Chikungunya virus, southeastern France.

In September 2010, autochthonous transmission of chikungunya virus was recorded in southeastern France, where the Aedes albopictus mosquito vector is present. Sequence analysis of the viral genomes of imported and autochthonous isolates indicated new features for the potential emergence and spread of the virus in Europe.

Identification of cellular proteome modifications in response to West Nile virus infection.

Flaviviruses are positive-stranded RNA viruses that are a public health problem because of their widespread distribution and their ability to cause a variety of diseases in humans. West Nile virus is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. Clinical manifestations of West Nile virus infection are diverse, and their pathogenic mechanisms depend on complex virus-cell interactions. In the present work, we used proteomics technology to analyze early Vero cell response to West Nile infection. The differential proteomes were resolved 24 h postinfection using two-dimensional DIGE followed by mass spectrometry identification. Quantitative analysis (at least 2-fold quantitative alteration, p < 0.05) revealed 127 differentially expressed proteins with 68 up-regulated proteins and 59 down-regulated proteins of which 93 were successfully identified. The implication for mammalian cellular responses to this neurotropic flavivirus infection was analyzed and made possible more comprehensive characterization of the virus-host interactions involved in pathogenesis. The present study thus provides large scale protein-related information that should be useful for understanding how the host metabolism is modified by West Nile infection and for identifying new potential targets for antiviral therapy.

Real-time reverse-transcription loop-mediated isothermal amplification for rapid detection of rift valley Fever virus.

The development and validation of a one-step, single-tube, real-time accelerated reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the L RNA segment of Rift Valley fever virus (RVFV) are described. The assay was performed at a constant temperature (63 degrees C), with a real-time follow-up using a LightCycler and a double-stranded-DNA-intercalating fluorochrome. The assay is highly sensitive and comparable to real-time RT-PCR, with a detection limit of approximately 10 RNA copies per assay. However, the RT-LAMP assay is much faster than traditional RT-PCR and generates results in <30 min for most diluted samples. The specificity of the primers was established using other, related arboviruses as well as virus-containing and virus-free sera. The RT-LAMP assay reported here is thus a valuable tool for the rapid detection of RVFV in field diagnostic laboratories.

Expression and biochemical characterization of nsP2 cysteine protease of Chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemic fever, rash and polyarthralgia in Africa and Asia. Although it is known since the 1950s, new epidemiological and clinical features reported during the recent outbreak in the Indian Ocean can be regarded as the emergence of a new disease. Numerous severe forms of the infection have been described that put emphasis on the lack of efficient antiviral therapy. Among the virus-encoded enzymes, nsP2 constitutes an attractive target for the development of antiviral drugs. It is a multifunctional protein of approximately 90 kDa with a helicase motif in the N-terminal portion of the protein while the papain-like protease activity resides in the C-terminal portion. The nsP2 proteinase is an essential enzyme whose proteolytic activity is critical for virus replication. In this work, a recombinant CHIKV nsP2pro and a C-terminally truncated variant were expressed in Escherichia coli and purified by metal-chelate chromatography. The enzymatic properties of the proteinase were then determined using specific synthetic fluorogenic substrates. This study constitutes the first characterization of a recombinant CHIKV nsP2 cysteine protease, which may be useful for future drug screening.

Improvement of the purification of Saint Louis encephalitis virus NS2B-NS3 recombinant protease expressed in Escherichia coli.

The NS2B-NS3 serine protease of Saint Louis Encephalitis virus (SLEV), a potential target for antiviral drug design, has been over-expressed as a recombinant His-tag protein in Escherichia coli for future structural determination. The production process resulted in a soluble protease with co-purification of DnaK, a bacterial molecular chaperone already described in E. coli protein expression. Two approaches were tested to remove this specific contaminant. The fusion protein bound to the purification resin was washed with MgATP plus soluble denatured E. coli proteins before elution, but this method proved to be poorly efficient due to a substantial loss of the targeted recombinant protease. After the immobilized metal affinity chromatography step, the use of gel permeation chromatography with addition of arginine in the mobile phase led to effective separation of the native viral protease from the DnaK aggregates. By this way, SLEV DeltaNS2B-NS3pro protease was purified as a functional protein with a purity greater than 90% suitable for crystallization attempts.

Unexpected altered specificity is responsible for St. Louis encephalitis virus recombinant protease autoproteolysis.

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.

Functional characterization of the NS2B/NS3 protease complex from seven viruses belonging to different groups inside the genus Flavivirus.

The genus Flavivirus, family Flaviviridae, comprises more than 70 viruses. Many of them cause severe, potentially fatal, human diseases. Human vaccines are available for only three viruses and no effective antiviral drug is available. In order to limit the consequences of infections with flaviviruses, a promising approach consists in developing specific compounds that target the virus-encoded NS2B/NS3 protease complex, which is crucial for the viral polyprotein processing. In order to develop such compounds active as antiviral drugs against several flaviviruses, identification of biochemical properties shared by proteases from different viruses is essential. In this work, the functional similarity between the proteases from seven flaviviruses belonging to different major groups was addressed by characterizing their enzymatic properties. For each virus, a catalytically active recombinant protease was designed and expressed as a hexahistidine-tagged protein. Chromogenic and fluorogenic substrates were used to identify optimal conditions for proteolysis. Our study identified important physico-chemical properties shared by all the seven proteases we studied (high pH value requirement for optimal activity, inhibition of substrate processing by salt). However, it also evidenced slight differences in biochemical properties of the flaviviral proteases, which could sustain heterogeneous sensitivity to future inhibitors.

Identification and enzymatic characterization of NS2B-NS3 protease of Alkhurma virus, a class-4 flavivirus.

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 developed system could help to identify or design inhibitors potentially active as antiviral drugs against ALKV and other pathogenic flaviviruses.

Development of a TaqMan RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses.

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.

Evidence for in vitro falsely-primed cDNAs that prevent specific detection of virus negative strand RNAs in dengue-infected cells: improvement by tagged RT-PCR.

The identification of cell types replicating dengue viruses is an important step towards the understanding of the pathophysiology of dengue severe forms. Since the detection of negative strand viral RNAs is the more reliable marker of active replication for single-strand positive sense RNA viruses, we reassessed the specificity of RT-PCR assays already developed to detect dengue negative strand RNAs. Studying mammalian Vero cells infected by a dengue-2 strain, it was shown that falsely-primed cDNAs are generated in vitro during the reverse transcription step and are amplified subsequently by PCR. Since this may compromise the specificity of existing RT-PCR systems, we developed a tagged RT-PCR assay and addressed the role of some critical factors in such a system. Optimization of the negative strand-specific tagged RT-PCR allowed to resolve the problems due to the PCR amplification of falsely-primed cDNAs. Using this assay it was possible to detect specifically negative strand RNAs as soon as 3h after Vero cells have been exposed to the dengue-2 strain and we showed that this system is highly specific. Thus, the present dengue negative strand-specific tagged RT-PCR assay may help to reassess viral replication in the context of dengue pathophysiology.

Genome-wide expression profiling deciphers host responses altered during dengue shock syndrome and reveals the role of innate immunity in severe dengue.

BACKGROUND: Deciphering host responses contributing to dengue shock syndrome (DSS), the life-threatening form of acute viral dengue infections, is required to improve both the differential prognosis and the treatments provided to DSS patients, a challenge for clinicians. METHODOLOGY/PRINCIPAL FINDINGS: Based on a prospective study, we analyzed the genome-wide expression profiles of whole blood cells from 48 matched Cambodian children: 19 progressed to DSS while 16 and 13 presented respectively classical dengue fever (DF) or dengue hemorrhagic fever grades I/II (DHF). Using multi-way analysis of variance (ANOVA) and adjustment of p-values to control the False Discovery Rate (FDR<10%), we identified a signature of 2959 genes differentiating DSS patients from both DF and DHF, and showed a strong association of this DSS-gene signature with the dengue disease phenotype. Using a combined approach to analyse the molecular patterns associated with the DSS-gene signature, we provide an integrative overview of the transcriptional responses altered in DSS children. In particular, we show that the transcriptome of DSS children blood cells is characterized by a decreased abundance of transcripts related to T and NK lymphocyte responses and by an increased abundance of anti-inflammatory and repair/remodeling transcripts. We also show that unexpected pro-inflammatory gene patterns at the interface between innate immunity, inflammation and host lipid metabolism, known to play pathogenic roles in acute and chronic inflammatory diseases associated with systemic vascular dysfunction, are transcriptionnally active in the blood cells of DSS children. CONCLUSIONS/SIGNIFICANCE: We provide a global while non exhaustive overview of the molecular mechanisms altered in of DSS children and suggest how they may interact to lead to final vascular homeostasis breakdown. We suggest that some mechanisms identified should be considered putative therapeutic targets or biomarkers of progression to DSS.

Circulation of Chikungunya virus in Gabon, 2006-2007.

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.

Enzymatic characterization of a trypsin-like serine protease encoded by the genome of cell fusing agent virus.

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).

Chikungunya virus, Cameroon, 2006.

We report the isolation of chikungunya virus from a patient during an outbreak of a denguelike syndrome in Cameroon in 2006. The virus was phylogenetically grouped in the Democratic Republic of the Congo cluster, indicating a continuous circulation of a genetically similar chikungunya virus population during 6 years in Central Africa.

O'nyong-nyong Virus, Chad.

We report the first laboratory-confirmed human infection with O'nyong-nyong virus in Chad. This virus was isolated from peripheral blood mononuclear cells of a patient with evidence of a seroconversion to a virus related to Chikungunya virus. Genome sequence was partly determined, and phylogenetic studies were conducted.

Mutagenesis analysis of the NS2B determinants of the Alkhurma virus NS2B-NS3 protease activation.

Alkhurma virus (ALKV) is a tick-borne class 4 flavivirus responsible for several human cases of haemorrhagic fever in Saudi Arabia, with no specific treatment currently available. The viral RNA encodes a serine protease (NS2B-NS3), essential for virus replication in infected cells, that constitutes an attractive target for antiviral compounds. In an attempt to identify residues and motifs on NS2B that are necessary for protease activity of the ALKV NS2B-NS3 complex, a series of modified NS2B-NS3 proteins was constructed, with point mutations on particular residues or with the NS2B domain derived from two different viruses. Four mutants and the two chimeric proteins exhibited reduction of protease activity against BAPNA (a p-nitroanilide substrate). The results demonstrate that tight complementarity of the protein sequences is necessary for NS2B-dependent activation of NS3. The results also determine residues in the ALKV NS2B cofactor essential for protease activation, giving new insights into protease function in flaviviruses.

Dengue type 3 virus, Saint Martin, 2003-2004.

We describe the spread of a dengue virus during an outbreak in Saint Martin island (French West Indies) during winter 2003-2004. Dengue type 3 viruses were isolated from 6 patients exhibiting clinical symptoms. This serotype had not been detected on the island during the preceding 3 years. Genome sequence determinations and analyses showed a common origin with dengue type 3 viruses isolated in Martinique 2 years earlier.

Genetic characterization of newly reintroduced dengue virus type 3 in Martinique (French West Indies).

Dengue type 3 viruses were isolated from patients in Martinique between 1999 and 2002. This serotype had not been detected on the island in the last 20 years. Genomic sequence determination and analysis showed great stability of the virus during the period studied.