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Despite progress in our knowledge of the internal organisation of influenza virus particles, little is known about the determinants of their morphology and, more particularly, of the actual abundance of structural proteins at the virion level. To address these issues, we used cryo-EM to focus on viral (and host) factors that might account for observed differences in virion morphology and characteristics such as size, shape and glycoprotein (GP) spike density. Twelve recombinant viruses were characterised in terms of their morphology, neuraminidase activity and virus growth. The genomic composition was shown to be important in determining the GP spike density. In particular, polymerase gene segments and especially PB1/PB2 were shown to have a prominent influence in addition to that for HA in determining GP spike density, a feature consistent with a functional link between these virus components important for virus fitness., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Neuraminidase inhibitors are thought to be efficacious in reducing the time to alleviation of symptoms in outpatients with seasonal influenza. The objective of this study was to compare the short-term virological efficacy of oseltamivir-zanamivir combination versus each monotherapy plus placebo., Methods and Findings: We conducted a randomized placebo-controlled trial with 145 general practitioners throughout France during the 2008-2009 seasonal influenza epidemic. Patients, general practitioners, and outcome assessors were all blinded to treatment assignment. Adult outpatients presenting influenza-like illness for less than 36 hours and a positive influenza A rapid test diagnosis were randomized to oseltamivir 75 mg orally twice daily plus zanamivir 10 mg by inhalation twice daily (OZ), oseltamivir plus inhaled placebo (O), or zanamivir plus oral placebo (Z). Treatment efficacy was assessed virologically according to the proportion of patients with nasal influenza reverse transcription (RT)-PCR below 200 copies genome equivalent (cgeq)/µl at day 2 (primary outcome), and clinically to the time to alleviation of symptoms until day 14. Overall 541 patients (of the 900 planned) were included (OZ,  =192; O, n=176; Z, n=173), 49% male, mean age 39 years. In the intention-to-treat analysis conducted in the 447 patients with RT-PCR-confirmed influenza A, 46%, 59%, and 34% in OZ (n=157), O (n=141), and Z (n=149) arms had RT-PCR<200 cgeq/µl (-13.0%, 95% confidence interval [CI] -23.1 to -2.9, p=0.025; +12.3%, 95% CI 2.39-22.2, p=0.028 for OZ/O and OZ/Z comparisons). Mean day 0 to day 2 viral load decrease was 2.14, 2.49, and 1.68 log(10) cgeq/µl (p=0.060, p=0.016 for OZ/O and OZ/Z). Median time to alleviation of symptoms was 4.0, 3.0, and 4.0 days (+1.0, 95% CI 0.0-4.0, p=0.018; +0.0, 95% CI -3.0 to 3.0, p=0.960 for OZ/O and OZ/Z). Four severe adverse events were observed. Nausea and/or vomiting tended to be more frequent in the combination arm (OZ, n=13; O, n=4; and Z, n=5 patients, respectively)., Conclusions: In adults with seasonal influenza A mainly H3N2 virus infection, the oseltamivir-zanamivir combination appeared less effective than oseltamivir monotherapy, and not significantly more effective than zanamivir monotherapy. Despite the theoretical potential for the reduction of the emergence of antiviral resistance, the lower effectiveness of this combination calls for caution in its use in clinical practice., Trial Registration: www.ClinicalTrials.govNCT00799760.

Influenza A(H1N1) viruses resistant to oseltamivir carboxylate (OC) emerged in 2007/2008 in the absence of antiviral pressure. These OC-resistant A(H1N1) viruses had a better fitness than the sensitive ones as they were 100% prevalent in 2008/2009. To better understand the role of the neuraminidase (NA) affinity in the emergence of these OC-resistant A(H1N1) viruses we compared the NA properties among A(H1N1) clinical isolates in south of France between 2005 and 2009 and reference strains from 1977 to 2007, using NA inhibition assays, kinetic analyses of NA activities, and sequence analysis of viral NA and hemagglutinin (HA). In 2007/2008, among 374 A(H1N1) isolates tested, 38% were resistant to OC with a mean IC50 of 564+/-357 nM. The mean Km of OC-sensitive isolates (H275) was significantly lower (22.6+/- 4.7 microM) than the Km of previous reference strains (44.9+/- 5 microM) and the mean Km of the OC-resistant isolates (Y275) (37.2 +/- 7.7 microM). The combination of different amino acid mutations in N1 particularly the D344N could explain the higher NA affinity of A/Brisbane/59/2007 related variants compared to the previous A(H1N1) strains and the H275Y mutation allowed to retrieve Km values near 40 microM.

Oseltamivir and zanamivir are two neuraminidase inhibitors (NAIs) active on A and B influenza viruses. These analogues have been developed from the structure of sialic acid, the neuraminidase (NA) substrate. Resistance to NAIs have been detected. They are mainly associated to mutations located on the NA gene. The use of these antiviral drugs remains low in the context of seasonal flu, even the duration of symptoms can be reduced of one day if an antiviral treatment is started within 48 hours after disease onset. NAIs also present a significant effect when used in postexposition prophylaxis. Resistance, mainly to oseltamivir, have been detected but remained rare until the spontaneous emergence in 2007-2008 winter of a seasonal A(H1N1) variant resistant to this drug. NAIs are also interesting for the treatment of severe flu infections, specially those associated to A(H5N1). Finally, because of the pandemic A(H1N1)2009 virus, NAIs use has largely increased for prophylactic and therapeutic treatment of severe and non severe infections. This large use may be associated to an increased risk of selection of resistant viruses. Up to now, this phenomenon remains fortunately limited but has to be closely monitored., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

The emerging A (H5N1) influenza virus is a clear pandemic threat. This avian virus is responsible for the largest epizootic event described so far. To date, 423 humans have been infected. In humans, this virus is responsible for a rapidly developing pneumonia, with an acute respiratory failure leading to death in 60% of the infected cases. The multi-organ failure seems to result from the cytokine storm. A (H5N1) infections are mainly reported in children and young adults. Different hypotheses have been proposed to explain this specific feature, including the lack of control of cytokine release during infection, or the presence of alternative cellular receptors. The specific susceptibility of children is more likely to be related with exposure to infected birds than to specific immune or physiological factors. The proper and efficient management of A (H5N1) infection is possible nowadays. Today, the pandemic threat is real and may be imminent because of the circulation of new A (H1N1). However, an active surveillance of A (H5N1) virus remains very important to monitor the genetic evolution of this changing and virulent virus.

Background: Neuraminidase inhibitors (NAIs) have been used since 2005 in France., Objective: Influenza viruses isolated in hospital and community cases in winter 2005-2006 were evaluated for their sensitivity to NAIs., Study Design: Isolates were tested in duplicate with a fluorescence-based neuraminidase assay. The IC50 for oseltamivir or zanamivir was calculated for each strain. Mean IC50 (+/-S.D.) are expressed in nM. Viruses with IC50 superior or very superior to the upper limit (mean IC50+2.5 S.D.) were considered as outliers or resistant viruses, respectively. HA and NA genes for outliers, resistant strains and for a few sensitive strains were sequenced., Results: Out of 225 B isolates, one was found resistant to both oseltamivir and zanamivir with a D197Y mutation in NA and eight isolates were outliers for oseltamivir and/or zanamivir. Out of 151 A (H1N1) isolates, one was found resistant to oseltamivir but sensitive to zanamivir with a H275Y mutation in NA, two isolates were resistant to zanamivir and three isolates were outliers for oseltamivir and/or zanamivir. New mutations were detected in outliers compared to sensitive viruses., Conclusion: Resistant influenza strains to NAIs are circulating at a stable and low level of 1% since the introduction of NAIs in clinical practice.

Background: Human rhinoviruses and enteroviruses (Picornaviridae) are suspected to be major viral etiological causes of bronchiolitis in infants., Objectives: In the present study, we assessed the potential role of the respiratory picornaviruses as causative agents of bronchiolitis in French infants., Study Design: From September 2001 to June 2002, we prospectively selected 192 infants < or =36 months of age and hospitalized for acute bronchiolitis. The detection of common respiratory viruses (respiratory syncytial virus, influenza virus A and B, parainfluenza virus 1, 2, 3 and adenovirus) was performed using classical immunofluorescence antigen and cell-culture detection assays on nasopharyngeal aspirates whereas the detection of human metapneumovirus (HMPV) was performed by a real-time RT-PCR assay. The presence of rhinovirus and/or enterovirus was assessed in respiratory samples by a picornavirus RT-PCR detection assay followed by a differential Southern blotting procedure., Results: A potential causative virus was detected in 72.5% of the 192 study infants. RSV (30%), rhinovirus (21%), enterovirus (9%), influenza virus A (6%) and human metapneumovirus (4%) were the most frequent causative agents detected. Rhinoviruses or enteroviruses were detected as the only evidence of respiratory viral tract infection in 57 (30%) of 192 infants, whereas rhinovirus or enterovirus occurred in mixed viral infection detected in 25 (13%) of 192 study cases (30% versus 13%, p<10(-3))., Conclusions: Our data suggest that respiratory picornaviruses are one of the leading etiological causes of bronchiolitis in French infants. These findings highlight the need to implement a rapid picornavirus RT-PCR detection assay for the clinical diagnosis of respiratory infections in pediatric patients with bronchiolitis.

Human metapneumovirus (HMPV) was the unique viral pathogen detected by a real-time reverse transcriptase PCR (RT-PCR) assay in 6 (6.4%) of 94 consecutive French children hospitalized for acute bronchiolitis from September 2001 to June 2002. This virus was identified as the third etiological cause of bronchiolitis, after respiratory syncytial virus and rhinovirus (35 [37%] and 21 [22%] of 94 cases, respectively). Phylogenetic analysis of F-gene sequences demonstrated the cocirculation of distinct HMPV genotypes during this study. These findings highlight the need to implement a rapid HMPV RT-PCR detection assay for the clinical diagnosis of respiratory infections in pediatric patients with bronchiolitis.