The role of PB1-F2 in adaptation of high pathogenicity avian influenza virus H7N7 in chickens.

Avian influenza viruses (AIV) of the H7N7 subtype are enzootic in the wild bird reservoir in Europe, cause infections in poultry, and have sporadically infected humans. The non-structural protein PB1-F2 is encoded in a second open frame in the polymerase segment PB1 and its sequence varies with the host of origin. While mammalian isolates predominantly carry truncated forms, avian isolates typically express full-length PB1-F2. PB1-F2 is a virulence factor of influenza viruses in mammals. It modulates the host immune response, causing immunopathology and increases pro-inflammatory responses. The role of full-length PB1-F2 in IAV pathogenesis as well as its impact on virus adaptation and virulence in poultry remains enigmatic. Here, we characterised recombinant high pathogenicity AIV (HPAIV) H7N7 expressing or lacking PB1-F2 in vitro and in vivo in chickens. In vitro, full-length PB1-F2 modulated viability of infected chicken fibroblasts by limiting apoptosis. In chickens, PB1-F2 promoted gastrointestinal tropism, as demonstrated by enhanced viral replication in the gut and increased cloacal shedding. PB1-F2's effects on cellular immunity however were marginal. Overall, chickens infected with full-length PB1-F2 virus survived for shorter periods, indicating that PB1-F2 is also a virulence factor in bird-adapted viruses.

Surface acoustic wave (SAW) real-time interaction analysis of influenza A virus hemagglutinins with sialylated neoglycolipids.

Real-time interaction analysis of H1 hemagglutinin from influenza A H1N1 (A/New York/18/2009) and H7 hemagglutinin from influenza A H7N7 (A/Netherlands/219/03) with sialylated neoglycolipids (neoGLs) was performed using the surface acoustic wave (SAW) technology. The produced neoGLs carried phosphatidylethanolamine (PE) as lipid anchor and terminally sialylated lactose (Lc2, Galß1-4Glc) or neolactotetraose (nLc4, Galß1-4GlcNAcß1-3Galß1-4Glc) harboring an N-acetylneuraminic acid (Neu5Ac). Using α2-6-sialylated neoGLs, H1 and H7 exhibited marginal attachment toward II6Neu5Ac-Lc2-PE, whereas Sambucus nigra lectin (SNL) exhibited strong binding and Maackia amurensis lectin (MAL) was negative in accordance with their known binding preference toward a distal Neu5Acα2-6Gal- and Neu5Acα2-3Gal-residue, respectively. H1 revealed significant binding toward IV6Neu5Ac-nLc4-PE when compared to weak interaction of H7, whereas SNL showed strong and MAL no attachment corresponding to their interaction specificities. Additional controls of MAL and SNL with α2-3-sialylated II3Neu5Ac-Lc2-PE and IV3Neu5Ac-nLc4-PE underscored the reliability of the SAW technology. Pre-exposure of model membranes spiked with α2-6-sialylated neoGLs to Vibrio cholerae neuraminidase substantially reduced the binding of the hemagglutinins and the SNL reference. Collectively, the SAW technology is capable of accurate measuring binding features of hemagglutinins toward neoGL-spiked lipid bilayers, which can be easily loaded to the functionalized biosensor gold surface thereby simulating biological membranes and suggesting promising clinical application for influenza virus research.

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.

Isolation and characterization of low pathogenic H7N7 avian influenza virus from a red-crowned crane in a zoo in South Korea.

BACKGROUND: South Korea conducts annual national surveillance programs to detect avian influenza (AI) in domestic poultry, live bird markets, and wild birds. In March 2017, an AIV was isolated from fecal samples in an outdoor aviary flight cage in a zoo in Korea. RESULTS: Nucleotide sequencing identified the isolate as low pathogenic avian influenza virus (LPAIV) H7N7, and DNA barcoding analysis identified the host species as red-crowned crane. This isolate was designated A/red-crowned crane/Korea/H1026/2017 (H7N7). Genetic analysis and gene constellation analysis revealed that A/red-crowned crane/Korea/H1026/2017 (H7N7) showed high similarity with four H7N7 LPAIVs isolated from wild bird habitats in Seoul and Gyeonggi in early 2017. CONCLUSIONS: Considering the genetic similarity and similar collection dates of the viruses, and the fact that zoo bird cages are vulnerable to AIV, it is likely that fecal contamination from wild birds might have introduced LPAIV H7N7 into the red-crowned crane at the zoo. Therefore, our results emphasize that enhanced biosecurity measures should be employed during the wild bird migration season, and that continued surveillance should be undertaken to prevent potential threats to avian species in zoos and to humans.

Virulence of three European highly pathogenic H7N1 and H7N7 avian influenza viruses in Pekin and Muscovy ducks.

BACKGROUND: There is paucity of data on the virulence of highly pathogenic (HP) avian influenza viruses (AIV) H7 in ducks compared to HPAIV H5. Here, the virulence of HPAIV H7N1 (designated H7N1-FPV34 and H7N1-It99) and H7N7 (designated H7N7-FPV27) was assessed in Pekin and/or Muscovy ducklings after intrachoanal (IC) or intramuscular (IM) infection. RESULTS: The morbidity rate ranged from 60 to 100% and mortality rate from 20 to 80% depending on the duck species, virus strain and/or challenge route. All Muscovy ducklings inoculated IC with H7N7-FPV27 or H7N1-FPV34 exhibited mild to severe clinical signs resulting in the death of 2/10 and 8/10 ducklings, respectively. Also, 2/10 and 6/9 of inoculated Muscovy ducklings died after IC or IM infection with H7N1-It99, respectively. Moreover, 5/10 Pekin ducklings inoculated IC or IM with H7N1-It99 died. The level of virus detected in the oropharyngeal swabs was higher than in the cloacal swabs. CONCLUSION: Taken together, HPAIV H7 cause mortality and morbidity in Muscovy and Pekin ducklings. The severity of disease in Muscovy ducklings depended on the virus strain and/or route of infection. Preferential replication of the virus in the respiratory tract compared to the gut merits further investigation.

Phylogenetic and Molecular Analysis of an H7N7 Avian Influenza Virus Isolated in East Dongting Lake in 2012.

OBJECTIVE: In March 2012, an H7N7 subtype avian influenza virus (AIV) named A/wild goose/Dongting/PC0360/2012 (H7N7) (DT/PC0360) was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization. METHODS: RNA was extracted from environment samples (including fecal samples from wild bird or domestic ducks, and water samples) for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the H1-H16 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed. RESULTS: Our results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in Mainland China in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus. CONCLUSION: Strengthening the surveillance of AIVs of wild waterfowl and poultry in this region is vital for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.

Novel H7N7 avian influenza viruses detected in migratory wild birds in eastern China between 2018 and 2020.

Wild birds are the natural reservoirs of avian influenza viruses, and surveillance and assessment of these viruses in wild birds provide valuable information for early warning and control of animal diseases. In this study, we isolated 19 H7N7 avian influenza viruses from wild bird between 2018 and 2020. Full genomic analysis revealed that these viruses bear a single basic amino acid in the cleavage site of their hemagglutinin gene, and formed four different genotypes by actively reassorting other avian influenza viruses circulating in wild birds and ducks. The H7N7 viruses bound to both avian-type and human-type receptors, although their affinity for human-type receptors was markedly lower than that for avian-type receptors. Moreover, we found that the H7N7 viruses could replicate efficiently in the upper respiratory tract and caecum of domestic ducks, and that the H5/H7 inactivated vaccine used in poultry in China provided complete protection against H7N7 wild bird virus challenge in ducks. Our findings demonstrate that wild bird H7N7 viruses pose a substantial threat to the poultry industry across the East Asian-Australian migratory flyway, emphasize the importance of influenza virus surveillance in both wild and domestic birds, and support the development of active control strategies against H7N7 virus.

An Outbreak of Highly Pathogenic Avian Influenza (H7N7) in Australia and the Potential for Novel Influenza A Viruses to Emerge.

In 2020, several geographically isolated farms in Victoria, Australia, experienced an outbreak of highly pathogenic avian influenza (HPAI) virus H7N7 and low pathogenic avian influenza (LPAI) viruses H5N2 and H7N6. Effective containment and control measures ensured the eradication of these viruses but the event culminated in substantial loss of livestock and significant economic impact. The avian HPAI H7N7 virus generally does not infect humans; however, evidence shows the ocular pathway presents a favourable tissue tropism for human infection. Through antigenic drift, mutations in the H7N7 viral genome may increase virulence and pathogenicity in humans. The Victorian outbreak also detected LPAI H7N6 in emus at a commercial farm. Novel influenza A viruses can emerge by mixing different viral strains in a host susceptible to avian and human influenza strains. Studies show that emus are susceptible to infections from a wide range of influenza viral subtypes, including H5N1 and the pandemic H1N1. The emu's internal organs and tissues express abundant cell surface sialic acid receptors that favour the attachment of avian and human influenza viruses, increasing the potential for internal genetic reassortment and the emergence of novel influenza A viruses. This review summarises the historical context of H7N7 in Australia, considers the potential for increased virulence and pathogenesis through mutations and draws attention to the emu as potentially an unrecognised viral mixing vessel.

Characterization of the low-pathogenic H7N7 avian influenza virus in Shanghai, China.

H7N7 avian influenza virus (AIV) can divided into low-pathogenic AIV and high-pathogenic AIV groups. It has been shown to infect humans and animals. Its prevalence state in wild birds in China remains largely unclear. In this study, a new strain of H7N7 AIV, designated CM1216, isolated from wild birds in Shanghai, China, was characterized. Phylogenetic and nucleotide sequence analyses of CM1216 revealed that HA, NA, PB1, NP, and M genes shared the highest nucleotide identity with the Japan H7 subtype AIV circulated in 2019; the PB2 and PA genes shared the highest nucleotide identity with the Korea H7 subtype AIV circulated in wild birds in 2018, while NS gene of CM1216 was 98.93% identical to that of the duck AIV circulating in Bangladesh, and they all belong to the Eurasian lineage. A Bayesian phylogenetic reconstruction of the 2 surface genes of CM1216 showed that multiple reassortments might have occurred in 2015. Mutations were found in HA (A135 T, T136S, and T160 A [H3 numbering]), M1 (N30D and T215 A), NS1 (P42S and D97 E), PB2 (R389 K), and PA (N383D) proteins; these mutations have been shown to be related to mammalian adaptation and changes in virulence of AIVs. Infection studies demonstrated that CM1216 could infect mice and cause symptoms characteristic of influenza virus infection and proliferate in the lungs without prior adaption. This study demonstrates the need for routine surveillance of AIVs in wild birds and detection of their evolution to become a virus with high pathogenicity and ability to infect humans.

An Overview of Equine Influenza in South America.

Equine influenza virus (EIV) is one of the most important respiratory pathogens of horses as outbreaks of the disease lead to significant economic losses worldwide. In this review, we summarize the information available on equine influenza (EI) in South America. In the region, the major events of EI occurred almost in the same period in the different countries, and the EIV isolated showed high genetic identity at the hemagglutinin gene level. It is highly likely that the continuous movement of horses, some of them subclinically infected, among South American countries, facilitated the spread of the virus. Although EI vaccination is mandatory for mobile or congregates equine populations in the region, EI outbreaks continuously threaten the equine industry. Vaccine breakdown could be related to the fact that many of the commercial vaccines available in the region contain out-of-date EIV strains, and some of them even lack reliable information about immunogenicity and efficacy. This review highlights the importance of disease surveillance and reinforces the need to harmonize quarantine and biosecurity protocols, and encourage vaccine manufacturer companies to carry out quality control procedures and update the EIV strains in their products.

Genetic Characterization and Pathogenicity of H7N7 and H7N9 Avian Influenza Viruses Isolated from South Korea.

H7 low pathogenic avian influenza viruses (LPAIVs) can mutate into highly pathogenic avian influenza viruses (HPAIVs). In addition to avian species, H7 avian influenza viruses (AIVs) also infect humans. In this study, two AIVs, H7N9 (20X-20) and H7N7 (34X-2), isolated from the feces of wild birds in South Korea in 2021, were genetically analyzed. The HA cleavage site of the two H7 Korean viruses was confirmed to be ELPKGR/GLF, indicating they are LPAIVs. There were no amino acid substitutions at the receptor-binding site of the HA gene of two H7 Korean viruses compared to that of A/Anhui/1/2013 (H7N9), which prefer human receptors. In the phylogenetic tree analysis, the HA gene of the two H7 Korean viruses shared the highest nucleotide similarity with the Korean H7 subtype AIVs. In addition, the HA gene of the two H7 Korean viruses showed high nucleotide similarity to that of the A/Jiangsu/1/2018(H7N4) virus, which is a human influenza virus originating from avian influenza virus. Most internal genes (PB2, PB1, PA, NP, NA, M, and NS) of the two H7 Korean viruses belonged to the Eurasian lineage, except for the M gene of 34X-2. This result suggests that active reassortment occurred among AIVs. In pathogenicity studies of mice, the two H7 Korean viruses replicated in the lungs of mice. In addition, the body weight of mice infected with 34X-2 decreased 7 days post-infection (dpi) and inflammation was observed in the peribronchiolar and perivascular regions of the lungs of mice. These results suggest that mammals can be infected with the two H7 Korean AIVs. Our data showed that even low pathogenic H7 AIVs may infect mammals, including humans, as confirmed by the A/Jiangsu/1/2018(H7N4) virus. Therefore, continuous monitoring and pathogenicity assessment of AIVs, even of LPAIVs, are required.

Amino acid substitutions involved in the adaptation of a novel H7N7 avian influenza virus in mice.

The H7N7 avian influenza viruses can infect humans and poses a great threat to human health. To identify the amino acid substitutions that are associated with adaptation of avian-origin H7N7 virus to mammals, adaptation of the H7N7 virus was carried out by serial lung-to-lung passage in mice. Genomic analysis of the mouse-adapted virus revealed amino acid changes in the PB2 (E525G, M645I, and D701N), NP (I475V), HA(D103N), and NA(K142E) proteins. The adapted H7N7 virus was more virulent in mice than the wild-type virus. Our results suggest that continued surveillance of poultry populations for these substitutions in the H7N7 virus is required.

The Emergence of H7N7 Highly Pathogenic Avian Influenza Virus from Low Pathogenicity Avian Influenza Virus Using an in ovo Embryo Culture Model.

Outbreaks of highly pathogenic avian influenza virus (HPAIV) often result in the infection of millions of poultry, causing up to 100% mortality. HPAIV has been shown to emerge from low pathogenicity avian influenza virus (LPAIV) in field outbreaks. Direct evidence for the emergence of H7N7 HPAIV from a LPAIV precursor with a rare di-basic cleavage site (DBCS) was identified in the UK in 2008. The DBCS contained an additional basic amino acid compared to commonly circulating LPAIVs that harbor a single-basic amino acid at the cleavage site (SBCS). Using reverse genetics, outbreak HPAIVs were rescued with a DBCS (H7N7DB), as seen in the LPAIV precursor or an SBCS representative of common H7 LPAIVs (H7N7SB). Passage of H7N7DB in chicken embryo tissues showed spontaneous evolution to a HPAIV. In contrast, deep sequencing of extracts from embryo tissues in which H7N7SB was serially passaged showed retention of the LPAIV genotype. Thus, in chicken embryos, an H7N7 virus containing a DBCS appears naturally unstable, enabling rapid evolution to HPAIV. Evaluation in embryo tissue presents a useful approach to study AIV evolution and allows a laboratory-based dissection of molecular mechanisms behind the emergence of HPAIV.

Two Single Incursions of H7N7 and H5N1 Low Pathogenicity Avian Influenza in U.K. Broiler Breeders During 2015 and 2016.

Low pathogenicity (LP) avian influenza viruses (AIVs) have a natural reservoir in wild birds. These cause few (if any) overt clinical signs, but include H5 and H7 LPAIVs, which are notifiable in poultry. In the European Union, notifiable avian disease (NAD) demands laboratory confirmation with prompt statutory interventions to prevent dissemination of infection to multiple farms. Crucially, for H5 and H7 LPAIVs, movement restrictions and culling limit the further risk of mutation to the corresponding highly pathogenic (HP) H5 and H7 AIVs in gallinaceous poultry. An H7N7 LPAIV outbreak occurred during February 2015 at a broiler breeder chicken premise in England. Full genome sequencing suggested an avian origin closely related to contemporary European H7 LPAIV wild bird strains with no correlates for human adaptation. However, a high similarity of PB2, PB1, and NA genes with H10N7 viruses from European seals during 2014 was observed. An H5N1 LPAIV outbreak during January 2016 affecting broiler breeder chickens in Scotland resulted in rapid within-farm spread. An interesting feature from this case was that although viral tropism occurred in heart and kidney endothelial cells, suggesting HPAIV infection, the H5N1 virus had the molecular cleavage site signature of an LPAIV belonging to an indigenous European H5 lineage. There was no genetic evidence for human adaptation or antiviral drug resistance. The source of the infection was also likely to be via indirect contact with wild birds mediated via fomite spread from the nearby environment. Both LPAIV outbreaks were preceded by local flooding events that attracted wild waterfowl to the premises. Prompt detection of both outbreaks highlighted the value of the "testing to exclude" scheme launched in the United Kingdom for commercial gallinaceous poultry in 2014 as an early warning surveillance mechanism for NAD.

Dos incursiones únicas de influenza aviar de baja patogenicidad H7N7 y H5N1 en criadores de pollos de engorde en el Reino Unido durante 2015 y 2016. Los virus de influenza aviar de baja patogenicidad tienen un reservorio natural en aves silvestres. Estos causan pocos (si es que se presentan) signos clínicos evidentes, pero se incluyen los virus de influenza de baja patogenicidad H5 y H7, que son notificables en avicultura. En la Unión Europea, las enfermedades aviares notificables (NAD, por sus siglas en inglés) requieren de confirmación de laboratorio con intervenciones reglamentarias rápidas para prevenir la diseminación de la infección a múltiples granjas. De manera crucial, para las los virus de baja patogenicidad H5 y H7, las restricciones de movimiento y el sacrificio limitan el riesgo adicional de mutación hacia los correspondientes virus H5 y H7 altamente patógenos en aves comerciales. Un brote de influenza aviar de baja patogenicidad H7N7 ocurrió en febrero del 2015 en una granja de pollos reproductores de pollos de engorde en Inglaterra. La secuenciación completa del genoma sugirió un origen aviar estrechamente relacionado con las cepas de aves silvestres contemporáneas europeas de baja patogenicidad H7 sin indicios para la adaptación humana. Sin embargo, se observó una alta similitud de los genes PB2, PB1 y NA con los virus H10N7 de focas europeas durante el 2014. Un brote de influenza aviar de baja patogenicidad por H5N1 en enero del 2016 que afectó a los pollos reproductores de pollos de engorde en Escocia resultó en una rápida propagación dentro de la granja. Una característica interesante de este caso fue que, aunque el tropismo viral ocurrió en las células endoteliales del corazón y el riñón, lo que sugería una infección por un virus de alta patogenicidad, el virus H5N1 tenía el sitio de disociación molecular característico de un virus de baja patogenicidad perteneciente a un linaje indígena H5 europeo. No se observó evidencia genética para la adaptación humana o la resistencia a los medicamentos antivirales. También es probable que la fuente de la infección fue a través del contacto indirecto con las aves silvestres mediadas a través de la propagación de fómites desde el entorno cercano. Ambos brotes de influenza aviar de baja patogenicidad fueron precedidos por inundaciones locales que atrajeron aves acuáticas silvestres a las instalaciones. La rápida detección de ambos brotes resaltó el valor del esquema de "Diagnóstico para Excluir" establecido en el Reino Unido para la avicultura comercial en el 2014 como un mecanismo de vigilancia de alerta temprana para las enfermedades aviares notificables.

Direct evidence of H7N7 avian influenza virus mutation from low to high virulence on a single poultry premises during an outbreak in free range chickens in the UK, 2008.

H5 and H7 subtypes of low pathogenicity avian influenza viruses (LPAIVs) have the potential to evolve into highly pathogenic avian influenza viruses (HPAIVs), causing high mortality in galliforme poultry with substantial economic losses for the poultry industry. This study provides direct evidence of H7N7 LPAIV mutation to HPAIV on a single poultry premises during an outbreak that occurred in June 2008 in free range laying hens in Oxfordshire, UK. We report the first detection of a rare di-basic cleavage site (CS) motif (PEIPKKRGLF), unique to galliformes, that has previously been associated with a LPAIV phenotype. Three distinct HPAIV CS sequences (PEIPKRKKRGLF, PEIPKKKKRGLF and PEIPKKKKKKRGLF) were identified in the infected sheds suggesting molecular evolution at the outbreak premises. Further evidence for H7N7 LPAIV preceding mutation to HPAIV was derived by examining clinical signs, epidemiological descriptions and analysing laboratory results on the timing and proportions of seroconversion and virus shedding at each infected shed on the premises. In addition to describing how the outbreak was diagnosed and managed via statutory laboratory testing, phylogenetic analysis revealed reassortant events during 2006-2008 that suggested likely incursion of a wild bird origin LPAIV precursor to the H7N7 HPAIV outbreak. Identifying a precursor LPAIV is important for understanding the molecular changes and mechanisms involved in the emergence of HPAIV. This information can lead to understanding how and why only some H7 LPAIVs appear to readily mutate to HPAIV.

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.

Genome-scale phylodynamics and evolution analysis of global H7N7 influenza viruses.

Previous studies lacked of comprehensive analysis about the evolutionary history and phylogeography of global H7N7 viruses. In this study, it is essential to undertake a genome-scale analysis to investigate the evolutionary processes in a global perspective. There was local phylogenetic divergence among eight trees based on individual segments of 132 strains. We detected four reassortments between four distinct groups of viruses divided by HA gene, suggesting intrasubtype reassortment could accelerate the emergence of highly pathogenic virus. The molecular clock estimated that H7N7 virus evolved at a slower evolutionary rate ranged from 1.03E-03 to 2.81E-03subs/site/year. And we also showed that all gene segments of the virus were under strong purifying selection. A total of 11 positively selected sites were detected by at least two out of three methods. We reconstructed the population dynamics of global H7N7 viruses spanning over a century, revealing that temporal trends of the effective population size were consistent with the major epidemics previously reported. Our study adopt a Bayesian phylogeographic approach to investigate the geographic spread of H7N7 viruses, which combined with temporal and spatial information of all sequences. We have confirmed several migration events between different geographic locations supported by higher values of Bayes factor. The diffusion patterns of H7N7 viruses reveal that the virus is more likely to evolve to expand their host ranges even cross the species.

Retrospective Analysis of the Equine Influenza Virus A/Equine/Kirgizia/26/1974 (H7N7) Isolated in Central Asia.

A retrospective phylogenetic characterization of the hemagglutinin, neuraminidase and nucleoprotein genes of equine influenza virus A/equine/Kirgizia/26/1974 (H7N7) which caused an outbreak in Kirgizia (a former Soviet Union republic, now Kyrgyzstan) in 1977 was conducted. It was defined that it was closely related to the strain London/1973 isolated in Europe and it shared a maximum nucleotide sequence identity at 99% with it. This Central Asian equine influenza virus isolate did not have any specific genetic signatures and can be considered as an epizootic strain of 1974 that spread in Europe. The absence of antibodies to this subtype EI virus (EIV) in recent research confirms its disappearance as of the 1990s when the antibodies were last found in unvaccinated horses.

Novel residues in the PA protein of avian influenza H7N7 virus affect virulence in mammalian hosts.

To evaluate the pathogenicity, a highly pathogenic avian influenza H7N7 virus (A/Netherlands/219/03) isolated from human was passaged in mice. A mutant virus (mH7N7) with attenuated virulence was isolated from mouse lung, which had a 3-log higher MLD50 than the wild-type virus (wH7N7). Sequence analysis and reverse genetics study revealed that mutations in PA account for the compromised viral replication in mammalian cells and mice. A mini-genome assay demonstrated that PA mutations P103H and S659L can cooperatively decrease polymerase activity. Actually, PA with double mutation P103H-S659L cannot sustain the generation of live virus by reverse genetics. Interestingly, the prior infection of mH7N7 virus provided mice with cross-protection against lethal challenge of other subtypes of influenza A virus including H1N1, H5N1 and H7N9. In conclusion, we demonstrated that PA mutations P103H and S659L can cooperatively reduce polymerase activity and viral replication in mammalian cells and attenuate pathogenicity in mice.