Search

Influenza A viral ribonucleoprotein (vRNP) complexes comprise the eight genomic negative-sense RNAs, each of which is bound to multiple copies of the vRNP and a trimeric viral polymerase complex. The influenza virus life cycle centres on the vRNPs, which in turn rely on host cellular processes to carry out functions that are necessary for the successful completion of the virus life cycle. In this Review, we discuss our current knowledge about vRNP trafficking within host cells and the function of these complexes in the context of the virus life cycle, highlighting how structure contributes to function and the crucial interactions with host cell pathways, as well as on the information gaps that remain. An improved understanding of how vRNPs use host cell pathways is essential to identify mechanisms of virus pathogenicity, host adaptation and, ultimately, new targets for antiviral intervention. [ABSTRACT FROM AUTHOR]

Background: Influenza is a common infectious disease caused by influenza viruses. Annual epidemics cause severe illnesses, deaths, and economic loss around the world. To better defend against influenza viral infection, it is essential to understand its mechanisms and associated host responses. Many studies have been conducted to elucidate these mechanisms, however, the overall picture remains incompletely understood. A systematic understanding of influenza viral infection in host cells is needed to facilitate the identification of influential host response mechanisms and potential drug targets. Description: We constructed a comprehensive map of the influenza A virus (‘IAV’) life cycle (‘FluMap’) by undertaking a literature-based, manual curation approach. Based on information obtained from publicly available pathway databases, updated with literature-based information and input from expert virologists and immunologists, FluMap is currently composed of 960 factors (i.e., proteins, mRNAs etc.) and 456 reactions, and is annotated with ~500 papers and curation comments. In addition to detailing the type of molecular interactions, isolate/strain specific data are also available. The FluMap was built with the pathway editor CellDesigner in standard SBML (Systems Biology Markup Language) format and visualized as an SBGN (Systems Biology Graphical Notation) diagram. It is also available as a web service (online map) based on the iPathways+ system to enable community discussion by influenza researchers. We also demonstrate computational network analyses to identify targets using the FluMap. Conclusion: The FluMap is a comprehensive pathway map that can serve as a graphically presented knowledge-base and as a platform to analyze functional interactions between IAV and host factors. Publicly available webtools will allow continuous updating to ensure the most reliable representation of the host-virus interaction network.The FluMap is available at http://www.influenza-x.org/flumap/. [ABSTRACT FROM AUTHOR]

Background: Influenza infection causes respiratory disease that can lead to death. The complex interplay between virus-encoded and host-specific pathogenicity regulators - and the relative contributions of each toward viral pathogenicity - is not well-understood. Results: By analyzing a collection of lung samples from mice infected by A/Vietnam/1203/2004 (H5N1; VN1203), we characterized a signature of transcripts and proteins associated with the kinetics of the host response. Using a new geometrical representation method and two criteria, we show that inoculation concentrations and four specific mutations in VN1203 mainly impact the magnitude and velocity of the host response kinetics, rather than specific sets of up- and down- regulated genes. We observed analogous kinetic effects using lung samples from mice infected with A/California/04/2009 (H1N1), and we show that these effects correlate with morbidity and viral titer. Conclusions: We have demonstrated the importance of the kinetics of the host response to H5N1 pathogenesis and its relationship with clinical disease severity and virus replication. These kinetic properties imply that time-matched comparisons of 'omics profiles to viral infections give limited views to differentiate host-responses. Moreover, these results demonstrate that a fast activation of the host-response at the earliest time points post-infection is critical for protective mechanisms against fast replicating viruses. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures. [ABSTRACT FROM AUTHOR]

The identification of host factors involved in virus replication is important to understand virus life cycles better. Accordingly, we sought host factors that interact with the influenza viral nonstructural protein 2 by using coimmunoprecipitation followed by mass spectrometry. Among proteins associating with nonstructural protein 2, we focused on the gi subunit of the F1Fo-ATPase. which received a high probability score in our mass spectrometry analysis. The siRNA-mediated down-regulation of the ~ subunit of the F1F0-ATPase reduced influenza virion formation and virus growth in cell culture. We further found that efficient influenza virion formation requires the ATPase activity of F1F0-ATPase and that plasma membrane-associated, but not mitochondrial. F1F0-ATPase is important for influenza virion formation and budding. Hence. our data identify plasma membrane-associated F1F0-ATPase as a critical host factor for efficient influenza virus replication. [ABSTRACT FROM AUTHOR]

Motivation: Protein-protein interaction (PPI) databases are widely used tools to study cellular pathways and networks; however, there are several databases available that still do not account for cell type-specific differences. Here, we evaluated the characteristics of six interaction databases, incorporated tissue-specific gene expression information and finally, investigated if the most popular proteins of scientific literature are involved in good quality interactions.Results: We found that the evaluated databases are comparable in terms of node connectivity (i.e. proteins with few interaction partners also have few interaction partners in other databases), but may differ in the identity of interaction partners. We also observed that the incorporation of tissue-specific expression information significantly altered the interaction landscape and finally, we demonstrated that many of the most intensively studied proteins are engaged in interactions associated with low confidence scores. In summary, interaction databases are valuable research tools but may lead to different predictions on interactions or pathways. The accuracy of predictions can be improved by incorporating datasets on organ- and cell type-specific gene expression, and by obtaining additional interaction evidence for the most ‘popular’ proteins.Contact: kitano@sbi.jpSupplementary information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Type I interferon (IFN) signaling is mediated through several signaling pathways, including the Janus kinase and signal transducer and activator (JAK-STAT) and p38 mitogen-activated protein (MAP) kinase pathways. The VP24 protein of Ebolavirus is an IFN antagonist, blocking type I IFN signaling through the JAK-STAT pathway. Here, we show that, in 293 cells, VP24 also interferes with the p38 MAP kinase pathway by blocking IFN-&bgr;-stimulated phosphorylation of p38-&agr;. Similar inhibition was not observed in HeLa cells, suggesting cell type-specific differences in signal transduction. [ABSTRACT FROM AUTHOR]

Please cite this paper as: Neumann G, Kawaoka Y. (2011) The first influenza pandemic of the new millennium. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750-2659.2011.00202.x. In the spring of 2009, a novel influenza A virus of the H1N1 subtype emerged that transmitted efficiently among humans; by June of 2009, the outbreak reached pandemic status. The pandemic virus possesses six viral RNA segments from so-called triple reassortant swine viruses that emerged in North American pig populations in the late 1990s and two viral RNA segments from Eurasian avian-like swine influenza viruses. Most human infections with the virus have been mild; however, severe and fatal infections occurred among certain risk groups, but also among those without any known risk factors. Here, we summarize the evolutionary, epidemiological, clinical, and molecular findings on the pandemic virus. We also discuss the arsenal of antiviral compounds and vaccines available to prevent and treat infections with the virus. [ABSTRACT FROM AUTHOR]

The spread of avian H5N1 influenza viruses around the globe has become a worldwide public health concern. To evaluate the pathogenic potential of reassortant viruses between currently cocirculating avian H5N1 and human H3N2 influenza viruses, we generated all the 254 combinations of reassortant viruses between A/chicken/South Kalimantan/UT6028/06 (5K06, H5N1) and Al Tokyo/Ut-Sk-1/07 (Tok07. H3N2) influenza viruses by reverse genetics. We found that the presence of Tok07 PB2 protein in the ribonucleoprotein (RNP) complex allowed efficient viral RNA tran- scription in a minigenome assay and that RNP activity played an essential role in the viability and replicative ability of the jeassortant viruses. When the pathogenicity of 75 reassortant H5 viruses was tested in mice, 22 were more pathogenic than the parental 51(06 virus, and three were extremely virulent. Strikingly, all 22 of these viruses obtained their PB2 segment from Tok07 virus. Further analysis showed that Tok07 PB 1 alone lacked the ability to enhance the pathogenicity of the reassortant viruses but could do so by cooper- ating with Tok07 PB2. Our data demonstrate that reassortment between an avian H5N1virus with low pathogenicity in mice and a human virus could result in highly pathogenic viruses and that the human virus PB2 segment functions in the background of an avian H5N1 virus, enhancing its virulence. Our findings highlight the importance of surveillance programs to monitor the emergence of human H5 reassortant viruses, especially those containing a PB2 segment of human origin. [ABSTRACT FROM AUTHOR]

All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortality rates. More importantly, influenza A viruses cause respiratory disease in humans with potentially fatal outcome. Local or global outbreaks in humans are typically characterized by excess hospitalizations and deaths. In 1997, highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Hong Kong that transmitted to humans, resulting in the first documented cases of human death by avian influenza virus infection. A new outbreak started in July 2003 in poultry in Vietnam, Indonesia, and Thailand, and highly pathogenic avian H5N1 influenza viruses have since spread throughout Asia and into Europe and Africa. These viruses continue to infect humans with a high mortality rate and cause worldwide concern of a looming pandemic. Moreover, H5N1 virus outbreaks have had devastating effects on the poultry industries throughout Asia. Since H5N1 virus outbreaks appear to originate from Southern China, we here examine H5N1 influenza viruses in China, with an emphasis on their biological properties. [ABSTRACT FROM AUTHOR]

The West African outbreak of Ebola virus (EBOV) is largely contained, but sporadic new cases continue to emerge. To assess the potential contribution of fomites to human infections with EBOV, we tested EBOV stability in human blood spotted onto Sierra Leonean banknotes and in syringe needles under hospital and environmental conditions. Under some of these conditions, EBOV remained infectious for >30 days, indicating that EBOV-contaminated items may pose a serious risk to humans. [ABSTRACT FROM AUTHOR]

Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. The emergence of new strains will continue to pose challenges to public health and the scientific communities. A prime example is the recent emergence of swine-origin H1N1 viruses that have transmitted to and spread among humans, resulting in outbreaks internationally. Efforts to control these outbreaks and real-time monitoring of the evolution of this virus should provide us with invaluable information to direct infectious disease control programmes and to improve understanding of the factors that determine viral pathogenicity and/or transmissibility. [ABSTRACT FROM AUTHOR]

M2SR (M2-deficient single replication) is an investigational live intranasal vaccine that protects against multiple influenza A subtypes in influenza-naïve and previously infected ferrets. We conducted a phase 1, first-in-human, randomized, dose-escalation, placebo-controlled study of M2SR safety and immunogenicity. Adult subjects received a single intranasal administration with either placebo or one of three M2SR dose levels (106, 107 or 108 tissue culture infectious dose (TCID50)) expressing hemagglutinin and neuraminidase from A/Brisbane/10/2007 (H3N2) (24 subjects per group). Subjects were evaluated for virus replication, local and systemic reactions, adverse events (AE), and immune responses post-vaccination. Infectious virus was not detected in nasal swabs from vaccinated subjects. At least one AE (most commonly mild nasal rhinorrhea/congestion) was reported among 29%, 58%, and 83% of M2SR subjects administered a low, medium or high dose, respectively, and among 46% of placebo subjects. No subject had fever or a severe reaction to the vaccine. Influenza-specific serum and mucosal antibody responses and B- and T-cell responses were significantly more frequent among vaccinated subjects vs. placebo recipients. The M2SR vaccine was safe and well tolerated and generated dose-dependent durable serum antibody responses against diverse H3N2 influenza strains. M2SR demonstrated a multi-faceted immune response in seronegative and seropositive subjects. [ABSTRACT FROM AUTHOR]

Ebola virus (EBOV), a public health concern in Africa and a potential biological weapon, is classified as a biosafety level-4 agent because of its high mortality rate and the lack of approved vaccines and antivirals. Basic research into the mechanisms of EBOV pathogenicity and the development of effective countermeasures are restricted by the current biosafety classification of EBOVs. We therefore developed biologically contained EBOV that express a reporter gene instead of the VP30 gene, which encodes an essential transcription factor. A Vero cell line that stably expresses VP30 provides this essential protein in trans and biologically confines the virus to its complete replication cycle in this cell line. This complementation approach is highly efficient because biologically contained EBOVs lacking the VP30 gene grow to titers similar to those obtained with wild-type virus. Moreover, EBOVs lacking the VP30 gene are indistinguishable in their morphology from wild-type virus and are genetically stable, as determined by sequence analysis after seven serial passages in VP30-expressing Vero cells. We propose that this system provides a safe means to handle EBOV outside a biosafety level-4 facility and will stimulate critical studies on the EBOV life cycle as well as large-scale screening efforts for compounds with activity against this lethal virus. [ABSTRACT FROM AUTHOR]

To facilitate an understanding of the molecular aspects of the pathogenesis of Zaire ebolavirus (ZEBOV) infection, we generated 2 different recombinant viruses expressing enhanced green fluorescent protein (eGFP) from additional transcription units inserted at different positions in the virus genome. These viruses showed in vitro phenotypes similar to that of wild-type ZEBOV (wt-ZEBOV) and were stable over multiple passages. Infection with one of the viruses expressing eGFP produced only mild disease in rhesus macaques, demonstrating a marked attenuation in this animal model. However, in mice lacking signal transducer and activator of transcription 1, both viruses expressing eGFP caused lethal cases of disease that were moderately attenuated, compared with that caused by wt-ZEBOV. In mice, viral replication could be easily tracked by the detection of eGFP-positive cells in tissues, by use of flow cytometry. These findings demonstrate that the incorporation of a foreign gene will attenuate ZEBOV in vivo but th these viruses still have potential for in vitro and in vivo research applications. [ABSTRACT FROM AUTHOR]

Influenza A viruses cause pandemics at random intervals. Pandemics are caused by viruses that contain a hemagglutinin (HA) surface glycoprotein to which human populations are immunologically naive. Such an HA can be introduced into the human population through reassortment between human and avian virus strains or through the direct transfer of an avian influenza virus to humans. The factors that determine the interspecies transmission and pathogenicity of influenza viruses are still poorly understood; however, the HA protein plays an important role in overcoming the interspecies barrier and in virulence in avian influenza viruses. Recently, the RNA polymerase (PB2) protein has also been recognized as a critical factor in host range restriction, while the nonstructural (NS1) protein affects the initial host immune responses. We summarize current knowledge of viral factors that determine host range restriction and pathogenicity of influenza A viruses. [ABSTRACT FROM AUTHOR]

The generation of vaccines for highly pathogenic avian influenza viruses, including those of the H5N1 subtype, relies on reverse genetics, which allows the production of influenza viruses from cloned cDNA. In the future, reverse genetics will likely be the method of choice for the generation of conventional influenza vaccine strains because gene reassortment by more traditional methods is cumbersome. Established systems for the artificial generation of influenza A viruses require transfection of cells with the eight to 12 plasmids that provide the eight influenza viral RNAs as well as the polymerase and nucleoproteins of the virus. However, cell lines appropriate for human vaccine production (e.g., Vero cells) cannot be transfected with high efficiencies. To overcome these problems, we established a reverse genetics system in which the eight RNA polymerase I transcription cassettes for viral RNA synthesis are combined on one plasmid. Similarly, two cassettes encoding the hemagglutinin and neuraminidase segments and six cassettes encoding the remaining proteins were combined. We also combined three RNA polymerase II transcription cassettes for the expression of the polymerase subunits. By combining these cassettes, we reduced the number of plasmids required for virus generation significantly and produced influenza A virus in Vero cells with higher efficiency than with the traditional 12 plasmid system. This new system is thus suitable for influenza virus vaccine production and may be applicable to other reverse genetics systems that rely on the introduction of several plasmids into eukaryotic cells. [ABSTRACT FROM AUTHOR]

Reverse genetics systems, i.e., systems for the generation of virus entirely from cloned cDNA, have been established for most nonsegmented negative-sense RNA viruses. In contrast, the generation of influenza A viruses (whose genome is composed of eight segments of negative-sense RNA) was not possible until 1999, likely due to the inherent technical difficulties of providing all eight viral RNAs as well as the four viral proteins required for replication and transcription. In 1999, we (Neumann et al., 1999, Proc. Natl. Acad. Sci. USA 96, 9345–9350) and others (Fodor et al., 1999, J. Virol. 73, 9679–9682) demonstrated the generation of influenza A virus from plasmids, relying on the cellular enzyme RNA polymerase I for the synthesis o influenza viral RNAs. In this review, we provide background on RNA polymerase I transcription and discuss its use for the generation of influenza virus from cloned cDNAs. [Copyright &y& Elsevier]

For nuclear export of proteins, the formation of a ternary export complex composed of the export substrate, a cellular export factor and Ran-GTP is crucial. CRM1 is a cellular export factor for proteins containing leucine-rich nuclear export signals (NESs). Although the NES sequence is crucial for nuclear export, its exact role in the formation of the ternary export complex is controversial. Here we demonstrate an interaction between human CRMI (hCRM1) and influenza A virus NS2 protein, which contains an NES motif in its N-terminal region. Replacement of the hydrophobic amino acids in the NES motif did not abolish NS2's interaction with hCRM1. Using our recently established systems for the generation of influenza virus or virus-like particles from cloned cDNAs, we found that NS2 is essential for nuclear export of influenza virus ribonucleoprotein (RNP) complexes, and that alteration of the NS2-NES abrogated this event and influenza virus generation. These findings suggest that the NS2-NES is not crucial for the interaction of this protein with hCRM1, but is for the formation of the ternary export complex with Ran-GTP. [ABSTRACT FROM AUTHOR]