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Avian influenza viruses impose serious public health burdens with significant mortality and morbidity not only in poultry but also in humans. While poultry susceptibility to avian influenza virus infection is well characterized, pigeons have been thought to have low susceptibility to these viruses. However, recent studies reported natural pigeon infections with highly pathogenic avian influenza H5N1 viruses. In Egypt, which is one of the H5N1 endemic areas for birds, pigeons are raised in towers built on farms in backyards and on house roofs, providing a potential risk for virus transmission from pigeons to humans. In this study, we performed genetic analysis of two H5N1 virus strains that were isolated from naturally infected pigeons in Egypt. Genetic and phylogenetic analyses showed that these viruses originated from Egyptian H5N1 viruses that were circulating in chickens or ducks. Several unique mutations, not reported before in any Egyptian isolates, were detected in the internal genes (i.e., polymerase residues PB1-V3D, PB1-K363R, PA-A369V, and PA-V602I; nucleoprotein residue NP-R38K; and nonstructural protein residues NS1-D120N and NS2-F55C). Our findings suggested that pigeons are naturally infected with H5N1 virus and can be a potential reservoir for transmission to humans, and showed the importance of genetic analysis of H5N1 internal genes.

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: A change in viral hemagglutinin (HA) receptor binding specificity from α2,3- to α2,6-linked sialic acid is necessary for highly pathogenic avian influenza (AI) virus subtype H5N1 to become pandemic. However, details of the human-adaptive change in the H5N1 virus remain unknown. Our database search of H5N1 clade 2.2.1 viruses circulating in Egypt identified multiple HA mutations that had been selected in infected patients. Using reverse genetics, we found that increases in both human receptor specificity and the HA pH threshold for membrane fusion were necessary to facilitate replication of the virus variants in human airway epithelia. Furthermore, variants with enhanced replication in human cells had decreased HA stability, apparently to compensate for the changes in viral receptor specificity and membrane fusion activity. Our findings showed that H5N1 viruses could rapidly adapt to growth in the human airway microenvironment by altering their HA properties in infected patients and provided new insights into the human-adaptive mechanisms of AI viruses., Importance: Circulation between bird and human hosts may allow H5N1 viruses to acquire amino acid changes that increase fitness for human infections. However, human-adaptive changes in H5N1 viruses have not been adequately investigated. In this study, we found that multiple HA mutations were actually selected in H5N1-infected patients and that H5N1 variants with some of these HA mutations had increased human-type receptor specificity and increased HA membrane fusion activity, both of which are advantageous for viral replication in human airway epithelia. Furthermore, HA mutants selected during viral replication in patients were likely to have less HA stability, apparently as a compensatory mechanism. These results begin to clarify the picture of the H5N1 human-adaptive mechanism., (Copyright © 2015 Watanabe et al.)

Highly pathogenic avian influenza virus H5N1 has spread across Eurasia and Africa, and outbreaks are now endemic in several countries, including Indonesia, Vietnam and Egypt. Continuous circulation of H5N1 virus in Egypt, from a single infected source, has led to significant genetic diversification with phylogenetically separable sublineages, providing an opportunity to study the impact of genetic evolution on viral phenotypic variation. In this study, we analysed the phylogeny of H5 haemagglutinin (HA) genes in influenza viruses isolated in Egypt from 2006 to 2011 and investigated the effect of conserved amino acid mutations in the HA genes in each of the sublineages on their antigenicity. The analysis showed that viruses in at least four sublineages still persisted in poultry in Egypt as of 2011. Using reverse genetics to generate HA-reassortment viruses with specific HA mutations, we found antigenic drift in the HA in two influenza virus sublineages, compared with the other currently co-circulating influenza virus sublineages in Egypt. Moreover, the two sublineages with significant antigenic drift were antigenically distinguishable. Our findings suggested that phylogenetically divergent H5N1 viruses, which were not antigenically cross-reactive, were co-circulating in Egypt, indicating that there was a problem in using a single influenza virus strain as seed virus to produce influenza virus vaccine in Egypt and providing data for designing more efficacious control strategies in H5N1-endemic areas.

Highly pathogenic avian influenza A virus subtype H5N1 has been endemic in some bird species since its emergence in 1996 and its ecology, genetics and antigenic properties have continued to evolve. This has allowed diverse virus strains to emerge in endemic areas with altered receptor specificity, including a new H5 sublineage with enhanced binding affinity to the human-type receptor. The pandemic potential of H5N1 viruses is alarming and may be increasing. We review here the complex dynamics and changing nature of the H5N1 virus that may contribute to the emergence of pandemic strains., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Highly pathogenic avian influenza A virus subtype H5N1 can potentially generate novel variants during replication of infected hosts. To determine which H5N1 variants predominate in wild birds, we determined the sequences of RT-PCR amplified viral genes from several organs of infected chickens and ducks from Egypt, where H5N1 outbreaks in birds are endemic. Comparison of the sequences in viruses from trachea, lung, brain and liver revealed diversification with different amino acid substitutions in different ducks, but no diversification in chickens. These specific amino acid substitutions were rare among viruses currently circulating in Egypt. In addition, the H5N1 variants showed distinct growth kinetics in duck, canine and human cells. Our findings suggested that ducks can generate H5N1 variants with novel amino acid substitutions that might serve as aetiological agents for new influenza virus outbreaks and epidemics.

The complete genome sequences of two isolates A/chicken/Egypt/CL6/07 (CL6/07) and A/duck/Egypt/D2br10/07 (D2br10/07) of highly pathogenic avian influenza virus (HPAI) H5N1 isolated at the beginning of 2007 outbreak in Egypt were determined and compared with all Egyptian HPAI H5N1 sequences available in the GenBank. Sequence analysis utilizing the RNA from the original tissue homogenate showed amino acid substitutions in seven of the viral segments in both samples. Interestingly, these changes were different between the CL6/07 and D2br10/07 when compared to other Egyptian isolates. Moreover, phylogenetic analysis showed independent sub-clustering of the two viruses within the Egyptian sequences signifying a possible differential adaptation in the two hosts. Further, pre-amplification analysis of H5N1 might be necessary for accurate data interpretation and identification of distinct factor(s) influencing the evolution of the virus in different poultry species.

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.

Inbred mice have been widely used for the study of influenza viruses as a mammalian model, while suitable cell lines derived from murine tissue have been limited. Here, we established several immortalized cell clones from respiratory regions of inbred mice (C57BL/6 and BALB/c) by transformation using simian virus 40 large T antigen expression vector. Twenty-five cell clones from C57/BL and BALB/c, designated as MRDC/C and MRDC/B series, respectively, showed different susceptibility to Thai isolates of influenza A virus H5N1. Two murine cell clones, C6 and B7 which were extensively studied expressed both SAα2,3 and SAα2,6 sialic acid receptors. Interestingly, the 6 Thai patient-derived H5N1 isolates examined showed varied virus propagation efficiency in murine cell clones, although there were only slight differences in their propagation in MDCK and A549 cell lines. The results indicate that the murine cell clones are useful for examining the propagation efficiency of H5N1 viruses in vitro., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Since December 1997, highly pathogenic avian influenza A H5N1 viruses have swept through poultry populations across Asian countries and been transmitted into African and European countries. We characterized 6 avian influenza H5N1 viruses isolated from humans in 2004 in Thailand. A highly pathogenic (HP) KAN353 strain showed faster replication and higher virulence in embryonated eggs compared to other strains, especially compared to the low pathogenic (LP) SP83 strain. HP KAN353 also showed strong cytopathogenicity compared to SP83 in Madin-Darby canine kidney cells. Interestingly, LP SP83 induced smaller plaques compared to other strains, especially HP KAN353. PB2 amino acid 627E may contribute to low virulence, whereas either PB2 amino acid 627 K or the combination of 627E/701N seems to be associated with high virulence. The in vitro assays used in this study may provide the basis for assessing the pathogenesis of influenza H5N1 viruses in vivo.

Hemagglutinin protein (HA) was considered to be the primary target for monoclonal antibody production. This protein not only plays an important role in viral infections, but can also be used to differentiate H5N1 virus from other influenza A viruses. Hence, for diagnostic and therapeutic applications, it is important to develop anti-HA monoclonal antibody (MAb) with high sensitivity, specificity, stability, and productivity. Nine unique Fab MAbs were generated from chimeric chicken/human Fab phage display library constructed from cDNA derived from chickens immunized with recombinant hemagglutinin protein constructed from H5N1 avian influenza virus (A/Vietnam/1203/04). The obtained Fab MAbs showed several characteristics for further optimization and development-three clones were highly specific to only H5N1 virus. This finding can be applied to the development of H5N1 diagnostic testing. Another clone showed neutralization activity that inhibited H5N1 influenza virus infection in Madin-Darby canine kidney (MDCK) cells. In addition, one clone showed strong reactivity with several of the influenza A virus subtypes tested. The conversion of this clone to whole IgG is a promising study for a cross-neutralization activity test., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The epitope sequences within the hemagglutinin (HA) of influenza A virus H3N2 at amino acid residues 173-181 and 227-239 that forms anti-parallel beta-sheet structure are similarly recognized by human monoclonal antibodies (HuMAbs), B-1 and D-1 that we recently obtained using the peripheral blood lymphocytes from two influenza-vaccinated volunteers. Both HuMAbs showed strong global neutralization of H3N2 strains. Here we show the significant conservation of the beta-sheet region consisting of the above-mentioned two epitope regions in H3N2. In addition, we also identified the corresponding regions with similar structure in other subtypes such as H1N1 and H5N1. These two regions are similarly located underneath the receptor-binding sites of individual subtypes. Analysis of those regions using sequences available from the Influenza Virus Resource at the National Center for Biotechnology Information revealed that compared with those in the known neutralizing epitopes A-E, those sequences were fairly conserved in human H3N2 (n=7955), swine H1N1 (n=360) and swine H3N2 (n=235); and highly conserved in human H1N1 (n=2722), swine-origin pandemic H1N1 (n=1474), human H5N1 (n=319) and avian H5N1 (n=2349). Phylogenetic tree for these regions formed clearly separable clusters for H1N1, H3N2 and H5N1, irrespective of different host origin. These data may suggest a possible significance of those regions for development of alternative vaccine that could induce neutralizing antibodies reactive against wide-range of influenza virus strains., (Copyright 2010 Elsevier Inc. All rights reserved.)

In this study, we show that the highly pathogenic H5N1 avian influenza virus (AIV) (A/crow/Kyoto/53/04 and A/chicken/Egypt/CL6/07) induced apoptosis in duck embryonic fibroblasts (DEF). In contrast, apoptosis was reduced among cells infected with low-pathogenic AIVs (A/duck/HK/342/78 [H5N2], A/duck/HK/820/80 [H5N3], A/wigeon/Osaka/1/01 [H7N7], and A/turkey/Wisconsin/1/66 [H9N2]). Thus, we investigated the molecular mechanisms of apoptosis induced by H5N1-AIV infection. Caspase-dependent and -independent pathways contributed to the cytopathic effects. We further showed that, in the induction of apoptosis, the hemagglutinin of H5N1-AIV played a major role and its cleavage sequence was not critical. We also observed outer membrane permeabilization and loss of the transmembrane potential of the mitochondria of infected DEF, indicating that mitochondrial dysfunction was caused by the H5N1-AIV infection. We then analyzed Ca(2+) dynamics in the infected cells and demonstrated an increase in the concentration of Ca(2+) in the cytosol ([Ca(2+)](i)) and mitochondria ([Ca(2+)](m)) after H5N1-AIV infection. Regardless, gene expression important for regulating Ca(2+) efflux from the endoplasmic reticulum did not significantly change after H5N1-AIV infection. These results suggest that extracellular Ca(2+) may enter H5N1-AIV-infected cells. Indeed, EGTA, which chelates extracellular free Ca(2+), significantly reduced the [Ca(2+)](i), [Ca(2+)](m), and apoptosis induced by H5N1-AIV infection. In conclusion, we identified a novel mechanism for influenza A virus-mediated cell death, which involved the acceleration of extracellular Ca(2+) influx, leading to mitochondrial dysfunction and apoptosis. These findings may be useful for understanding the pathogenesis of H5N1-AIV in avian species as well as the impact of Ca(2+) homeostasis on influenza A virus infection.

Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb specifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody recognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis demonstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses.

The possible emergence of a pandemic influenza virus from the avian influenza virus (AIV) has become a serious threat. The isolation of viruses will be crucial for further virological analysis and the development of vaccines. However, currently, there is no simple method for facilitating the isolation of infectious AIV. Here, we have developed a simple method of capturing AIV using anionic magnetic beads. The method employed the capture of AIV (H5N1, H5N2, and H5N3) from liquid samples such as allantoic fluid (AF) and cell culture medium (CM) using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydride). After their incubation with AIV-containing samples, the magnetic beads were separated from the supernatant by applying a magnetic field. The absorption of AIV on the beads was confirmed by immunochromatography and an enzyme-linked immunosorbent assay, which indicated the presence of hemagglutinin, neuraminidase, and nucleoprotein of AIV. Furthermore, the infectivity in chicken eggs of AIV captured by magnetic beads was similar to that of the starting materials. The capture of AIV using magnetic beads coated with anionic polymers will contribute to the sufficient recovery of infectious AIV and approach for potential pandemic influenza viruses.

In recent years, the highly pathogenic avian influenza virus H5N1 has raised serious worldwide concern about an influenza pandemic; however, the biology of H5N1 pathogenesis is largely unknown. To elucidate the mechanism of H5N1 pathogenesis, we prepared primary airway epithelial cells from alveolar tissues from 1-year-old pigs and measured the growth kinetics of three avian H5 influenza viruses (A/Crow/Kyoto/53/2004 [H5N1], A/Duck/Hong Kong/342/78 [H5N2], and A/Duck/Hong Kong/820/80 [H5N3]), the resultant cytopathicity, and possible associated mechanisms. H5N1, but not the other H5 viruses, strongly induced cell death in porcine alveolar epithelial cells (pAEpC), although all three viruses induced similar degrees of cytopathicity in chicken embryonic fibroblasts. Intracellular viral growth and the production of progeny viruses were comparable in pAEpC infected with each H5 virus. In contrast, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive cells were detected only in H5N1-infected pAEpC, and the activities of caspases 3, 8, and 9 were significantly elevated in pAEpC infected with H5N1, but not with H5N2 and H5N3. These results suggest that only H5N1 induces apoptosis in pAEpC. H5N1 cytopathicity was inhibited by adding the caspase inhibitor z-VAD-FMK; however, there were no significant differences in viral growth or release of progeny viruses. Further investigations using reverse genetics demonstrated that H5N1 hemagglutinin protein plays a critical role in inducing caspase-dependent apoptosis in infected pAEpC. H5N1-specific cytopathicity was also observed in human primary airway epithelial cells. Taken together, these data suggest that avian H5N1 influenza virus leads to substantial cell death in mammalian airway epithelial cells due to the induction of apoptosis.

Patients infected with H5N1 influenza A virus, who had a severe or fatal outcome, exhibited several characteristic clinical manifestations including lymphopenia. In this study, human CD4(+) T-cell lines and healthy donor-derived peripheral blood mononuclear cells (PBMCs) were examined for susceptibility to infection with Thai isolates of H5N1 in comparison to those of H1N1. Although cellular levels were variable between H5N1 and H1N1 in T-cell lines and PBMCs, rates of production of progeny virions were significantly higher in H5N1 infections, suggesting a more efficient release of virions. In addition, cytopathogenicity in PBMCs, leading to a decline in CD4(+) T-cell numbers, were much severer with H5N1 than H1N1. Thus, human T cells could be an important target for infection with H5N1.

A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation. [ABSTRACT FROM AUTHOR]

Since December 1997, highly pathogenic avian influenza A H5N1viruses have swept through poultry populations across Asian countries and been transmitted into African and European countries. We characterized 6 avian influenza H5N1 viruses isolated from humans in 2004 in Thailand. A highly pathogenic (HP) KAN353 strain showed faster replication and higher virulence in embryonated eggs compared to other strains, especially compared to the low pathogenic (LP) SP83 strain. HP KAN353 also showed strong cytopathogenicity compared to SP83 in Madin-Darby canine kidney cells. Interestingly, LP SP83 induced smaller plaques compared to other strains, especially HP KAN353. PB2 amino acid 627E may contribute to low virulence, whereas either PB2 amino acid 627 K or the combination of 627E/701N seems to be associated with high virulence. The in vitro assays used in this study may provide the basis for assessing the pathogenesis of influenza H5N1 viruses in vivo. [ABSTRACT FROM AUTHOR]

In this study, we show that the highly pathogenic H5N1 avian influenza virus (AIV) (A/crow/Kyoto/53/04 and A/chicken/Egypt/CL6/07) induced apoptosis in duck embryonic fibroblasts (DEF). In contrast, apoptosis was reduced among cells infected with low-pathogenic AIVs (A/duck/HK/342/78 [H5N2], A/duck/HK/820/80 [H5N3], A/wigeon/Osaka/1/01 [H7N7], and A/turkey/Wisconsin/1/66 [H9N2]). Thus, we investigated the molecular mechanisms of apoptosis induced by H5N1-AIV infection. Caspase-dependent and -independent pathways contributed to the cytopathic effects. We further showed that, in the induction of apoptosis, the hemagglutinin of H5N1-AIV played a major role and its cleavage sequence was not critical. We also observed outer membrane permeabilization and loss of the transmembrane potential of the mitochondria of infected DEF, indicating that mitochondrial dysfunction was caused by the H5N1-AIV infection. We then analyzed Ca2+ dynamics in the infected cells and demonstrated an increase in the concentration of Ca2+ in the cytosol ([Ca2+]i) and mitochondria ([Ca2+]m) after H5N1-AIV infection. Regardless, gene expression important for regulating Ca2+ efflux from the endoplasmic reticulum did not significantly change after H5N1-AIV infection. These results suggest that extracellular Ca2+ may enter H5N1-AIV-infected cells. Indeed, EGTA, which chelates extracellular free Ca2+, significantly reduced the [Ca2+]i, [Ca2+]m, and apoptosis induced by H5N1-AIV infection. In conclusion, we identified a novel mechanism for influenza A virus-mediated cell death, which involved the acceleration of extracellular Ca2+ influx, leading to mitochondrial dysfunction and apoptosis. These findings may be useful for understanding the pathogenesis of H5N1-AIV in avian species as well as the impact of Ca2+ homeostasis on influenza A virus infection. [ABSTRACT FROM AUTHOR]