Differential Effect of Nucleotide Substitutions in the 3′ Arm of the Influenza A Virus vRNA Promoter on Transcription/Replication by Avian and Human Polymerase Complexes Is Related to the Nature of PB2 Amino Acid 627

AbstractUsing a genetic system that allows the in vivo reconstitution of active ribonucleoproteins, the ability to ensure transcription/replication of a viral-like reporter RNA harboring the G3 → A3, U5 → C5, and C8 → U8 mutations (triple 3-5-8 mutations) in the 3′ arm of the promoter was examined with core proteins from human or avian strains of influenza A viruses. The efficiency of transcription/replication of the viral-like RNA with the triple 3-5-8 mutations in COS-1 cells was found to be slightly decreased as compared to the wild-type RNA when the polymerase was derived from a human virus. In contrast, it was found to be considerably increased when the polymerase was derived from an avian virus, in agreement with published observations using the avian A/FPV/Bratislava virus (G. Neumann and G. Hobom, 1995, J. Gen. Virol. 76, 1709–1717). This increase could be attributed to the compensation of the defect in transcription/replication activity in the COS-1 mammalian cell line due to the presence of a glutamic acid at PB2 residue 627, characteristic of avian strains of influenza viruses. Our results thus suggest that PB2 and/or cellular proteins interacting with PB2 could be involved in RNA conformational changes during the process of transcription/replication

Non coding extremities of the seven influenza virus type C vRNA segments: effect on transcription and replication by the type C and type A polymerase complexes

<p>Abstract</p> <p>Background</p> <p>The transcription/replication of the influenza viruses implicate the terminal nucleotide sequences of viral RNA, which comprise sequences at the extremities conserved among the genomic segments as well as variable 3' and 5' non-coding (NC) regions. The plasmid-based system for the <it>in vivo </it>reconstitution of functional ribonucleoproteins, upon expression of viral-like RNAs together with the nucleoprotein and polymerase proteins has been widely used to analyze transcription/replication of influenza viruses. It was thus shown that the type A polymerase could transcribe and replicate type A, B, or C vRNA templates whereas neither type B nor type C polymerases were able to transcribe and replicate type A templates efficiently. Here we studied the importance of the NC regions from the seven segments of type C influenza virus for efficient transcription/replication by the type A and C polymerases.</p> <p>Results</p> <p>The NC sequences of the seven genomic segments of the type C influenza virus C/Johannesburg/1/66 strain were found to be more variable in length than those of the type A and B viruses. The levels of transcription/replication of viral-like vRNAs harboring the NC sequences of the respective type C virus segments flanking the CAT reporter gene were comparable in the presence of either type C or type A polymerase complexes except for the NS and PB2-like vRNAs. For the NS-like vRNA, the transcription/replication level was higher after introduction of a U residue at position 6 in the 5' NC region as for all other segments. For the PB2-like vRNA the CAT expression level was particularly reduced with the type C polymerase. Analysis of mutants of the 5' NC sequence in the PB2-like vRNA, the shortest 5' NC sequence among the seven segments, showed that additional sequences within the PB2 ORF were essential for the efficiency of transcription but not replication by the type C polymerase complex.</p> <p>Conclusion</p> <p>In the context of a PB2-like reporter vRNA template, the sequence upstream the polyU stretch plays a role in the transcription/replication process by the type C polymerase complex.</p

Anti-influenza chemotherapies

The recent outbreaks of avian influenza A (H5N1) virus have called attention to the need for antiviral treatments to use in the event of pandemic influenza. The goal of antiviral treatments is also to reduce symptoms and complications associated with seasonal epidemics. Two classes of antiviral drugs, M2 proton channel inhibitors (amantadine, rimantadine) and neuraminidase inhibitors (zanamivir, oseltamivir), are effective for the chemoprophylaxis and treatment of influenza. Antiviral resistance is especially frequent with treatment with M2 inhibitors, and limits their clinical use. Resistance to oseltamivir during treatment has been described recently in several Vietnamese patients infected with H5N1. A close monitoring of antiviral resistance is needed, as is further research into the development of new agents, potentially targeting other viral proteins such as hemagglutinin or polymerase, and which could be used in combination chemotherapies.L'actuelle épizootie de grippe A (H5N1) souligne la nécessité de traitements antiviraux pour faire face à une éventuelle pandémie grippale. Les traitements anti-influenza ont aussi pour objectif de réduire les symptômes et complications survenant lors des épidémies saisonnières. Deux classes d'antiviraux, les inhibiteurs du canal à protons M2 (amantadine, rimantadine), et les inhibiteurs de neuraminidase (zanamivir, oseltamivir), ont une efficacité prophylactique et thérapeutique. L'émergence de virus résistants est particulièrement fréquente lors du traitement avec les inhibiteurs de M2, et limite leur utilisation. Le développement d'une résistance à l'oseltamivir a été décrit chez plusieurs patients infectés avec le virus H5N1. Une surveillance étroite de la résistance aux anti-viraux s'impose, ainsi que le développement de nouveaux composés, pouvant cibler éventuellement d'autres protéines virales telles que l'hémagglutinine ou la polymérase, et pouvant être utilisés en polychimiothérapies

H9N2 avian influenza virus in a Mediterranean gull

International audienc

Seasonal Genetic Drift of Human Influenza A Virus Quasispecies Revealed by Deep Sequencing

After a pandemic wave in 2009 following their introduction in the human population, the H1N1pdm09 viruses replaced the previously circulating, pre-pandemic H1N1 virus and, along with H3N2 viruses, are now responsible for the seasonal influenza type A epidemics. So far, the evolutionary potential of influenza viruses has been mainly documented by consensus sequencing data. However, like other RNA viruses, influenza A viruses exist as a population of diverse, albeit related, viruses, or quasispecies. Interest in this quasispecies nature has increased with the development of next generation sequencing (NGS) technologies that allow a more in-depth study of the genetic variability. NGS deep sequencing methodologies were applied to determine the whole genome genetic heterogeneity of the three categories of influenza A viruses that circulated in humans between 2007 and 2012 in France, directly from clinical respiratory specimens. Mutation frequencies and single nucleotide polymorphisms were used for comparisons to address the level of natural intrinsic heterogeneity of influenza A viruses. Clear differences in single nucleotide polymorphism profiles between seasons for a given subtype also revealed the constant genetic drift that human influenza A virus quasispecies undergo

Enhancement of the influenza A hemagglutinin (HA)-mediated cell-cell fusion and virus entry by the viral neuraminidase (NA).

International audienceBACKGROUND: The major role of the neuraminidase (NA) protein of influenza A virus is related to its sialidase activity, which disrupts the interaction between the envelope hemagglutinin (HA) protein and the sialic acid receptors expressed at the surface of infected cells. This enzymatic activity is known to promote the release and spread of progeny viral particles following their production by infected cells, but a potential role of NA in earlier steps of the viral life cycle has never been clearly demonstrated. In this study we have examined the impact of NA expression on influenza HA-mediated viral membrane fusion and virion infectivity. METHODOLOGY/PRINCIPAL FINDINGS: The role of NA in the early stages of influenza virus replication was examined using a cell-cell fusion assay that mimics HA-mediated membrane fusion, and a virion infectivity assay using HIV-based pseudoparticles expressing influenza HA and/or NA proteins. In the cell-cell fusion assay, which bypasses the endocytocytosis step that is characteristic of influenza virus entry, we found that in proper HA maturation conditions, NA clearly enhanced fusion in a dose-dependent manner. Similarly, expression of NA at the surface of pseudoparticles significantly enhanced virion infectivity. Further experiments using exogenous soluble NA revealed that the most likely mechanism for enhancement of fusion and infectivity by NA was related to desialylation of virion-expressed HA. CONCLUSION/SIGNIFICANCE: The NA protein of influenza A virus is not only required for virion release and spread but also plays a critical role in virion infectivity and HA-mediated membrane fusion

Efficacy of Oseltamivir-Zanamivir Combination Compared to Each Monotherapy for Seasonal Influenza: A Randomized Placebo-Controlled Trial

Analysis of virological and clinical outcomes from a randomized trial that was terminated early suggest that combined treatment of seasonal influenza in adult outpatients with oseltamivir plus zanamivir is no more effective than either oseltamivir or zanamivir monotherapy

Swine influenza: Epidemiological situation in France

Serological and virus identification studies have been carried out in France to assess the epidemiological situation of the swine population. At the end of the 70’s, the serological profile in pig farms was dominated by the presence of A/H3N2 antibodies, associated with epidemics of human influenza. Since then, epizootic outbreaks have succeeded one another in the pig. The disease is now both enzootic and epizootic. Since the early 2000s, swine influenza in France occurs mainly in Brittany, where pig density is the highest. Its economic impact is considerable in pig farms of that area. The disease is caused by the influenza A/H1 virus of avian origin (A/H1N1) or by reassortants (A/H1N2). As influenza viruses are unstable, detection tools need permanent updating to guarantee an effective epidemiological surveillance.La situation épidémiologique du cheptel porcin français est appréhendée au travers d'études sérologiques ainsi que de recherches virales. À la fin des années 1970,le profil sérologique des élevages est dominé par la présence d'anticorps A/H3N2 correspondant à des épidémies de grippe humaine. Par la suite, des vagues épizootiques ont déferlé. La maladie se présente désormais sous une forme enzootique et épizootique. Depuis le début des années 2000, la grippe du porc en France concerne avant tout les élevages de Bretagne, où la densité porcine est la plus élevée. Elle a un impact économique considérable dans les élevages de cette région. L'activité grippale est le fait de virus A/H1 d'origine aviaire (A/H1N1) ou de réassortants (A/H1N2). L'instabilité des virus grippaux suppose d'adapter régulièrement les outils de détection afin de permettre une épidémiosurveillance efficace

Highly Pathogenic Avian Influenza Virus (H5N1) Outbreak in Captive Wild Birds and Cats, Cambodia

From December 2003 through January 2004, the Phnom Tamao Wildlife Rescue Centre, Cambodia, was affected by the highly pathogenic influenza virus (H5N1). Birds from 26 species died. Influenza virus subtype H5N1 was detected in 6 of 7 species tested. Cats from 5 of 7 species were probably infected; none died

Characterization of pandemic influenza immune memory signature after vaccination or infection

International audienceThe magnitude, quality, and maintenance of immunological memory after infection or vaccination must be considered for future design of effective influenza vaccines. In 2009, the influenza pandemic produced disease that ranged from mild to severe, even fatal, illness in infected healthy adults and led to vaccination of a portion of the population with the adjuvanted, inactivated influenza A(H1N1)pdm09 vaccine. Here, we have proposed a multi-parameter quantitative and qualitative approach to comparing adaptive immune memory to influenza 1 year after mild or severe infection or vaccination. One year after antigen encounter, severely ill subjects maintained high levels of humoral and polyfunctional effector/memory CD4$^+$ T cells responses, while mildly ill and vaccinated subjects retained strong cellular immunity, as indicated by high levels of mucosal homing and degranulation markers on IFN-$\gamma^+$ antigen-specific T cells. A principal component analysis distinguished 3 distinct clusters of individuals. The first group comprised vaccinated and mildly ill subjects, while clusters 2 and 3 included mainly infected individuals. Each cluster had immune memory profiles that differed in magnitude and quality. These data provide evidence that there are substantial similarities between the antiinfluenza response that mildly ill and vaccinated individuals develop and that this immune memory signature is different from that seen in severely ill individuals