Influenza A viruses cause recurrent outbreaks of local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On June 11, 2009, the WHO declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses1. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) appear to be mild; however, more than 50% of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To better assess the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in nonhuman primates, causes more severe pathologic lesions in the lungs of infected mice, ferrets, and nonhuman primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting these compounds as a first line of defence against the recently declared S-OIV pandemic.

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.

The persistence of H5N1 avian influenza viruses in many Asian countries and their ability to cause fatal infections in humans have raised serious concerns about a global flu pandemic. Here we report the isolation of an H5N1 virus from a Vietnamese girl that is resistant to the drug oseltamivir, which is an inhibitor of the viral enzyme neuraminidase and is currently used for protection against and treatment of influenza. Further investigation is necessary to determine the prevalence of oseltamivir-resistant H5N1 viruses among patients treated with this drug.

The RNA genome of influenza A virus, which forms viral ribonucleoprotein complexes (vRNPs) with viral polymerase subunit proteins (PA, PB1, and PB2) and nucleoprotein (NP), is transcribed and replicated in the nucleus. NP, the major component of vRNPs, has at least two amino acid sequences that serve as nuclear localization signals (NLSs): an unconventional NLS (residues 3 to 13; NLS1) and a bipartite NLS (residues 198 to 216; NLS2). Although both NLSs are known to play a role in nuclear transport, their relative contributions to viral replication are poorly understood. We therefore investigated their contributions to NP subcellular/ subnuclear localization, viral RNA (vRNA) transcription, and viral replication. Abolishing the unconventional NLS caused NP to localize predominantly to the cytoplasm and affected its activity in vRNA transcription. However, we were able to create a virus whose NP contained amino acid substitutions in NLS1 known to abolish its nuclear localization function, although this virus was highly attenuated. These results indicate that while the unconventional NLS is not essential for viral replication, it is necessary for efficient viral mRNA synthesis. On the other hand, the bipartite NLS, whose contribution to the nuclear transport of NP is limited, was essential for vRNA transcription and NP's nucleolar accumulation. A virus with nonfunctional NLS2 could not be generated. Thus, the bipartite NLS, but not the unconventional NLS, of NP is essential for influenza A virus replication.Influenza A virus, a member of the family Orthomyxoviridae, is characterized by segmented RNA genomes of negative polarity (16). The viral genome encodes at least 11 proteins (4, 16) and consists of eight single-stranded RNA segments. These genomic RNAs are incorporated into virions as viral ribonucleoprotein complexes (vRNPs) that comprise viral RNA (vRNA), heterotrimeric viral polymerase subunit proteins (PA, PB1, and PB2), and nucleoprotein (NP). A unique property of influenza viruses among RNA viruses is that every step of vRNA synthesis take place in the nucleus by use of the nuclear machinery of the host cells (16). Therefore, newly synthesized proteins required for the vRNPs must be transported into the nuclei of the cells.The transport of proteins (larger than 50 kDa) from the cytoplasm into the nucleus is regulated by signal-mediated processes. Peptide motifs that allow the proteins to be imported through the nuclear pore complex are referred to as nuclear localization signals (NLSs). They are rich in basic amino acids and bind to NLS receptors (e.g., karyopherin family members), which are responsible for the nuclear translocation of target proteins (12,31). Of the influenza A virus proteins, NLSs have been found in three polymerase subunits, the matrix M1 protein (which associates with vRNP complexes), the nonstructural NS1 protein, and NP, the primary component of vRNPs (for a review, see reference 6).NP has at least two NLS sequences. An unconventional NLS (termed here NLS1) is located between residu...

Ebolavirus is responsible for highly lethal hemorrhagic fever. Like all viruses, it must reproduce its various components and assemble them in cells in order to reproduce infectious virions and perpetuate itself. To generate infectious Ebolavirus, a viral genome-protein complex called the nucleocapsid (NC) must be produced and transported to the cell surface, incorporated into virions, and then released from cells. To further our understanding of the Ebolavirus life cycle, we expressed the various viral proteins in mammalian cells and examined them ultrastructurally and biochemically. Expression of nucleoprotein alone led to the formation of helical tubes, which likely serve as a core for the NC. The matrix protein VP40 was found to be critical for transport of NCs to the cell surface and for the incorporation of NCs into virions, where interaction between nucleoprotein and the matrix protein VP40 is likely essential for these processes. Examination of virus-infected cells revealed that virions containing NCs mainly emerge horizontally from the cell surface, whereas empty virions mainly bud vertically, suggesting that horizontal budding is the major mode of Ebolavirus budding. These data form a foundation for the identification and development of potential antiviral agents to combat the devastating disease caused by this virus.