1 single-virus infection of human airway cells. Most of the HA variants had increased fitness to infect human airway cells but incurred a fitness cost of less HA stability. To our knowledge, this is the first report to characterize the adaptive changes of AI virus quasispecies produced by infection of human airway cells. These results provide a better perspective on AI virus adaptation to infect humans., (Copyright © 2018 American Society for Microbiology.)

We have developed pandemic live attenuated influenza vaccines (pLAIVs) against clade 1 H5N1 viruses on an Ann Arbor cold-adapted ( ca ) backbone that induced long-term immune memory. In 2015, many human infections caused by a new clade (clade 2.2.1.1) of goose/Guangdong (gs/GD) lineage H5N1 viruses were reported in Egypt, which prompted updating of the H5N1 pLAIV. We explored two strategies to generate suitable pLAIVs. The first approach was to modify the hemagglutinin gene of a highly pathogenic wild-type ( wt ) clade 2.2.1.1 virus, A/Egypt/N03434/2009 (Egy/09) (H5N1), with its unmodified neuraminidase (NA) gene; this virus was designated Egy/09 ca The second approach was to select a low-pathogenicity avian influenza H5 virus that elicited antibodies that cross-reacted with a broad range of H5 viruses, including the Egypt H5N1 viruses, and contained a novel NA subtype for humans. We selected the low-pathogenicity A/duck/Hokkaido/69/2000 (H5N3) (dk/Hok/00) virus for this purpose. Both candidate vaccines were attenuated and immunogenic in ferrets, inducing antibodies that neutralized homologous and heterologous H5 viruses with different degrees of cross-reactivity; Egy/09 ca vaccine antisera were more specific for the gs/GD lineage viruses but did not neutralize recent North American isolates (clade 2.3.4.4), whereas antisera from dk/Hok/69 ca -vaccinated ferrets cross-reacted with clade 2.3.4.4 and 2.2.1 viruses but not clade 1 or 2.1 viruses. When vaccinated ferrets were challenged with homologous and heterologous H5 viruses, challenge virus replication was reduced in the respiratory tract. Thus, the two H5 pLAIV candidates are suitable for clinical development to protect humans from infection with different clades of H5 viruses. IMPORTANCE In response to the continuing evolution of H5N1 avian influenza viruses and human infections, new candidate H5 live attenuated vaccines were developed by using two different approaches: one targeted a specific circulating strain in Egypt, and the other was based on a virus that elicits broadly cross-reactive antibodies against a wide range of H5 viruses. Both candidate vaccines were immunogenic and exhibited protective efficacy in ferrets. Our study permits a comparison of the two approaches, and the data support the further development of both vaccine viruses to optimally prepare for the further spread of clade 2.2.1 or 2.3.4.4 viruses., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: The novel H7N9 avian influenza virus (AIV) was demonstrated to cause severe human respiratory infections in China. Here, we examined poultry specimens from live bird markets linked to human H7N9 infection in Hangzhou, China. Metagenomic sequencing revealed mixed subtypes (H5, H7, H9, N1, N2, and N9). Subsequently, AIV subtypes H5N9, H7N9, and H9N2 were isolated. Evolutionary analysis showed that the hemagglutinin gene of the novel H5N9 virus originated from A/Muscovy duck/Vietnam/LBM227/2012 (H5N1), which belongs to clade 2.3.2.1. The neuraminidase gene of the novel H5N9 virus originated from human-infective A/Hangzhou/1/2013 (H7N9). The six internal genes were similar to those of other H5N1, H7N9, and H9N2 virus strains. The virus harbored the PQRERRRKR/GL motif characteristic of highly pathogenic AIVs at the HA cleavage site. Receptor-binding experiments demonstrated that the virus binds α-2,3 sialic acid but not α-2,6 sialic acid. Identically, pathogenicity experiments also showed that the virus caused low mortality rates in mice. This newly isolated H5N9 virus is a highly pathogenic reassortant virus originating from H5N1, H7N9, and H9N2 subtypes. Live bird markets represent a potential transmission risk to public health and the poultry industry., Importance: This investigation confirms that the novel H5N9 subtype avian influenza A virus is a reassortant strain originating from H5N1, H7N9, and H9N2 subtypes and is totally different from the H5N9 viruses reported before. The novel H5N9 virus acquired a highly pathogenic H5 gene and an N9 gene from human-infecting subtype H7N9 but caused low mortality rates in mice. Whether this novel H5N9 virus will cause human infections from its avian host and become a pandemic subtype is not known yet. It is therefore imperative to assess the risk of emergence of this novel reassortant virus with potential transmissibility to public health., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Because of their rapid evolution, genetic diversity, broad host range, ongoing circulation in birds, and potential human-to-human transmission, H5N1 influenza viruses remain a major global health concern. Their high degree of genetic diversity also poses enormous burdens and uncertainties in developing effective vaccines. To overcome this, we took a new approach, i.e., the development of immunogens based on a comprehensive serologic study. We constructed DNA plasmids encoding codon-optimized hemagglutinin (HA) from 17 representative strains covering all reported clades and subclades of highly pathogenic avian influenza H5N1 viruses. Using DNA plasmids, we generated the corresponding H5N1 pseudotypes and immune sera. We performed an across-the-board pseudotype-based neutralization assay and determined antigenic clusters by cartography. We then designed a triclade DNA vaccine and evaluated its immunogenicity and protection in mice. We report here that (sub)clades 0, 1, 3, 4, 5, 6, 7.1, and 9 were grouped into antigenic cluster 1, (sub)clades 2.1.3.2, 2.3.4, 2.4, 2.5, and 8 were grouped into another antigenic cluster, with subclade 2.2.1 loosely connected to it, and each of subclades 2.3.2.1 and 7.2 was by itself. Importantly, the triclade DNA vaccine encoding HAs of (sub)clades 0, 2.3.2.1, and 7.2 elicited broadly neutralizing antibody responses against all H5 clades and subclades and protected mice against high-lethal-dose heterologous H5N1 challenge. Thus, we conclude that broadly neutralizing antibodies against all H5 clades and subclades can indeed be elicited with immunogens on the basis of a comprehensive serologic study. Further evaluation and optimization of such an approach in ferrets and in humans is warranted.

Human infections of H5N1 highly pathogenic avian influenza virus have continued to occur in China without corresponding outbreaks in poultry, and there is little conclusive evidence of the source of these infections. Seeking to identify the source of the human infections, we sequenced 31 H5N1 viruses isolated from humans in China (2005 to 2010). We found a number of viral genotypes, not all of which have similar known avian virus counterparts. Guided by patient questionnaire data, we also obtained environmental samples from live poultry markets and dwellings frequented by six individuals prior to disease onset (2008 and 2009). H5N1 viruses were isolated from 4 of the 6 live poultry markets sampled. In each case, the genetic sequences of the environmental and corresponding human isolates were highly similar, demonstrating a link between human infection and live poultry markets. Therefore, infection control measures in live poultry markets are likely to reduce human H5N1 infection in China.

The molecular basis of pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in chickens remains largely unknown. H5N1 A/chicken/Yamaguchi/7/2004 virus (CkYM7) replicates rapidly in macrophages and vascular endothelial cells in chickens, causing sudden death without fever or gross lesions, while H5N1 A/duck/Yokohama/aq10/2003 virus (DkYK10) induces high fever, severe gross lesions, and a prolonged time to death, despite the 98% amino acid identity between the two viruses. To explore the molecular basis of this difference in pathogenicity, a series of eight single-gene reassortant viruses from these HPAI viruses were compared for pathogenicity in chickens. Two reassortants possessing the NP or PB2 gene from DkYK10 in the CkYM7 background reduced pathogenicity compared to other reassortants or CkYM7. Inversely, reassortants possessing the NP or PB2 gene of CkYM7 in the DkYK10 background (rgDkYK-PB2(Ck), rgDkYK-NP(Ck)) replicated quickly and reached higher titers than DkYK10, accompanied by more rapid and frequent apoptosis of macrophages. The rgDkYK-NP(Ck) and rgDkYK-PB2(Ck) reassortants also replicated more rapidly in chicken embryo fibroblasts (CEFs) than did rgDkYK10, but replication of these viruses was similar to that of CkYM7 and DkYK10 in duck embryo fibroblasts. A comparison of pathogenicities of seven rgDkYK10 mutants with a single amino acid substitution in NP(Dk) demonstrated that valine at position 105 in the NP(Ck) was responsible for the increased pathogenicity in chickens. NP(Ck), NP(105V), and PB2(Ck) enhanced the polymerase activity of DkYK10 in CEFs. These results indicate that both NP and PB2 contribute to the high pathogenicity of the H5N1 HPAI viruses in chickens, and valine at position 105 of NP may be one of the determinants for adaptation of avian influenza viruses from ducks to chickens.

The nature of influenza virus to randomly mutate and evolve into new types with diverse antigenic determinants is an important challenge in the control of influenza infection. Particularly, variations within the amino acid sequences of major neutralizing epitopes of influenza virus hemagglutinin (HA) hindered the development of universal vaccines against H5N1 lineages. Based on distribution analyses of the identified major neutralizing epitopes of hemagglutinin, we selected three vaccine strains that cover the entire variants in the neutralizing epitopes among the H5N1 lineages. HA proteins of selected vaccine strains were expressed on the baculovirus surface (BacHA), and the preclinical efficacy of the vaccine formulations was evaluated in a mouse model. The combination of three selected vaccine strains could effectively neutralize viruses from clades 1, 2.1, 2.2, 4, 7, and 8 of influenza H5N1 viruses. In contrast, a vaccine formulation containing only adjuvanted monovalent BacHA (mono-BacHA) or a single strain of inactivated whole viral vaccine was able to neutralize only clade 1 (homologous), clade 2.1, and clade 8.0 viruses. Also, the trivalent BacHA vaccine was able to protect 100% of the mice against challenge with three different clades (clade 1.0, clade 2.1, and clade 7.0) of H5N1 strains compared to mono-BacHA or inactivated whole viral vaccine. The present findings provide a rationale for the development of a universal vaccine against H5N1 lineages. Furthermore, baculoviruses displaying HA will serve as an ideal choice for a vaccine in prepandemic or pandemic situations and expedite vaccine technology without the requirement of high-level-biocontainment facilities or tedious protein purification processes.

Despite substantial efforts to control H5N1 avian influenza viruses (AIVs), the viruses have continued to evolve and cause disease outbreaks in poultry and infections in humans. In this report, we analyzed 51 representative H5N1 AIVs isolated from domestic poultry, wild birds, and humans in China during 2004 to 2009, and 21 genotypes were detected based on whole-genome sequences. Twelve genotypes of AIVs in southern China bear similar H5 hemagglutinin (HA) genes (clade 2.3). These AIVs did not display antigenic drift and could be completely protected against by the A/goose/Guangdong/1/96 (GS/GD/1/96)-based oil-adjuvanted killed vaccine and recombinant Newcastle disease virus vaccine, which have been used in China. In addition, antigenically drifted H5N1 viruses, represented by A/chicken/Shanxi/2/06 (CK/SX/2/06), were detected in chickens from several provinces in northern China. The CK/SX/2/06-like viruses are reassortants with newly emerged HA, NA, and PB1 genes that could not be protected against by the GS/GD/1/96-based vaccines. These viruses also reacted poorly with antisera generated from clade 2.2 and 2.3 viruses. The majority of the viruses isolated from southern China were lethal in mice and ducks, while the CK/SX/2/06-like viruses caused mild disease in mice and could not replicate in ducks. Our results demonstrate that the H5N1 AIVs circulating in nature have complex biological characteristics and pose a continued challenge for disease control and pandemic preparedness.

Highly pathogenic A/H5N1 avian influenza (HPAI H5N1) viruses have seriously affected the Nigerian poultry industry since early 2006. Previous studies have identified multiple introductions of the virus into Nigeria and several reassortment events between cocirculating lineages. To determine the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. We identified a major new subclade in Nigeria that is phylogenetically distinguishable from all previously identified sublineages, as well as two novel reassortment events. A detailed analysis of viral phylogeography identified two major source populations for the HPAI H5N1 virus in Nigeria, one in a major commercial poultry area (southwest region) and one in northern Nigeria, where contact between wild birds and backyard poultry is frequent. These findings suggested that migratory birds from Eastern Europe or Russia may serve an important role in the introduction of HPAI H5N1 viruses into Nigeria, although virus spread through the movement of poultry and poultry products cannot be excluded. Our study provides new insight into the genesis and evolution of H5N1 influenza viruses in Nigeria and has important implications for targeting surveillance efforts to rapidly identify the spread of the virus into and within Nigeria.

The preparation of high-yield prepandemic influenza virus H5N1 strains has presented a challenge to both researchers and vaccine manufacturers. The reasons for the relatively low yield of the H5N1 strains are not fully understood, but it might be partially dependent on the interactions between the hemagglutinin (HA) or neuraminidase (NA) surface glycoprotein and other influenza virus proteins. In this study, we have constructed chimeras between the A/Puerto Rico/8/34 (PR8) NA gene and the A/Vietnam/1203/2004 (VN1203) NA gene that have resulted in an increase in the virus yield of the reassortant viruses without a significant loss of NA activity. By combining the amino terminus of NA from the PR8 strain with the carboxy terminus of NA from VN1203, the surface epitopes unique to the H5N1 VN1203 NA glycoprotein are maintained. This reassortant virus had a higher titer and total protein yield in eggs, grew to a higher titer, produced large plaques on MDCK cells, and retained NA activity. This work describes a novel recombinant technique designed to increase the yields of vaccine candidates for the production of pandemic influenza virus vaccines. The relationship between the infectivity and protein yield of the reassortants also is discussed.

Following circulation of avian influenza H5 and H7 viruses in poultry, the hemagglutinin (HA) can acquire additional glycosylation sites, and the neuraminidase (NA) stalk becomes shorter. We investigated whether these features play a role in the pathogenesis of infection in mammalian hosts. From 1996 to 2007, H5N1 viruses with a short NA stalk have become widespread in several avian species. Compared to viruses with a long-stalk NA, viruses with a short-stalk NA showed a decreased capacity to elute from red blood cells and an increased virulence in mice, but not in chickens. The presence of additional HA glycosylation sites had less of an effect on virulence than did NA stalk length. The short-stalk NA of H5N1 viruses circulating in Asia may contribute to virulence in humans.

The transmission of highly pathogenic avian influenza H5N1 virus to Southeast Asian countries triggered the first major outbreak and transmission wave in late 2003, accelerating the pandemic threat to the world. Due to the lack of influenza surveillance prior to these outbreaks, the genetic diversity and the transmission pathways of H5N1 viruses from this period remain undefined. To determine the possible source of the wave 1 H5N1 viruses, we recently conducted further sequencing and analysis of samples collected in live-poultry markets from Guangdong, Hunan, and Yunnan in southern China from 2001 to 2004. Phylogenetic analysis of the hemagglutinin and neuraminidase genes of 73 H5N1 isolates from this period revealed a greater genetic diversity in southern China than previously reported. Moreover, results show that eight viruses isolated from Yunnan in 2002 and 2003 were most closely related to the clade 1 virus sublineage from Vietnam, Thailand, and Malaysia, while two viruses from Hunan in 2002 and 2003 were most closely related to viruses from Indonesia (clade 2.1). Further phylogenetic analyses of the six internal genes showed that all 10 of those viruses maintained similar phylogenetic relationships as the surface genes. The 10 progenitor viruses were genotype Z and shared high similarity (>/=99%) with their corresponding descendant viruses in most gene segments. These results suggest a direct transmission link for H5N1 viruses between Yunnan and Vietnam and also between Hunan and Indonesia during 2002 and 2003. Poultry trade may be responsible for virus introduction to Vietnam, while the transmission route from Hunan to Indonesia remains unclear.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries, resulting in repeated outbreaks in poultry and increased cases of human infection. The immediate precursor of these HPAI viruses is believed to be A/goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses first detected in Guangdong, China, in 1996. From 2000 onwards, many novel reassortant H5N1 influenza viruses or genotypes have emerged in southern China. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low-pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also show reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza virus gene pool among poultry in Eurasia.