Alkhurma virus (ALKV) is a recently discovered class-4 flavivirus that was responsible for severa... more Alkhurma virus (ALKV) is a recently discovered class-4 flavivirus that was responsible for several cases of severe haemorrhagic fever in humans in Saudi Arabia. It has been shown for other flaviviruses that processing of the viral polyprotein is partly due to the virus-encoded NS2B/NS3 trypsin-like serine protease. As the viral proteinase plays a critical role in the virus replication cycle, it represents one of the main targets for antiviral therapy against members of the Flavivirus genus. We report here on the identification of the ALKV NS2B and NS3 domains and the expression and purification of a catalytically active viral protease as a hexahistidine recombinant protein. Its enzymatic properties were characterized in vitro using a para-nitroanilide substrate. This constitutes the first characterization of the proteinase from a class-4 flavivirus. Our results indicate that the association of NS3 with a short segment of NS2B is necessary and sufficient for protease activity. The de...
Emergence of new viruses is dependent on the intrinsic and extrinsic constraints exerting on vira... more Emergence of new viruses is dependent on the intrinsic and extrinsic constraints exerting on viral evolution. Intrinsic constraints are semantic and grammatical in nature. They are analysed here in reference to Hamming's spaces, driving to a new interpretation of the evolution of the quasispecies of Manfred Eigen. Extrinsic constraints are relevant to the fact that viral evolution is always a co-evolution story, with two or three partners implicated (the viruses, their hosts and eventually their vectors). They imply that viral phylogenies are disconnected, and viruses constitute a polyphyletic system. A possible consequence is that potential viral families are already known, or at least are present in nature, in such a manner that the frames for future viral evolution are already determined and that the probability for the emergence of a new frame is nil. Nevertheless, the emergence of new pathogens in the existing frames remain possible.
Three strains of yellow fever virus (YFV) were isolated in 1982 in The Ivory Coast, one from a hu... more Three strains of yellow fever virus (YFV) were isolated in 1982 in The Ivory Coast, one from a human case and two from Aedes luteocephalus, during and subsequent to an epidemic. The complete genomic sequence of the human strain was determined and compared to that of the 1927 Asibi strain of YFV. The divergence observed was on average of 8.3%, ranging from 5.5 to 11.7% in the coding region. The transitions to transversions ratio was 5.9. Most mutations (84.3%) occurred on the third position of the codons, with synonymous mutations representing 92.5%. However, when partial sequences representing 60% of each genome were compared, homology between the three Ivory Coast strains was greater than 99%. These results demonstrate the homogeneity of the virus strains circulating in different hosts and vectors in a limited geographical region and validate the concept of topotype in viral quasi-species.
Médecine tropicale : revue du Corps de santé colonial, 2005
This study was carried out in 2003 to detected serological evidence of West Nile virus infection ... more This study was carried out in 2003 to detected serological evidence of West Nile virus infection in 190 Army horses kept nearby French troops stationed in Southeast France and in Africa (Chad, Côte d'Ivoire and Senegal). Both IgG and IgM antibodies were searched for using an ELISA assay. Specifiity of IgG antibodies was determined by western blot and plaque reduction seroneutraization. Finding showed that 79% of the Army horses (n=96) tested in Africa presented specific IgG antibodies. All horses that were seropositive for IgG were seronegative for IgM. None of the Army horses (n=94) tested in the Southeast France were seropositive for West Nile virus. This study indicates that West Nile virus has circulated in all three African countries but not recently. It also underscores the value of western blotting as a rapid, specific confirmation technique that could eliminate the need to use plaque reduction seroneutralization.
Cell fusing agent virus (CFAV) is a positive strand RNA insect virus first isolated from a mosqui... more Cell fusing agent virus (CFAV) is a positive strand RNA insect virus first isolated from a mosquito cell line. Based on viral morphology, phenotypic and phylogenetic studies, CFAV had been tentatively assigned to the genus Flavivirus (family Flaviviridae). The determination of the CFAV polyprotein complete sequence showed a putative serine protease domain analogue to the flaviviral NS2B/NS3 complex. This complex had been extensively studied, because it represented one of the main targets for antiflavivirus therapy development. We report herein the biochemical characterization of CFAV DeltaNS2B-NS3pro protease complex. CFAV polyprotein sequence was computationally analysed to identify the amino-acid regions involved in protease activity. We designed, expressed and purified a catalytically active protease whose enzymatic properties were determined using fluorogenic substrates. Our results showed that, despite the low level of conservation of its amino-acid sequence, CFAV protease exhibited physico-chemical properties of other flaviviruses (high pH value requirement for optimal activity, inhibition by salt and preference for substrates featuring a basic residue at P(1) position).
Toscana virus (TOSV) is a sandfly-borne phlebovirus causing meningitis and encephalitis during th... more Toscana virus (TOSV) is a sandfly-borne phlebovirus causing meningitis and encephalitis during the summer period. A significant proportion of infection results in asymptomatic or pauci-symptomatic forms. Although seroprevalence studies had been conducted in Italy, Spain, Greece, and Cyprus, no data were available from France at the outset of this study. We present here results of seroprevalence studies conducted in volunteer blood donors and in patients presenting with central nervous system (CNS) infections. Twelve percent of sera from blood donors and 18.9% of sera from patients hospitalized for CNS infection contained immunoglobulin G (IgG) reacting against TOSV or TOSV-related phleboviruses. This study confirms that TOSV and possibly TOSV-related phleboviruses actively circulates in southeastern France and demonstrates that a significant proportion of healthy blood donors and patients with CNS infections have a history of TOSV or TOSV-related phlebovirus infection.
We report herein the study of the cleavage fragments generated by autoproteolysis of the St. Loui... more We report herein the study of the cleavage fragments generated by autoproteolysis of the St. Louis encephalitis virus recombinant protease. The cleavage sites leading to truncated forms were identified by microsequencing, which revealed an unexpected altered specificity of the recombinant proteinase towards unusual sequences.
A large chikungunya virus (CHIKV) outbreak emerged in 2005-2006 in the Indian Ocean islands, incl... more A large chikungunya virus (CHIKV) outbreak emerged in 2005-2006 in the Indian Ocean islands, including Comoros, Mayotte, Mauritius, the Seychelles, and particularly in Reunion Island where 35% of 770,000 inhabitants were infected in 6 months. More recently, circulation of the virus has been documented in Madagascar and in India where CHIKV is spreading rapidly. CHIKV-infected visitors have returned home to nonendemic regions from these islands. We conducted a 14-month prospective observational study on the clinical aspects of CHIKV infection imported to Marseilles, France, in travelers returning from the Indian Ocean islands. A total of 47 patients have been diagnosed with imported CHIKV infection confirmed by serology, reverse transcription-polymerase chain reaction, and/or viral culture. At the early stage of the disease (within 10 days of the disease onset), fever was present in 45 of 47 patients. A rash was present in the first week in 25 cases. All patients suffered with arthritis. The most frequently affected joints were fingers, wrists, toes, and ankles. Eight patients were hospitalized during the acute stage, including 2 severe life-threatening cases. A total of 38 patients remained symptomatic after the tenth day with chronic peripheral rheumatism, characterized by severe joint pain and multiple tenosynovitis, with a dramatically limited ability to ambulate and carry out activities in daily life. Three patients were hospitalized at this stage for severe persistent handicap. Follow-up demonstrated slow improvement in joint pain and stiffness despite symptomatic treatment, mainly antiinflammatory and analgesic drugs. In the current series we describe 2 stages of the disease, an initial severe febrile and eruptive polyarthritis, followed by disabling peripheral rheumatism that can persist for months. We point out the possibility of transitory peripheral vascular disorders during the second stage and the occasional benefit of short-term corticosteroids. As CHIKV could spread throughout the world, all physicians should be prepared to encounter this arboviral infection.
Chikungunya virus (CHIKV), a member of the alphavirus genus, is of considerable public health con... more Chikungunya virus (CHIKV), a member of the alphavirus genus, is of considerable public health concern in Southeast Asian and African countries. However, despite serological evidence, the diagnosis of this arthropod-borne human disease is confirmed infrequently and needs to be improved. In fact, illness caused by CHIKV can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms, and laboratory confirmation of suspected cases is required to launch control measures during an epidemic. Moreover, no quantitative molecular tool is described to study CHIKV replication or detection in clinical samples and cell culture supernatants. In this study, a specific and sensitive CHIKV one-step TaqMan RT-PCR assay was developed as a tool for the diagnosis of African CHIKV as well as a rapid indicator of active infection by quantifying viral load. This study also showed that a simple heat viral RNA release during the reverse transcription step constituted an alternative to the conventional RNA extraction method.
This study reports the first isolation and partial genetic characterization of Chikungunya virus ... more This study reports the first isolation and partial genetic characterization of Chikungunya virus (CHIKV) from patients during a 2006-2007 dengue-like syndrome outbreak in Gabon. The isolated viruses were phylogenetically close to strains isolated in the Democratic Republic of the Congo 7 years ago and to strains isolated more recently in Cameroon. These results indicate a continuing circulation of a genetically stable CHIKV population during 7 years in Central Africa.
Dengue shock syndrome (DSS), the major life threatening outcome of severe dengue disease, which o... more Dengue shock syndrome (DSS), the major life threatening outcome of severe dengue disease, which occurs in some patients in the course of dengue infection, is the consequence of plasma leakage in the microvascular territories. Data from clinical and in vitro studies suggest that an inadequate immunological response is partly responsible for the pathophysiology of DSS, but few is known concerning the consequences of direct infection of endothelial cells by dengue virus per se. In this study, an attempt was made to study the response of two microvascular human cell lines originating, respectively, from liver and dermis to infection by a dengue type 2 virus, by analyzing the virus-induced modulation of functional markers. It is shown that the two microvascular cell lines exhibit both common and specific behaviors upon infection. In particular, LSEC and HMEC-1 replicate efficiently the low-passage virus and respond to infection by over-producing inflammatory mediators involved in the cross talk with circulating immune cells. However, direct infection modulates differently the cell surface expression of molecules critically involved in the interactions between endothelial and inflammatory cells. ICAM-1 and HLA-I are up regulated as a consequence of infection in LSEC whereas direct infection results in downregulation of ICAM-1 in HMEC-1. The present results show that infection of human microvascular cells by unadapted dengue virus results in both common and specific activation patterns depending likely on the tissue origin of the cells, thus suggesting that endothelia from different territories may contribute differently to the pathophysiological events in the course of dengue infection.
Alkhurma virus (ALKV) is a recently discovered class-4 flavivirus that was responsible for severa... more Alkhurma virus (ALKV) is a recently discovered class-4 flavivirus that was responsible for several cases of severe haemorrhagic fever in humans in Saudi Arabia. It has been shown for other flaviviruses that processing of the viral polyprotein is partly due to the virus-encoded NS2B/NS3 trypsin-like serine protease. As the viral proteinase plays a critical role in the virus replication cycle, it represents one of the main targets for antiviral therapy against members of the Flavivirus genus. We report here on the identification of the ALKV NS2B and NS3 domains and the expression and purification of a catalytically active viral protease as a hexahistidine recombinant protein. Its enzymatic properties were characterized in vitro using a para-nitroanilide substrate. This constitutes the first characterization of the proteinase from a class-4 flavivirus. Our results indicate that the association of NS3 with a short segment of NS2B is necessary and sufficient for protease activity. The de...
Emergence of new viruses is dependent on the intrinsic and extrinsic constraints exerting on vira... more Emergence of new viruses is dependent on the intrinsic and extrinsic constraints exerting on viral evolution. Intrinsic constraints are semantic and grammatical in nature. They are analysed here in reference to Hamming's spaces, driving to a new interpretation of the evolution of the quasispecies of Manfred Eigen. Extrinsic constraints are relevant to the fact that viral evolution is always a co-evolution story, with two or three partners implicated (the viruses, their hosts and eventually their vectors). They imply that viral phylogenies are disconnected, and viruses constitute a polyphyletic system. A possible consequence is that potential viral families are already known, or at least are present in nature, in such a manner that the frames for future viral evolution are already determined and that the probability for the emergence of a new frame is nil. Nevertheless, the emergence of new pathogens in the existing frames remain possible.
Three strains of yellow fever virus (YFV) were isolated in 1982 in The Ivory Coast, one from a hu... more Three strains of yellow fever virus (YFV) were isolated in 1982 in The Ivory Coast, one from a human case and two from Aedes luteocephalus, during and subsequent to an epidemic. The complete genomic sequence of the human strain was determined and compared to that of the 1927 Asibi strain of YFV. The divergence observed was on average of 8.3%, ranging from 5.5 to 11.7% in the coding region. The transitions to transversions ratio was 5.9. Most mutations (84.3%) occurred on the third position of the codons, with synonymous mutations representing 92.5%. However, when partial sequences representing 60% of each genome were compared, homology between the three Ivory Coast strains was greater than 99%. These results demonstrate the homogeneity of the virus strains circulating in different hosts and vectors in a limited geographical region and validate the concept of topotype in viral quasi-species.
Médecine tropicale : revue du Corps de santé colonial, 2005
This study was carried out in 2003 to detected serological evidence of West Nile virus infection ... more This study was carried out in 2003 to detected serological evidence of West Nile virus infection in 190 Army horses kept nearby French troops stationed in Southeast France and in Africa (Chad, Côte d'Ivoire and Senegal). Both IgG and IgM antibodies were searched for using an ELISA assay. Specifiity of IgG antibodies was determined by western blot and plaque reduction seroneutraization. Finding showed that 79% of the Army horses (n=96) tested in Africa presented specific IgG antibodies. All horses that were seropositive for IgG were seronegative for IgM. None of the Army horses (n=94) tested in the Southeast France were seropositive for West Nile virus. This study indicates that West Nile virus has circulated in all three African countries but not recently. It also underscores the value of western blotting as a rapid, specific confirmation technique that could eliminate the need to use plaque reduction seroneutralization.
Cell fusing agent virus (CFAV) is a positive strand RNA insect virus first isolated from a mosqui... more Cell fusing agent virus (CFAV) is a positive strand RNA insect virus first isolated from a mosquito cell line. Based on viral morphology, phenotypic and phylogenetic studies, CFAV had been tentatively assigned to the genus Flavivirus (family Flaviviridae). The determination of the CFAV polyprotein complete sequence showed a putative serine protease domain analogue to the flaviviral NS2B/NS3 complex. This complex had been extensively studied, because it represented one of the main targets for antiflavivirus therapy development. We report herein the biochemical characterization of CFAV DeltaNS2B-NS3pro protease complex. CFAV polyprotein sequence was computationally analysed to identify the amino-acid regions involved in protease activity. We designed, expressed and purified a catalytically active protease whose enzymatic properties were determined using fluorogenic substrates. Our results showed that, despite the low level of conservation of its amino-acid sequence, CFAV protease exhibited physico-chemical properties of other flaviviruses (high pH value requirement for optimal activity, inhibition by salt and preference for substrates featuring a basic residue at P(1) position).
Toscana virus (TOSV) is a sandfly-borne phlebovirus causing meningitis and encephalitis during th... more Toscana virus (TOSV) is a sandfly-borne phlebovirus causing meningitis and encephalitis during the summer period. A significant proportion of infection results in asymptomatic or pauci-symptomatic forms. Although seroprevalence studies had been conducted in Italy, Spain, Greece, and Cyprus, no data were available from France at the outset of this study. We present here results of seroprevalence studies conducted in volunteer blood donors and in patients presenting with central nervous system (CNS) infections. Twelve percent of sera from blood donors and 18.9% of sera from patients hospitalized for CNS infection contained immunoglobulin G (IgG) reacting against TOSV or TOSV-related phleboviruses. This study confirms that TOSV and possibly TOSV-related phleboviruses actively circulates in southeastern France and demonstrates that a significant proportion of healthy blood donors and patients with CNS infections have a history of TOSV or TOSV-related phlebovirus infection.
We report herein the study of the cleavage fragments generated by autoproteolysis of the St. Loui... more We report herein the study of the cleavage fragments generated by autoproteolysis of the St. Louis encephalitis virus recombinant protease. The cleavage sites leading to truncated forms were identified by microsequencing, which revealed an unexpected altered specificity of the recombinant proteinase towards unusual sequences.
A large chikungunya virus (CHIKV) outbreak emerged in 2005-2006 in the Indian Ocean islands, incl... more A large chikungunya virus (CHIKV) outbreak emerged in 2005-2006 in the Indian Ocean islands, including Comoros, Mayotte, Mauritius, the Seychelles, and particularly in Reunion Island where 35% of 770,000 inhabitants were infected in 6 months. More recently, circulation of the virus has been documented in Madagascar and in India where CHIKV is spreading rapidly. CHIKV-infected visitors have returned home to nonendemic regions from these islands. We conducted a 14-month prospective observational study on the clinical aspects of CHIKV infection imported to Marseilles, France, in travelers returning from the Indian Ocean islands. A total of 47 patients have been diagnosed with imported CHIKV infection confirmed by serology, reverse transcription-polymerase chain reaction, and/or viral culture. At the early stage of the disease (within 10 days of the disease onset), fever was present in 45 of 47 patients. A rash was present in the first week in 25 cases. All patients suffered with arthritis. The most frequently affected joints were fingers, wrists, toes, and ankles. Eight patients were hospitalized during the acute stage, including 2 severe life-threatening cases. A total of 38 patients remained symptomatic after the tenth day with chronic peripheral rheumatism, characterized by severe joint pain and multiple tenosynovitis, with a dramatically limited ability to ambulate and carry out activities in daily life. Three patients were hospitalized at this stage for severe persistent handicap. Follow-up demonstrated slow improvement in joint pain and stiffness despite symptomatic treatment, mainly antiinflammatory and analgesic drugs. In the current series we describe 2 stages of the disease, an initial severe febrile and eruptive polyarthritis, followed by disabling peripheral rheumatism that can persist for months. We point out the possibility of transitory peripheral vascular disorders during the second stage and the occasional benefit of short-term corticosteroids. As CHIKV could spread throughout the world, all physicians should be prepared to encounter this arboviral infection.
Chikungunya virus (CHIKV), a member of the alphavirus genus, is of considerable public health con... more Chikungunya virus (CHIKV), a member of the alphavirus genus, is of considerable public health concern in Southeast Asian and African countries. However, despite serological evidence, the diagnosis of this arthropod-borne human disease is confirmed infrequently and needs to be improved. In fact, illness caused by CHIKV can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms, and laboratory confirmation of suspected cases is required to launch control measures during an epidemic. Moreover, no quantitative molecular tool is described to study CHIKV replication or detection in clinical samples and cell culture supernatants. In this study, a specific and sensitive CHIKV one-step TaqMan RT-PCR assay was developed as a tool for the diagnosis of African CHIKV as well as a rapid indicator of active infection by quantifying viral load. This study also showed that a simple heat viral RNA release during the reverse transcription step constituted an alternative to the conventional RNA extraction method.
This study reports the first isolation and partial genetic characterization of Chikungunya virus ... more This study reports the first isolation and partial genetic characterization of Chikungunya virus (CHIKV) from patients during a 2006-2007 dengue-like syndrome outbreak in Gabon. The isolated viruses were phylogenetically close to strains isolated in the Democratic Republic of the Congo 7 years ago and to strains isolated more recently in Cameroon. These results indicate a continuing circulation of a genetically stable CHIKV population during 7 years in Central Africa.
Dengue shock syndrome (DSS), the major life threatening outcome of severe dengue disease, which o... more Dengue shock syndrome (DSS), the major life threatening outcome of severe dengue disease, which occurs in some patients in the course of dengue infection, is the consequence of plasma leakage in the microvascular territories. Data from clinical and in vitro studies suggest that an inadequate immunological response is partly responsible for the pathophysiology of DSS, but few is known concerning the consequences of direct infection of endothelial cells by dengue virus per se. In this study, an attempt was made to study the response of two microvascular human cell lines originating, respectively, from liver and dermis to infection by a dengue type 2 virus, by analyzing the virus-induced modulation of functional markers. It is shown that the two microvascular cell lines exhibit both common and specific behaviors upon infection. In particular, LSEC and HMEC-1 replicate efficiently the low-passage virus and respond to infection by over-producing inflammatory mediators involved in the cross talk with circulating immune cells. However, direct infection modulates differently the cell surface expression of molecules critically involved in the interactions between endothelial and inflammatory cells. ICAM-1 and HLA-I are up regulated as a consequence of infection in LSEC whereas direct infection results in downregulation of ICAM-1 in HMEC-1. The present results show that infection of human microvascular cells by unadapted dengue virus results in both common and specific activation patterns depending likely on the tissue origin of the cells, thus suggesting that endothelia from different territories may contribute differently to the pathophysiological events in the course of dengue infection.
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Papers by Hugues Tolou