An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus.

International audienceThe genome of influenza A viruses (IAV) is split into eight viral RNAs (vRNAs) that are encapsidated as viral ribonucleoproteins. The existence of a segment-specific packaging mechanism is well established, but the molecular basis of this mechanism remains to be deciphered. Selective packaging could be mediated by direct interaction between the vRNA packaging regions, but such interactions have never been demonstrated in virions. Recently, we showed that the eight vRNAs of a human H3N2 IAV form a single interaction network in vitro that involves regions of the vRNAs known to contain packaging signals in the case of H1N1 IAV strains. Here, we show that the eight vRNAs of an avian H5N2 IAV also form a single network of interactions in vitro, but, interestingly, the interactions and the regions of the vRNAs they involve differ from those described for the human H3N2 virus. We identified the vRNA sequences involved in five of these interactions at the nucleotide level, and in two cases, we validated the existence of the interaction using compensatory mutations in the interacting sequences. Electron tomography also revealed significant differences in the interactions taking place between viral ribonucleoproteins in H5N2 and H3N2 virions, despite their canonical '7 + 1' arrangement

Gene Expression Signature-Based Screening Identifies New Broadly Effective Influenza A Antivirals

Classical antiviral therapies target viral proteins and are consequently subject to resistance. To counteract this limitation, alternative strategies have been developed that target cellular factors. We hypothesized that such an approach could also be useful to identify broad-spectrum antivirals. The influenza A virus was used as a model for its viral diversity and because of the need to develop therapies against unpredictable viruses as recently underlined by the H1N1 pandemic. We proposed to identify a gene-expression signature associated with infection by different influenza A virus subtypes which would allow the identification of potential antiviral drugs with a broad anti-influenza spectrum of activity. We analyzed the cellular gene expression response to infection with five different human and avian influenza A virus strains and identified 300 genes as differentially expressed between infected and non-infected samples. The most 20 dysregulated genes were used to screen the connectivity map, a database of drug-associated gene expression profiles. Candidate antivirals were then identified by their inverse correlation to the query signature. We hypothesized that such molecules would induce an unfavorable cellular environment for influenza virus replication. Eight potential antivirals including ribavirin were identified and their effects were tested in vitro on five influenza A strains. Six of the molecules inhibited influenza viral growth. The new pandemic H1N1 virus, which was not used to define the gene expression signature of infection, was inhibited by five out of the eight identified molecules, demonstrating that this strategy could contribute to identifying new broad anti-influenza agents acting on cellular gene expression. The identified infection signature genes, the expression of which are modified upon infection, could encode cellular proteins involved in the viral life cycle. This is the first study showing that gene expression-based screening can be used to identify antivirals. Such an approach could accelerate drug discovery and be extended to other pathogens

A supramolecular assembly formed by influenza A virus genomic RNA segments

The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common ‘transition zone’ located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5′ region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals

Parainfluenza virus type 5 (PIV-5) morphology revealed by cryo-electron microscopy.

International audienceThe knowledge of parainfluenza type 5 (PIV-5) virion morphology is essentially based on the observation of negatively stained preparations in conventional transmission electron microscopy (CTEM). In this study, the ultrastructure of frozen-hydrated intact PIV-5 was examined by cryo-electron microscopy (cryo-EM). Cryo-EM revealed a majority of spherical virions (70%), with a lower pleiomorphy than originally observed in CTEM. Phospholipid bilayer thickness, spike length and glycoprotein spikes density were measured. About 2000 glycoprotein spikes were present in an average-sized spherical virion. Altogether, these data depict a more precise view of PIV-5 morphology

Protective role of protease-activated-receptor 2 against influenza viruses occurs through an ERK independent pathway: implication for new influenza therapy

International audienceTo date there is an urgent need to develop new antivirals against influenza. Most of the molecules reported target influenza proteins that acquire rapid mutations of resistance. The development of new molecules that have a broad antiviral activity and are not subjected to influenza mutation is of particular interest. Our laboratory and others recently showed that proteases can participate to the innate immune response in the airways through the activation of a family of receptors called PAR. In particular, through the release of interferon, PAR2 agonists curbed viral replication significantly in infected cells. In this study, since ERK activation is crucial for virus replication, we investigated whether PAR2 could inhibit virus replication through inhibition of the ERK pathway. Results showed that while influenza A infection alone or PAR2 stimulation alone induced ERK activation, PAR2 stimulation does not inhibit ERK activation in influenza infected cells. Thus, PAR2 agonists may be a potential new drug against influenza viruses that could be used in combination with other anti flu therapy such as the inhibition of the ERK pathway

Supplemental treatment of air in airborne infection isolation rooms using high-throughput in-room air decontamination units.

BACKGROUND: Evidence has recently emerged indicating that in addition to large airborne droplets, fine aerosol particles can be an important mode of influenza transmission that may have been hitherto underestimated. Furthermore, recent performance studies evaluating airborne infection isolation (AII) rooms designed to house infectious patients have revealed major discrepancies between what is prescribed and what is actually measured. METHODS: We conducted an experimental study to investigate the use of high-throughput in-room air decontamination units for supplemental protection against airborne contamination in areas that host infectious patients. The study included both intrinsic performance tests of the air-decontamination unit against biological aerosols of particular epidemiologic interest and field tests in a hospital AII room under different ventilation scenarios. RESULTS: The unit tested efficiently eradicated airborne H5N2 influenza and Mycobacterium bovis (a 4- to 5-log single-pass reduction) and, when implemented with a room extractor, reduced the peak contamination levels by a factor of 5, with decontamination rates at least 33% faster than those achieved with the extractor alone. CONCLUSION: High-throughput in-room air treatment units can provide supplemental control of airborne pathogen levels in patient isolation rooms

Antiviral Activity of Active Materials: Standard and Finger-Pad-Based Innovative Experimental Approaches

Environmental surfaces, including high-touch surfaces (HITS), bear a high risk of becoming fomites and can participate in viral dissemination through contact and transmission to other persons, due to the capacity of viruses to persist on such contaminated surface before being transferred to hands or other supports at sufficient concentration to initiate infection through direct contact. Interest in the development of self-decontaminating materials as additional safety measures towards preventing viral infectious disease transmission has been growing. Active materials are expected to reduce the viral charge on surfaces over time and consequently limit viral transmission capacity through direct contact. In this study, we compared antiviral activities obtained using three different experimental procedures by assessing the survival of an enveloped virus (influenza virus) and non-enveloped virus (feline calicivirus) over time on a reference surface and three active materials. Our data show that experimental test conditions can have a substantial impact of over 1 log10 on the antiviral activity of active material for the same contact period, depending on the nature of the virus. We then developed an innovative and reproducible approach based on finger-pad transfer to evaluate the antiviral activity of HITS against a murine norovirus inoculum under conditions closely reflecting real-life surface exposure

Fate of premalignant clones during the asymptomatic phase preceding lymphoid malignancy.

peer reviewedAlmost all cancers are preceded by a prolonged period of clinical latency during which a combination of cellular events helps move carcinogen-exposed cells towards a malignant phenotype. Hitherto, investigating the fate of premalignant cells in vivo remained strongly hampered by the fact that these cells are usually indistinguishable from their normal counterparts. Here, for the first time, we have designed a strategy able to reconstitute the replicative history of the bona fide premalignant clone in an animal model, the sheep experimentally infected with the lymphotropic bovine leukemia virus. We have shown that premalignant clones are early and clearly distinguished from other virus-exposed cells on the basis of their degree of clonal expansion and genetic instability. Detectable as early as 0.5 month after the beginning of virus exposure, premalignant cells displayed a two-step pattern of extensive clonal expansion together with a mutation load approximately 6 times higher than that of other virus-exposed cells that remained untransformed during the life span of investigated animals. There was no fixation of somatic mutations over time, suggesting that they regularly lead to cellular death, partly contributing to maintain a normal lymphocyte count during the prolonged premalignant stage. This equilibrium was finally broken after a period of 18.5 to 60 months of clinical latency, when a dramatic decrease in the genetic instability of premalignant cells coincided with a rapid increase in lymphocyte count and lymphoma onset