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1.
Antiviral Res ; 118: 75-81, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25796972

ABSTRACT

Crimean-Congo hemorrhagic virus (CCHFV) causes hemorrhagic fever with high case mortality rates and is endemic in south-eastern Europe, Africa, and Asia. The limited catalog of specific treatment, highlight the necessity to look for additional therapeutic solutions. Previous experiments suggested that CCHFV enters the cells via a clathrin dependent pathway. Therefore, we have evaluated the potential anti-CCHFV activity of several molecules targeting this entry possibility. We identified two molecules chloroquine and chlorpromazine. Neutralization and virus yield reduction assays were tested in Vero E6 and Huh7 cells on two different CCHFV strains. Several combinations, including ribavirin, were assayed to test a potential synergistic effect. The two molecules inhibited CCHFV, and depending on the virus and the cell lines, the 50% inhibitory concentration (IC50) values for chloroquine and chlorpromazine ranged from 28 to 43 and 10.8-15.7 µM, respectively. Time-of-addition studies demonstrated that these molecules had a direct effect on CCHFV infectivity and spread. The antiviral activity of the two molecules was still effective even when added up to 6h post-infection and up to 24h. The selectivity index ranging from 3 to 35 lead us to evaluate combinations with ribavirin. Combinations of ribavirin and chloroquine or chlorpromazine were synergistic against CCHFV. Though the low chlorpromazine selectivity index suggests the need for a chemical improvement, our present study highlights chloroquine as the main drug having the potential for drug repurposing.


Subject(s)
Antiviral Agents/pharmacology , Chloroquine/pharmacology , Chlorpromazine/pharmacology , Drug Repositioning , Hemorrhagic Fever Virus, Crimean-Congo/drug effects , Virus Internalization/drug effects , Animals , Cell Line , Drug Synergism , Hemorrhagic Fever Virus, Crimean-Congo/physiology , Humans , Inhibitory Concentration 50 , Microbial Sensitivity Tests , Ribavirin/pharmacology
3.
Virology ; 404(2): 215-24, 2010 Sep 01.
Article in English | MEDLINE | ID: mdl-20627352

ABSTRACT

Among a panel of 788 clinical influenza H3N2 isolates, two isolates were characterized by an oseltamivir-resistant phenotype linked to the absence of any detectable NA activity. Here, we established that the two H3NA- isolates lack any detectable full-length NA segment, and one of these could be rescued by reverse genetics in the absence of any NA segment sequence. We found that the absence of NA segment induced a moderate growth defect of the H3NA- viruses as on cultured cells. The glycoproteins density at the surface of H3NA- virions was unchanged as compared to H3N2 virions. The HA protein as well as residues 188 and 617 of the PB1 protein were shown to be strong determinants of the ability of H3NA- viruses to grow in the absence of the NA segment. The significance of these findings about naturally occurring seven-segment influenza A viruses is discussed.


Subject(s)
Influenza A virus/genetics , Neuraminidase/genetics , Virus Replication/physiology , Amino Acid Sequence , Animals , Antiviral Agents/pharmacology , Cell Line , Cryoelectron Microscopy , Dogs , Drug Resistance, Viral/genetics , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Viral/physiology , Humans , Influenza A Virus, H3N2 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/enzymology , Influenza A Virus, H3N2 Subtype/genetics , Influenza A virus/drug effects , Influenza A virus/enzymology , Influenza A virus/physiology , Models, Molecular , Neuraminidase/antagonists & inhibitors , Neuraminidase/chemistry , Oseltamivir/pharmacology , Protein Conformation , Sequence Alignment , Virion/ultrastructure
4.
Pathol Biol (Paris) ; 58(2): e69-78, 2010 Apr.
Article in French | MEDLINE | ID: mdl-20303677

ABSTRACT

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.


Subject(s)
Antiviral Agents/therapeutic use , Influenza A virus/drug effects , Influenza, Human/drug therapy , Neuraminidase/antagonists & inhibitors , Oseltamivir/therapeutic use , Viral Proteins/antagonists & inhibitors , Zanamivir/therapeutic use , Adult , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Child , Clinical Trials as Topic , Disease Outbreaks , Double-Blind Method , Drug Resistance, Viral/genetics , Drug Therapy, Combination , Female , Humans , Immunocompromised Host , Influenza A virus/enzymology , Influenza A virus/genetics , Influenza, Human/epidemiology , Influenza, Human/prevention & control , Male , Models, Molecular , Molecular Structure , Mutation, Missense , Neuraminidase/chemistry , Neuraminidase/genetics , Oseltamivir/administration & dosage , Oseltamivir/chemistry , Oseltamivir/pharmacology , Point Mutation , Pregnancy , Pregnancy Complications, Infectious/drug therapy , Pregnancy Complications, Infectious/virology , Protein Conformation , Viral Proteins/chemistry , Viral Proteins/genetics , Zanamivir/administration & dosage , Zanamivir/chemistry , Zanamivir/pharmacology
5.
Epidemiol Infect ; 138(9): 1274-80, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20092669

ABSTRACT

In 2006 an outbreak of avian influenza A(H5N1) in Turkey caused 12 human infections, including four deaths. We conducted a serological survey to determine the extent of subclinical infection caused by the outbreak. Single serum samples were collected from five individuals with avian influenza whose nasopharyngeal swabs tested positive for H5 RNA by polymerase chain reaction, 28 family contacts of the cases, 95 poultry cullers, 75 individuals known to have had contact with diseased chickens and 81 individuals living in the region with no known contact with infected chickens and/or patients. Paired serum samples were collected from 97 healthcare workers. All sera were tested for the presence of neutralizing antibodies by enzyme-linked immunoassay, haemagglutination inhibition and microneutralization assays. Only one serum sample, from a parent of an avian influenza patient, tested positive for H5N1 by microneutralization assay. This survey shows that there was minimal subclinical H5N1 infection among contacts of human cases and infected poultry in Turkey in 2006. Further, the low rate of subclinical infection following contact with diseased poultry gave further support to the reported low infectivity of the virus.


Subject(s)
Disease Outbreaks , Influenza A Virus, H5N1 Subtype/pathogenicity , Influenza in Birds/epidemiology , Influenza in Birds/virology , Influenza, Human/epidemiology , Influenza, Human/virology , Adolescent , Adult , Aged , Animals , Antibodies, Viral/blood , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay , Female , Humans , Infant , Influenza A Virus, H5N1 Subtype/immunology , Influenza in Birds/immunology , Influenza in Birds/transmission , Influenza, Human/immunology , Influenza, Human/transmission , Male , Middle Aged , Neutralization Tests , Polymerase Chain Reaction , Poultry/virology , Turkey/epidemiology
6.
Curr Microbiol ; 61(1): 25-8, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20033691

ABSTRACT

The A(H5N1) influenza virus pandemic may be the result of avian H5N1 adapting to humans, leading to massive human to human transmission in a context of a lack of pre-existing immunity. As A(H1N1) and A(H5N1) share the same neuraminidase subtype, anti-N1 antibodies subsequent to H1N1 infections or vaccinations may confer some protection against A(H5N1). We analysed, by microneutralization assay, the A/Vietnam/1194/04 (H5N1) anti-N1 cross-protection acquired either during A/New-Caledonia/20/99 (H1N1) infection or vaccination. In cases with documented H1N1 infection, H5N1 cross-protection could be observed only in patients born between 1930 and 1950. No such protection was detected in the sera of vaccinated individuals.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Cross Protection , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/immunology , Influenza, Human/immunology , Neuraminidase/immunology , Adult , Aged , Aged, 80 and over , Aging/immunology , Humans , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Influenza, Human/virology , Middle Aged , Neutralization Tests , Vaccination , Young Adult
7.
J Clin Virol ; 45(2): 119-24, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19406687

ABSTRACT

BACKGROUND: Respiratory infections caused by viruses are major causes of upper and lower respiratory tract infections. They account for an important mortality and morbidity worldwide. Amongst these viruses, influenza viruses and paramyxoviruses are major pathogens. Their transmission is mainly airborne, by direct transmission through droplets from infected cases. OBJECTIVES: In the context of an influenza pandemic, as well as for the reduction of nosocomial infections, systems that can reduce or control virus transmission will reduce the burden of this disease. It may also be part of the strategy for pandemic mitigation. STUDY DESIGN: A new system based on physical decontamination of surface and air has been developed. This process generates cold oxygen plasma (COP) by subjecting air to high-energy deep-UV light. To test its efficiency, we have developed an experimental device to assess for the decontamination of nebulized respiratory viruses. High titer suspensions of influenza virus type A, human parainfluenza virus type 3 and RSV have been tested. RESULTS: Different experimental conditions have been evaluated against these viruses. The use of COP with an internal device allowed the best results against all viruses tested. We recorded a reduction of 6.5, 3.8 and 4 log(10) TCID50/mL of the titre of the hPIV-3, RSV and influenza virus A (H5N2) suspensions. CONCLUSIONS: The COP technology is an efficient and innovative strategy to control airborne virus dissemination. It could successfully control nosocomial diffusion of respiratory viruses in hospital setting, and could be useful for the reduction of influenza transmission in the various consultation settings implemented for the management of cases during a pandemic.


Subject(s)
Disinfectants/pharmacology , Disinfection/methods , Environmental Microbiology , Influenza A Virus, H5N2 Subtype/drug effects , Microbial Viability/drug effects , Oxygen/pharmacology , Parainfluenza Virus 3, Human/drug effects , Animals , Cell Line , Dogs , Haplorhini
8.
J Clin Virol ; 43(1): 86-92, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18579437

ABSTRACT

BACKGROUND: Human parainfluenza viruses (hPIV) are respiratory pathogens responsible for upper and lower respiratory tract infections. In most labs, the clinical diagnosis of hPIV is routinely done using techniques based on the detection of viral antigens such as immunofluorescence assay or/and viral isolation. STUDY DESIGN: Five hPIV-2 isolated from respiratory samples exhibited unusual phenotypic and antigenic characteristics. These isolates showed important syncytial cytopathic effect and failed to react with one specific monoclonal antibody. These variant strains were subsequently compared with hPIV-2 prototype strain by cellular and molecular techniques. RESULTS: Both variant and prototype strains showed similar growth kinetics. Observation of plaque formation and syncytia assay indicated a more important fusogenic activity for the variant strains. Sequencing of fusion (F) and hemagglutinin-neuraminidase (HN) genes showed differences between the "atypical" hPIV-2 isolates and the Greer hPIV-2 prototype strain. These differences were analyzed with molecular modelling and structure prediction soft wares. A potential new glycosylation site in HN, in addition to minor changes observed in the predicted structure for the variant strains could explain their antigenic variation. Genetic changes in the fusion peptide and the cleavage site of F could also explain the difference observed in the fusion activity. CONCLUSIONS: Continuous global viral surveillance is essential to monitor antigenic changes that may occur in nature particularly with regards to the implementation of diagnostic assays. The differences observed in F and HN between the prototype strain and clinical hPIV-2 variants could also provide new data for the analysis of Paramyxovirus fusion mechanisms and their pathogenesis.


Subject(s)
HN Protein/genetics , Parainfluenza Virus 2, Human/physiology , RNA, Viral , Rubulavirus Infections/virology , Viral Fusion Proteins/genetics , Adult , Amino Acid Sequence , Animals , Antigenic Variation , Cell Line , Child , Glycosylation , HN Protein/chemistry , HN Protein/immunology , Haplorhini , Humans , Models, Molecular , Molecular Sequence Data , Parainfluenza Virus 2, Human/classification , Parainfluenza Virus 2, Human/genetics , Parainfluenza Virus 2, Human/isolation & purification , Phylogeny , Protein Conformation , Reverse Transcriptase Polymerase Chain Reaction , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/immunology , Viral Plaque Assay
9.
Antiviral Res ; 68(1): 43-8, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16125799

ABSTRACT

Influenza virus neuraminidase inhibitors (NAIs) were introduced in clinical practice in various parts of the world since 1999 but were only scarcely distributed in France. Prior to the generalization of zanamivir and oseltamivir utilization in our country, we decided to test a large panel of influenza strains to establish the baseline sensitivity of these viruses to anti-neuraminidase drugs, based upon a fluorometric neuraminidase enzymatic test. Our study was performed on clinical samples collected by practitioners of the GROG network (Groupe Régional d'Observation de la Grippe) in the south of France during the 2002-2003 influenza season. Out of 355 isolates tested in the fluorometric neuraminidase activity assay, 267 isolates could be included in inhibition assay against anti-neuraminidase drugs. Differences in IC50 range were found according to the subtype and the anti-neuraminidase drug. Influenza B and A/H1N1 viruses appeared to be more sensitive to zanamivir than to oseltamivir (mean B IC50 values: 4.19 nM versus 13 nM; mean H1N1 IC50 values: 0.92 nM versus 1.34 nM), while A/H1N2 and A/H3N2 viruses were more sensitive to oseltamivir than to zanamivir (mean H3N2 IC50 values: 0.67 nM versus 2.28 nM; mean H1N2 IC50 values: 0.9 nM versus 3.09 nM). Out of 128 N2 carrying isolates, 10 isolates had zanamivir or oseltamivir IC50 values in upper limits compared to their respective data range. Sequencing of the neuraminidase of these outliers N2 highlighted several mutations, but none of them were associated with resistance to neuraminidase inhibitors.


Subject(s)
Acetamides/pharmacology , Enzyme Inhibitors/pharmacology , Guanidines/pharmacology , Influenza A virus/drug effects , Influenza B virus/drug effects , Neuraminidase/drug effects , Pyrans/pharmacology , Sialic Acids/pharmacology , France , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H3N2 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/growth & development , Influenza A virus/enzymology , Influenza B virus/enzymology , Influenza, Human/virology , Neuraminidase/antagonists & inhibitors , Oseltamivir , Zanamivir
10.
Dev Biol (Basel) ; 115: 75-83, 2003.
Article in English | MEDLINE | ID: mdl-15088778

ABSTRACT

Anti-neuraminidase (NA) antibodies (Ab) play a role in protection against influenza and in combination with anti-HA Ab they increase the protection in mice. To control the NA content of vaccines, which should improve vaccine standardisation and may benefit vaccine efficacy, a series of questions must be addressed: 1) The antigenic characterization of NA in vaccine strains and seed lots is based on the measurement of the enzymatic (E) activity using fetuin as substrate. The antigenic profile is established by inhibiting the E activity with post infectious ferret antisera. Overnight incubation ensures sensitivity, and fetuin substrate gives specificity by detection of variant specific antibodies. Several difficulties have to be overcome, such as the low level of E activity in MDCK grown viruses, and the lability of N1. 2) The NA protein content of the vaccines (in bulk or final product) can be measured by an ELISA capture test but the lability of the NA proteins at 4 degrees C must be checked. 3) The anti NA Ab response can be measured using a neuraminidase inhibition test. --The steric hindrance by HI antibodies does not exceed a titre of 20 in human sera. --Triton treatment of viruses reduces the steric hindrance in polyclonal sera and monoclonal antibodies but unmasks epitopes. 4) The correlations between neuraminidase inhibition, neutralization and protection, has been established in the mouse model, but remains to be shown in humans. 5) The use of a small fluorescent (MUN) or chemiluminescent (NA-STAR) substrate can be used for the rapid differentiation of N1 from N2 and NB, but not for the titration of protective NI antibodies.


Subject(s)
Antibodies, Viral/immunology , Neuraminidase/immunology , Animals , Enzyme-Linked Immunosorbent Assay , Influenza Vaccines/immunology , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/immunology
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