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M Bouloy

Publications and source records attributed to M Bouloy.

At least 19 recordsLinked to original sources
PubMed2005

Production of monoclonal antibodies against Rift Valley fever virus Application for rapid diagnosis tests (virus detection and ELISA) in human sera.

This paper describes the production and characterization of RVFV monoclonal antibodies. The characteristics of 32 out of 55 ELISA and/or IFA positive monoclonal antibodies were determined, including the RVFV components against which they are directed. One monoclonal antibody recognized the nucleoprotein, 15 the Gc and 16 the Gn. Among the latter ones, five monoclonal antibodies possess another specificity and recognized both Gn and either the nucleoprotein (four of them) or the NSs protein (one). To validate the use of these monoclonal antibodies for diagnosis tests, a pool of monoclonal antibodies reacting with the structural proteins was prepared and used successfully to detect RVFV from cell culture as well as viral antigen-antibody complex in ELISA.

Animals↗
PubMed2005

Evaluation of a Crimean-Congo hemorrhagic fever virus recombinant antigen expressed by Semliki Forest suicide virus for IgM and IgG antibody detection in human and animal sera collected in Iran.

Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans by ticks or by direct contact with infected blood. It causes severe, often fatal, hemorrhagic diseases in humans but infection in animals is asymptomatic. CCHFV can spread from person to person and has caused many nosocomial outbreaks. Because the virus is very pathogenic for humans it must be manipulated in a biosafety level 4 (BSL4) laboratory, rendering the production of antigen for serological diagnosis difficult. To replace the native antigen, we produced a recombinant nucleoprotein expressed in mammalian cells via the recombinant Semliki Forest alphavirus replicon and developed an indirect immunofluorescence assay (IFA) as well as an enzyme-linked immunosorbent assay (ELISA) by immunocapture to detect IgM and IgG in human and animal serum. Using these methods, we analyzed clinical samples from human patients and sera from domestic animals collected in Iran and we show that this novel antigen provides a novel, sensitive and specific tool for CCHF diagnosis.

Animals↗
PubMed2003

Expression of the nucleoprotein of the Puumala virus from the recombinant Semliki Forest virus replicon: characterization and use as a potential diagnostic tool.

Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), micro -capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.

Animals↗
PubMed2002

Use of reverse transcriptase PCR in early diagnosis of Rift Valley fever.

Reverse transcriptase PCR (RT-PCR) for diagnosis of Rift Valley fever (RVF) was evaluated by using 293 human and animal sera sampled during an RVF outbreak in Mauritania in 1998. Results of the RT-PCR diagnostic method were compared with those of virus isolation (VI) and detection of immunoglobulin M (IgM) antibodies. Our results showed that RT-PCR is a specific, sensitive tool for RVF diagnosis in the early phase of the disease and that its results do not differ significantly from those obtained by VI. Moreover, the combined results of RT-PCR and IgM antibody detection were in 100% concordance with the results of VI.

Animals↗
PubMed2002

[Rift Valley fever: sporadic infection of French military personnel outside currently recognized epidemic zones].

For three years the arbovirus surveillance unit of the Tropical Medicine Institute of the French Army Medical Corps (French acronym IMTSSA) in Marseille, France has been investigating causes of benign non-malarial febrile syndromes in French military personnel serving outside mainland France. The methodology used in N'Djamena consisted of sending frozen specimens collected concomitant with viremia, to Marseille for culture. During the rainy season of 2001, specimens were collected from a total of 50 febrile soldiers. Cultures allowed isolation and identification of two strains of Rift Valley virus. The risk of contamination exists not only in the field but also in mainland hospital departments treating infected patients. Routine serological diagnosis for Rift Valley fever must be DISCUSSED for all patients in the field or returning from Africa.

Adult↗
PubMed2001

Single-tube and nested reverse transcriptase-polymerase chain reaction for detection of Rift Valley fever virus in human and animal sera.

Rift Valley fever (RVF) is an anthropozoonosis caused by a Phlebovirus (Bunyaviridae family) that has re-emerged recently in East and West Africa in 1997-1998. This emphasizes the need for early and rapid detection of the virus and an efficient surveillance system. To this goal, a single tube or a nested reverse transcriptase-polymerase chain reaction (RT-PCR) method focusing on the NSs coding region of the S segment was developed and used to detect the RVF virus (RVFV) genome, resulting respectively in the synthesis of 810 and 662 bp DNA amplimers. The assay was specific for RVFV and did not amplify any other phleboviruses known to circulate in sub-Saharan Africa. When serial dilutions of RVFV were artificially mixed with human normal serum, the minimal detection limits were 50 and 0.5 plaque forming units respectively using the simple and the nested RT-PCR. The RT-PCR method was efficient for the detection of RVFV RNA in the blood from experimentally RVFV-infected mice and lamb and the nested RT-PCR was found more sensitive than the virus isolation method. Additionally, this detection method was applied successfully for the diagnosis of human cases during the 1998 Mauritanian outbreak.

Animals↗
PubMed2001

Highly sensitive Taqman PCR detection of Puumala hantavirus.

An increasing number of clinical cases of Hantavirus infections have been reported from various regions in Asia, Europe and North America. Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped and possess a single-stranded trisegmented RNA genome of negative polarity. Rodents or insectivores are natural hosts of hantaviruses and transmit the virus to humans chiefly by aerosolisation. These viruses are the causative agents of haemorrhagic fever with renal and pulmonary syndromes. In the northeast of France, Puumala hantavirus causes, every year, more than 150 mild forms of haemorrhagic fever with a renal syndrome known as nephropathia epidemica. Serological tests may lack sensitivity for diagnosing early stages of infection and virus isolation is limited because it grows poorly in cell culture. Since reverse transcription (RT)-PCR amplification is an efficient method for detecting viral genomes in patient specimens, we developed an assay using a Taqman probe and compared it with the classical RT-PCR amplification. To achieve this goal, a Puumala strain was grown in Vero E6 cells and RNA extracted from the culture supernatant. We found that the semi-nested RT-PCR detected a minimal amount of 300 TCID(50) mL(-1), while the Taqman PCR allowed detection of less than 10 TCID(50) mL(-1 )and provided a quantitative analysis.

Animals↗
PubMed2001

Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds.

The Rift Valley fever virus (RVFV), a member of the genus Phlebovirus (family Bunyaviridae) is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using TaqMan technology and targeting the nonstructural protein-coding region was developed, and primers and a probe were designed. After optimization of the amplification reaction and establishment of a calibration curve with synthetic RNA transcribed in vitro from a plasmid containing the gene of interest, real-time RT-PCR was assessed with samples consisting of RVFV from infected Vero cells. The method was found to be specific for RVFV, and it was successfully applied to the detection of the RVFV genome in animal sera infected with RVFV as well as to the assessment of the efficiency of various drugs (ribavirin, alpha interferon, 6-azauridine, and glycyrrhizin) for antiviral activity. Altogether, the results indicated a strong correlation between the infectious virus titer and the amount of viral genome assayed by real time RT-PCR. This novel method could be of great interest for the rapid diagnosis and screening of new antiviral compounds, as it is sensitive and time saving and does not require manipulation of infectious material.

Animals↗
PubMed2001

Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs.

Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR(-/-) mice lacking the alpha/beta interferon (IFN-alpha/beta) receptor but remained attenuated in IFN-gamma receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-alpha/beta production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-alpha/beta and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-alpha/beta production, we infected susceptible IFNAR(-/-) mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-alpha/beta production. These results demonstrate that the ability of RVFV to inhibit IFN-alpha/beta production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist.

Animals↗
PubMed2000

Pathogen-specific resistance to Rift Valley fever virus infection is induced in mosquito cells by expression of the recombinant nucleoprotein but not NSs non-structural protein sequences.

Rift Valley fever virus (RVFV) is an arbovirus of the BUNYAVIRIDAE: family, causing recurrent disease outbreaks in Africa. Natural vertebrate hosts include cattle and humans. Several mosquito species belonging to the AEDES: and CULEX: genera act as vectors of this phlebovirus. To test whether pathogen-derived resistance against RVFV could be induced by expressing genomic sequences in mosquito cells, as has been shown for La Crosse and dengue 2 viruses, we generated various recombinant Semliki Forest viruses expressing the S segment (or its genes) in the genomic or antigenomic sense. Expression of the N but not the NSs gene interfered with the production of RVFV in mosquito cells and this phenomenon was RNA- but not protein-dependent. These results raise questions on the molecular mechanisms involved in virus resistance.

Aedes↗
PubMed2000

The S segment of rift valley fever phlebovirus (Bunyaviridae) carries determinants for attenuation and virulence in mice.

Unlike all the other Rift Valley fever virus strains (Bunyaviridae, Phlebovirus) studied so far, clone 13, a naturally attenuated virus, does not form the filaments composed of the NSs nonstructural protein in the nuclei of infected cells (R. Muller, J. F. Saluzzo, N. Lopez, T. Drier, M. Turell, J. Smith, and M. Bouloy, Am. J. Trop. Med. Hyg. 53:405-411, 1995). This defect is correlated with a large in-frame deletion in the NSs coding region of the S segment of the tripartite genome. Here, we show that the truncated NSs protein of clone 13 is expressed and remains in the cytoplasm, where it is degraded rapidly by the proteasome. Through the analysis of reassortants between clone 13 and a virulent strain, we localized the marker(s) of attenuation in the S segment of this attenuated virus. This result raises questions regarding the role of NSs in pathogenesis and highlights, for the first time in the Bunyaviridae family, a major role of the S segment in virulence and attenuation, possibly associated with a defect in the nonstructural protein.

Animals↗
PubMed2000

Infections by viruses of the families Bunyaviridae and Filoviridae.

Rift Valley fever is the most important bunyaviral disease of animals in Africa. The virus, transmitted by mosquitoes, causes abortions and mortality in young animals in addition to haemorrhagic fevers in humans. Although vaccines against this virus are available, the uses of these vaccines are limited because of deleterious effects or incomplete protection, justifying further studies to improve the existing vaccines or to develop others. Nairobi sheep disease is transmitted by ticks. The disease is endemic in East Africa and sporadic cases are reported in India and Sri Lanka. Other viruses transmitted by mosquitoes or midges are teratogenic in cattle or sheep, these include Akabane and related viruses in Asia, Australia and the Middle East, and Cache Valley in North America. The Marburg and Ebola viruses of the genus Filovirus are associated with epidemics in Central Africa with high fatality rates in humans; some outbreaks were related to contact with monkeys. Another subtype of Ebola virus was first described in a quarantine facility in the United States of America among cynomolgus monkeys (Macaca fascicularis) from the Philippines. The reservoir of these viruses remains unknown.

Animals↗
PubMed2000

[Hantavirus: recent data and review of the literature].

Hantavirus (Bunyaviridae family) are present worldwide and are the causative agent of haemorrhagic fever with renal or pulmonary syndrome. These viruses are transmitted by rodents with asymptomatic infection which thus act as an excellent reservoir. Molecular epidemiology indicates that Hantavirus may have evolved in three ways: mutations within the genome, reassortment of the segmented genome between two closely related Hantavirus, and genomic recombination, a relatively rare phenomenon among negative stranded RNA viruses. Each virus is carried primarily by a specific rodent host. This observation is confirmed by phylogenetic analyses: the evolution tree of the different Hantavirus, based on viral genomic sequences has been shown to mirror the evolution tree of their specific rodents, based on sequences of mitochondrial DNA. Altogether these data suggest that an ancestral Hantavirus infected a specific rodent, early during evolution, and was subsequently submitted to the same evolutionary pressure as the rodent host.

Animals↗
PubMed1999

The Rift Valley fever virus nonstructural protein NSs is phosphorylated at serine residues located in casein kinase II consensus motifs in the carboxy-terminus.

The S segment of Rift Valley fever virus (Bunyaviridae, Phlebovirus) codes for two proteins, the nucleoprotein N and the nonstructural protein NSs. The NSs protein is a phosphoprotein of unknown function that is localized in the cytoplasm and the nuclei of infected cells where it forms filamentous structures. To characterize further the protein expressed in VC10 cells infected with the MP12 strain, we analyzed its phosphorylation states and showed that phosphorylated forms were found in both compartments. Cytoplasmic and nuclear NSs were phosphorylated only at serine residues. Phosphopeptide mapping and molecular analysis of mutants obtained by site-directed mutagenesis allowed us to map the major phosphorylation sites of nuclear and cytoplasmic forms of NSs to serine residues 252 and 256, located at the carboxy-terminus in consensus sequences for casein kinase II. A similar map was obtained when the protein was purified from mosquito cells infected with MP12. In addition, we showed that the purified unphosphorylated NSs protein expressed from pET-NSs plasmid in a coupled transcription-translation reaction containing Escherichia coli S30 extracts did not possess autophosphorylation activity but was phosphorylated in vitro after incubation with recombinant casein kinase II.

Amino Acid Motifs↗
PubMed1999

Transient gene expression in mammalian and mosquito cells using a recombinant Semliki Forest virus expressing T7 RNA polymerase.

In this report, we describe a novel recombinant Semliki Forest virus (SFV) expressing T7 RNA polymerase (T7-RP), which was shown to drive transient expression of the chloramphenicol acetyltransferase (cat) gene in mammalian and mosquito cells after transfection of plasmids carrying the reporter gene under the control of the T7 promoter. To our knowledge, this is the first description of a T7-RP-based expression in mosquito cells. Expression of the cat gene was significantly enhanced in mammalian cells by inserting the sequence of the encephalomyocarditis virus internal ribosome entry site between the T7 promoter and the 5' end of the cat gene. In mosquito cells, the level of chloramphenicol acetyltransferase activity was increased by flanking the cat gene with the Autographa californica baculovirus p10 gene 5' and 3' untranslated regions. SFV expressing T7-RP appears, therefore, to be an alternative to other virus-based gene-expression systems in mammalian cells and a powerful tool for protein expression in mosquito cells.

Aedes↗
PubMed1999

Genetic reassortment of Rift Valley fever virus in nature.

Rift Valley fever virus (RVFV), a phlebovirus of the Bunyaviridae family, is an arthropod-borne virus which emerges periodically throughout Africa, emphasizing that it poses a major threat for animal and human populations. To assess the genetic variability of RVFV, several isolates from diverse localities of Africa were investigated by means of reverse transcription-PCR followed by direct sequencing of a region of the small (S), medium (M), and large (L) genomic segments. Phylogenetic analysis showed the existence of three major lineages corresponding to geographic variants from West Africa, Egypt, and Central-East Africa. However, incongruences detected between the L, M, and S phylogenies suggested that genetic exchange via reassortment occurred between strains from different lineages. This hypothesis, depicted by parallel phylogenies, was further confirmed by statistical tests. Our findings, which strongly suggest exchanges between strains from areas of endemicity in West and East Africa, strengthen the potential existence of a sylvatic cycle in the tropical rain forest. This also emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines in areas of endemicity.

Genetic Variation↗
PubMed1999

The carboxy-terminal acidic domain of Rift Valley Fever virus NSs protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein.

The ambisense S segment of Rift Valley fever (RVF) virus (a phlebovirus in the Bunyaviridae family) codes for two proteins: the viral complementary-sense RNA for the N nucleoprotein and the genomic-sense RNA for the nonstructural protein NSs. Except for the fact that the NSs protein is phosphorylated and forms filamentous structures in the nuclei of infected cells (R. Swanepoel and N. K. Blackburn, J. Gen. Virol. 34:557-561, 1977), its role is poorly understood, especially since the replication cycle of all these viruses takes place in the cytoplasm. To investigate the mechanisms involved in filament formation, we expressed NSs in mammalian cells via a recombinant Semliki Forest virus and demonstrated that the protein alone was able to form structures similar to those observed in RVF virus-infected cells, indicating that the presence of other RVF virus proteins is not required for filament formation. The yeast two-hybrid system was used to show that the protein interacts with itself and to map the interacting domains. Various deletion and substitution mutants were constructed, and the mutant proteins were analyzed by immunoprecipitation, Western blotting and immunofluorescence. These experiments indicated that the 10 to 17 amino acids of the carboxy-terminal domain were involved in self-association of the protein and that deletion of this acidic carboxy-terminal domain prevents the protein from forming filaments but does not affect its nuclear localization. The role of two phosphorylation sites present in this domain was also investigated, but they were not found to have a major influence on the formation of the nuclear filament.

Amino Acid Sequence↗