Search

Chikungunya virus (CHIKV) is an alphavirus which causes chronic and incapacitating arthralgia in humans. Although previous studies have shown that antibodies against the virus are produced during and after infection, the fine specificity of the antibody response against CHIKV is not known. Here, using plasma from patients at different times postinfection, we characterized the antibody response against various proteins of the virus. We have shown that the E2 and E3 glycoproteins and the capsid and nsP3 proteins are targets of the anti-CHIKV antibody response. Moreover, we have identified the different regions in these proteins which contain the linear epitopes recognized by the anti-CHIKV antibodies and determined their structural localization. Data also illustrated the effect of a single K 252 Q amino acid change at the E2 glycoprotein that was able to influence antibody binding and interaction between the antibodies and epitope because of the changes of epitope-antibody binding capacity. This study provides important knowledge that will not only aid in the understanding of the immune response to CHIKV infection but also provide new knowledge in the design of modern vaccine development. Furthermore, these pathogen-specific epitopes could be used for future seroepidemiological studies that will unravel the molecular mechanisms of human immunity and protection from CHIKV disease.

BACKGROUND: Chikungunya virus (CHIKV) is an arbovirus with a high potential to spread globally. We investigated whether CHIKV is transmittable via corneal grafts. METHODS: Serum specimens from 69 potential corneal donors living in La Reunion during the 2005–2006 outbreak of CHIKV infection were screened for anti-CHIKV antibodies. Serum specimens and corneoscleral rims were subjected to quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) for detection of CHIKV. CHIKV isolation and immunolabeling were performed on eye tissue specimens. Viral transmission via the ocular route was assessed in an animal model of human CHIKV infection. RESULTS: Twelve apparently uninfected donors were viremic and/or positive for immunoglobulin M (IgM) and/or immunoglobulin G. Eye tissue specimens from 12 donors who were or were not viremic and were or were not seropositive were investigated. qRT-PCR detected CHIKV RNA in corneoscleral rims from 4 patients: 1 patient was viremic, 2 were viremic and IgM positive, and 1 was IgM positive. Infectious CHIKV was isolated from all qRT-PCR–positive samples, and antigens were detected in corneal and scleral specimens, the iris, the ciliary body, and oculomotor muscles. CONCLUSIONS: One-third of eligible corneas (4 of 12) from donors apparently uninfected with CHIKV were infected with CHIKV during the study period. CHIKV infects the human cornea and can be transmitted via the ocular route. In the absence of systematic CHIKV screening in donors, cornea donation should be banned in areas where CHIKV circulates.

International audience; 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.

Dengue virus infection is the most frequent arthropod-borne infection affecting humans in the world. Our understanding of the pathophysiological events leading to mild or severe outcomes of the disease remains limited by the fact that viral target cells in the human body are poorly characterized. One of the most sensitive strategies for detecting cells supporting active replication of this positive-strand RNA virus is the search for the replicative intermediate, an antigenome of negative polarity, by RT-PCR. However, a phenomenon described as 'false priming' of the reverse transcriptase (RT) prevents strand-specific detection. The results of the current study showed that this event corresponds to cDNA synthesis that is independent of any primer addition. This property was general to all RNAs tested and was not associated with small free nucleic acids, such as tRNAs and microRNAs. Rather, it corresponded to initiation of cDNA synthesis from the 3' end of the RNA template, and a model is proposed in which the template RNA snaps back upon itself and creates a transient RNA primer suitable for the RT. Such a property would explain why many assays proposed for detection of a replicative intermediate are not specific, and may help in the development of a molecular biology protocol that could allow replication studies of RNA viruses of human interest, such as dengue virus, hepatitis C virus and enteroviruses.

Resume Introduction La dengue est la principale maladie vectorielle emergente dans le monde. On estime que deux cinquiemes de la population mondiale sont exposes. Un vecteur potentiel (Aedes albopictus) est present dans quatre departements du Sud-Est de la France metropolitaine. Methode Nous avons cherche a documenter le nombre de cas importes de dengue diagnostiques au cours des annees 2001 a 2006 incluse, ainsi que leurs principales caracteristiques. Resultats Entre 12 et 28 cas de dengue d’importation ont ete diagnostiques par mois durant cette periode (huit a 18 cas par mois si l’on exclut les annees 2001–2002 durant lesquelles une importante epidemie a ete decrite aux Antilles). Pres de 40 % des cas diagnostiques l’ont ete de juin a septembre inclus, periode d’activite du vecteur en metropole. Conclusions Ces donnees soulignent le lien particulierement etroit qui existe entre la situation en zone d’endemie (Martinique, temoignant de la situation dans les departements francais d’Amerique, DFA) et l’importation de cas en metropole. L’existence de ce « corridor d’importation » implique la necessite de renforcer le systeme de detection et de gestion des cas importes de dengue en metropole des lors qu’une epidemie de dengue est detectee dans les DFA.

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.

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.

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.

International audience; 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.

International audience; Mayaro (MAY) virus is a member of the genus Alphavirus in the family Togaviridae. Alphaviruses are distributed throughout the world and cause a wide range of diseases in humans and animals. Here, we determined the complete nucleotide sequence of MAY from a viral strain isolated from a French Guianese patient. The deduced MAY genome was 11,429 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. Nucleotide and amino acid homologies, as well as phylogenetic analyses of the obtained sequence confirmed that MAY is not a recombinant virus and belongs to the Semliki Forest complex according to the antigenic complex classification. Furthermore, analyses based on the E1 region revealed that MAY is closely related to Una virus, the only other South American virus clustering with the Old World viruses. On the basis of our results and of the alphaviruses diversity and pathogenicity, we suggest that alphaviruses may have an Old World origin.

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.

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.

Recombination events are known to occur in non-segmented RNA viruses like polioviruses or alphaviruses. Analysis of the subgenomic sequences of dengue virus type 1 (DENV-1) structural genes has recently allowed the identification of possible recombination breakpoints. Because DENV is a major human pathogen, this discovery might have important implications for virus pathogenicity, vaccine safety and efficiency, or diagnosis and, therefore, requires clear confirmation. We report the complete sequence determination of one Asian and two African strains of DENV-1 isolated from human patients. Rigorous sequence analysis provided strong evidence for the occurrence of intragenomic recombination events between DENV-1 strains belonging to different lineages. Singapore S275/90 strain appears to be the evolutionary product of a recombination event between viruses belonging to two distinct lineages: one lineage includes an African strain isolated in Abidjan (Ivory Coast) and the other includes isolates from Djibouti and Cambodia. The ‘Recombination Detection Program’, bootscanning and analysis of diversity plots provided congruent results concerning the existence of a two-switch recombination event and the localization of recombination breakpoints. Thus, the 5′ and 3′ genomic ends of the Singapore S275/90 strain were inherited from a Djibouti/Cambodia lineage ancestor and an internal fragment located in the envelope/NS1 region originated from an Abidjan lineage ancestor.

A Dutch couple, presenting with persisting arthralgias, temporary fever and rash after a stay in Surinam were diagnosed with Mayaro virus infection. Mayaro virus is a relatively unknown South American Alphavirus responsible for dengue-like clinical features and persisting arthralgias. An important, but probably underappreciated cross-reactivity with other Alphaviruses like Chikungunya virus is present, which may become of clinical importance in the event the various Alphaviruses will have overlapping geographical distributions and in seroprevalence studies. (C) 2010 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Severe forms of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome, were not prominent in the Americas until the epidemic of Cuba in 1981. Since that time, they have spread to other countries in Central and South America, correlating with the spread of dengue type 2 viruses related to Southeast Asian strains. We report here the complete genomic sequence of a dengue type 2 virus isolated during the epidemic in La Martinique in 1998. This constitutes the first complete genetic characterization of a dengue virus strain from French West Indies, and also the first molecular identification in this region of a dengue 2 strain phylogenetically related to the emerging American type 2 dengue viruses.

Yellow fever was presumably imported to the Americas from West-Africa from the 16th to the 19th century. American and African genotypes of the virus are distinguishable, indicating separate evolution in different vector/host cycles. The complete nucleotide sequence of the yellow fever virus strain TRINID79A, isolated in Trinidad in 1979, has been established. It exhibits extensive homology with those of current West-African strains and attenuated strain FNV. However, a unique deletion of the 3' non-coding region (NCR) of the viral RNA has been identified. It indicates that RYF1 and RYF2 repeated sequences of the 3' NCR are not necessary to the replication of the virus.

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.

RESUME Depuis quelques decennies, Marseille est devenue la plus grande ville comorienne hors de l’archipel du fait des flux migratoires. Elle est aussi le siege d’une faculte de medecine qui a fait de l’infectiologie au sens large l’un de ses poles d’excellence. Cette conjonction a permis a la cite phoceenne d’etre au cours des deux dernieres annees a la pointe de la reponse de la France metropolitaine face a la crise generee par l’emergence de la fievre a virus Chikungunya dans l’Ocean Indien, en particulier a La Reunion et a Mayotte. L’Hopital d’instruc- tion des armees (HIA) Laveran a pris en charge une des plus grandes cohortes de patients de metropole. Ses equipes ont precise la grande richesse clinique de la maladie au stade tardif, decrivant notamment l’incidence elevee des tenosynovites et de la polyarthrite distale invalidante, la survenue d’un acro-syndrome transitoire aux deuxieme et troisieme mois chez pres d’un malade sur quatre. Elles ont egalement identifie la presence d’une cryoglobuline mixte chez plus de 90 % des patients, evoluant en parallele des symptomes cliniques et sensible a une corticotherapie generale. Cette decouverte ouvre des voies importantes de recherche pour une meilleure comprehension de la physiopathologie de la maladie. Le laboratoire de virologie tropicale de l’Institut de medecine tropicale du Service de sante des armees (IMTSSA), laboratoire associe au Centre national de reference (CNR) des arboviroses, a developpe de nouveaux outils de diagnostic en particulier en matiere de RT-PCR. Il est avec le CNR, le producteur et fournisseur d’antigenes pour la serologie du Chikungunya de toute la France metropolitaine et d’Outre-mer. Il a pris en compte la presence de cryoglobulines, susceptibles d’induire des faux negatifs chez les patients infectes, et adapte la technique serologique, permettant d’augmenter considerablement la rentabilite diagnostique. Ses recherches fondamentales sont axees sur la caracterisation genomique des variants du virus isoles chez l’homme et le vecteur et sur l’approche de l’expression proteasique du virus Chikungunya pour des etudes fonctionnelles et le criblage d’antiviraux. Il travaille en cooperation avec les equipes cliniques reunionnaises et metropolitaines sur la reponse immunitaire humorale et cellulaires des patients et la description des formes cliniques. Le Departement d’epidemiologie et de sante publique de l’IMTSSA a realise l’etude epidemiologique d’une population particuliere constituee de l’integralite des gendarmes en poste a La Reunion a la fin de l’epidemie (juin 2006). Cette etude, menee en partenariat avec le laboratoire de virologie tropicale et le CNRS, permet de completer la description clinique des patients victimes de l’epidemie, sur une population non biaisee. Elle debouchera en 2007 sur une etude de cohorte prospective visant a suivre ces patients pendant plusieurs annees pour mieux decrire la phase chronique de la maladie dans une population de patients beneficiant d’un haut niveau d’accessibilite aux soins. Enfin ce departement a fourni aux autorites civiles une expertise et un renfort dans la conduite des operations de lutte a La Reunion. Dans cette participation a la gestion de crise, la place de la communication a ete importante, montrant combien il est devenu indispensable aux acteurs de sante de develop- per une expertise dans ce domaine loin de la pratique clinique. Dans cette tâche, le Service de sante des armees a Marseille n’a jamais ete isole ni coupe de ses partenaires universitai- res, qu’il s’agisse de collaborations cliniques entre l’HIA Laveran et le CHU Nord (Assis- tance Publique des Hopitaux de Marseille) ou virologiques entre les laboratoires de l’IMTSSA et de l’Etablissement francais du sang (EFS), cette experience augurant bien du partenariat que renforce la creation a Marseille d’un Reseau thematique de recherche de soins (RTRS) consacre aux maladies infectieuses emergentes et aux maladies tropicales.

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.

To evaluate the presence and extension of West Nile virus where French soldiers are stationed in Africa, specific antibody prevalence was determined by using ELISA and Western blot. Among 245 horses living in close proximity to the soldiers, seroprevalence was particularly high in Chad (97%) and Senegal (92%).

Twenty one mouse monoclonal antibodies reacting or cross-reacting with the Plasmodium falciparum RhopH3 protein reacted with Ag44, a recombinant antigen expressing the 134 C-terminal RhopH3 residues. Using overlapping peptides scanning this region, two major binding sites were identified. The first one, recognised by eight anti-RhopH3 and seven cross-reacting mAbs, was mapped to the sequence Thr Asp Asn Thr Tyr or Thr Asp Asn Thr Tyr Lys (aa 823–828), depending on the support used for synthesis. Binding specificity and affinity were investigated for a subset of four mAbs reacting with this epitope, including one growth inhibitory mAb. Systematic replacements showed that the various mAbs had similar requirements. The inhibitory mAb presented a higher affinity for this sequence and bound to the adjacent sequence, Tyr Lys Glu Met Glu Leu (aa 827–832). A 2nd binding site, located around residue 850, was recognised by two anti-RhopH3 mAbs, which reacted exclusively with the 110 kDa RhopH3 polypeptide, unlike the other mAbs, which reacted with the 110 and 105 kDa RhopH3 antigens. This suggested that the 105 kDa RhopH3 polypeptide derives from the 110 kDa by C-terminal processing. Experimental evidence substantiating this conclusion was provided by the observation that antisera raised to peptides located upstream of the putative cleavage site reacted with both the 110 kDa and 105 kDa polypeptides, whereas antisera raised to the 45 C-terminal amino acids of RhopH3 reacted exclusively with the larger, 110 kDa product. The biological significance of this processing is discussed.

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.

To the Editor: The Polio Eradication Campaign was started in Thailand in 1990, and the last polio case was reported in April 1997. Although no new cases have been reported, the Polio Eradication Campaign continues with 4 prevention strategies: high coverage with 3 doses of oral polio in children

Phlebotomine sand flies are known to transmit Leishmania, bacteria, and viruses that affect humans and animals in many countries worldwide. Sand fly-borne viruses belong mainly to the Phlebovirus, Vesiculovirus, and Orbivirus genera, and some of them are associated with outbreaks or sporadic human cases in the Mediterranean Europe. Up to now, Toscana virus is the only phlebovirus of medical importance identified in France. To study the diversity of the sand fly population living in the southeastern France, an entomological study was conducted from May to October, 2007. Most of the trapped sand flies belonged to Phlebotomus perniciosus (82.0%) and Sergentomyia minuta (17.3%) species; only three specimens were Phlebotomus ariasi. Molecular characterization of the P. perniciosus specimen based on the mitochondrial cytochrome b gene demonstrated different subpopulations living in the same areas. Most of the specimens belonged to the haplotypes pern01 and pern09, already described in France, but some belonged to original new haplotypes. The detection of one viral sequence clustering with Massilia/Granada virus, and of four sequences corresponding to two potential new phleboviruses (proposed names Olbia and Provencia viruses, respectively), revealed an unexpected diversity of phlebovirus species in France.

A seroepidemiology survey of nine zoonoses was carried out in 2007 on 90 healthy adult volunteers in Viljujsk, a northern city in the Republic of Sakha (Eastern Siberia). The seroprevalence of Lyme borreliosis was 3.3% by immunofluorescence. None of the subjects displayed a positive enzyme-linked immunosorbent assay/Western blot result for alveolar or cystic echinococcosis. The seroprevalence of toxocariasis by Western blot was 4.4%, and 8.9% of the subjects had anti-Toxoplasma IgG. By enzyme-linked immunosorbent assay, the seroprevalence of trichinellosis was 4.4%. Three subjects were simultaneously positive for tick-borne encephalitis and West Nile infection, so no clear diagnostic conclusion could be reached for these flavivirus diseases. Interestingly, Crimea-Congo hemorrhagic fever had an 11.1% seroprevalence rate, indicating that Viljujsk is the most northern focus of this infection. Additionally, this finding suggests a potential involvement of Crimea-Congo hemorrhagic fever agent, or of another member of the Bunyaviridae family, in the genesis of the so-called Viljujsk encephalomyelitis.

R J Hassing1,2, I Leparc-Goffart3, H Tolou3, G van Doornum4, P J van Genderen (p.van.genderen@havenziekenhuis.nl)1 1. Department of Internal Medicine, Harbour Hospital and Institute for Tropical Diseases, Rotterdam, the Netherlands 2. Department of Internal Medicine, Erasmus University Hospital Rotterdam, Rotterdam, the Netherlands 3. Institute of Tropical Medicine, Virology Unit, Army Biomedical Research Institute, Marseille, France 4. Department of Virology, Erasmus University Hospital Rotterdam, Rotterdam, the Netherlands

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. To date, interactions between the immune system and the different stages of the virus life cycle remain poorly defined. We demonstrated for the first time that CHIKV Ags could be detected in vivo in the monocytes of acutely infected patients. Using in vitro experimental systems, whole blood and purified monocytes, we confirmed that monocytes could be infected and virus growth could be sustained. CHIKV interactions with monocytes, and with other blood leukocytes, induced a robust and rapid innate immune response with the production of specific chemokines and cytokines. In particular, high levels of IFN-α were produced rapidly after CHIKV incubation with monocytes. The identification of monocytes during the early phase of CHIKV infection in vivo is significant as infected monocyte/macrophage cells have been detected in the synovial tissues of chronically CHIKV-infected patients, and these cells may behave as the vehicles for virus dissemination. This may explain the persistence of joint symptoms despite the short duration of viremia. Our results provide a better understanding on the basic mechanisms of infection and early antiviral immune responses and will help in the development of future effective control strategies.

Alphaviruses, including Chikungunya virus (CHIKV), produce a transient illness in humans, but severe forms leading to chronic incapacitating arthralgia/arthritis have been reported by mechanisms largely ill-characterized. The pathogenesis of CHIKV was addressed in a prospective cohort study of 49 hospitalized patients from Reunion Island subsequently categorized into two distinct groups at 12 mo postinfection. Comprehensive analyses of the clinical and immunological parameters throughout the disease course were analyzed in either the “recovered” or the “chronic” groups to identify prognostic markers of arthritis-like pathology after CHIKV disease. We found that the chronic group consisted mainly of more elderly patients (>60 y) and with much higher viral loads (up to 1010 viruses per milliliter of blood) during the acute phase. Remarkably, a rapid innate immune antiviral response was demonstrated by robust dendritic/NK/CD4/CD8 cell activation and accompanied by a rather weak Th1/Th2 cytokine response in both groups. Interestingly, the antiviral immune response witnessed by high levels of IFN-α mRNA in PBMCs and circulating IL-12 persisted for months only in the chronic group. CHIKV (RNA and proteins) was found in perivascular synovial macrophages in one chronic patient 18 mo postinfection surrounded by infiltrating NK and T cells (CD4++ but rare cytotoxic CD8). Fibroblast hyperplasia, strong angiogenesis, tissue lesions given the high levels of matrix metalloproteinase 2, and acute cell death [high cleaved poly(ADP-ribose) polymerase staining] were observed in the injured synovial tissue. These observed cellular and molecular events may contribute to chronic arthralgia/arthritis targeted by methotrexate used empirically for effective treatment but with immunosuppressive function in a context of viral persistence.

BackgroundDeciphering 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 findingsBased 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 (FDRConclusions/significanceWe 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.

International audience; 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.

Background Chikungunya virus (CHIKV), an arbovirus, is responsible for a two-stage disabling disease, consisting of an acute febrile polyarthritis for the first 10 days, frequently followed by chronic rheumatisms, sometimes lasting for years. Up to now, the pathophysiology of the chronic stage has been elusive. Considering the existence of occasional peripheral vascular disorders and some unexpected seronegativity during the chronic stage of the disease, we hypothesized the role of cryoglobulins. Methods From April 2005 to May 2007, all travelers with suspected CHIKV infection were prospectively recorded in our hospital department. Demographic, clinical and laboratory findings (anti-CHIKV IgM and IgG, cryoglobulin) were registered at the first consultation or hospitalization and during follow-up. Results Among the 66 travelers with clinical suspicion of CHIKV infection, 51 presented anti-CHIKV IgM. There were 45 positive with the serological assay tested at room temperature, and six more, which first tested negative when sera were kept at 4°C until analysis, became positive after a 2-hour incubation of the sera at 37°C. Forty-eight of the 51 CHIKV-seropositive patients were screened for cryoglobulinemia; 94% were positive at least once during their follow-up. Over 90% of the CHIKV-infected patients had concomitant arthralgias and cryoglobulinemia. Cryoglobulin prevalence and level drop with time as patients recover, spontaneously or after short-term corticotherapy. In some patients cryoglobulins remained positive after 1 year. Conclusion Prevalence of mixed cryoglobulinemia was high in CHIKV-infected travelers with long-lasting symptoms. No significant association between cryoglobulinemia and clinical manifestations could be evidenced. The exact prognostic value of cryoglobulin levels has yet to be determined. Responsibility of cryoglobulinemia was suspected in unexpected false negativity of serological assays at room temperature, leading us to recommend performing serology on pre-warmed sera., Author Summary Chikungunya virus is present in tropical Africa and Asia and is transmitted by mosquito bites. The disease is characterized by fever, headache, severe joint pain and transient skin rash for about a week. Most patients experience persisting joint pain and/or stiffness for months to years. In routine practice, diagnosis is based upon serology. Since 2004 there has been an ongoing giant outbreak of Chikungunya fever in East Africa, the Indian Ocean Islands, India and East Asia. In parallel, more than 1,000 travelers were diagnosed with imported Chikungunya infection in most developed countries. Considering the clinical features of our patients (joint pain), we hypothesized that cryoglobulins could be involved in the pathophysiology of the disease as observed in chronic hepatitis C infection. Cryoglobulins, which are immunoglobulins that precipitate when temperature is below 37°C, can induce rheumatic and vascular disorders. From April 2005 through May 2007, we screened all patients with possible imported Chikungunya infection for cryoglobulins. They were present in over 90% of patients, and possibly responsible for the unexpected false negativity of serological assays. Cryoglobulin frequency and levels decreased with time in recovering patients.

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°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 ∼10 RNA copies per assay. However, the RT-LAMP assay is much faster than traditional RT-PCR and generates results in

In recent decades Marseilles, through immigration, has become the largest Comorian city outside the archipelago. It is also home to a faculty of medicine that has made infectious diseases one of its fields of excellence. During the last two years, Marseilles has spearheaded the metropolitan French response to the Chikungunya crisis in the Indian Ocean region, and especially in the Reunion Island and Mayotte. Laveran military teaching hospital (Hôpital d'instruction des armées, HIA) has managed one of the largest metropolitan cohorts. Its teams have also reported the broad clinical spectrum of the disease in its later stages, and especially the high incidence of incapacitating tenosynovitis and distal arthritis, as well as the occurrence of a transient acrosyndrome during the second and third months in nearly one-quarter of patients. Importantly, they have also identified a mixed cryoglobulin in more than 90% of patients, the level of which matches clinical symptoms and is sensitive to systemic steroid therapy. This discovery opens the way to a better understanding of the pathophysiology of this viral disease. The Tropical Virology laboratory of the Tropical Medicine Institute of the Army health service (IMTSSA), which has close links with the national references center (CNRS) arbovirus laboratory, has developed new diagnostic tools, notably based on RT-PCR. Together with national reference center (CNRS), the laboratory produces and supplies antigens for Chikungunya serological tests in metropolitan France and overseas. It has taken into account the presence of cryoglobulins, which can lead to false-negative results in infected patients, and has considerably increased the diagnostic yield of serological techniques. The laboratory's fundamental research focuses on genomic characterization of viral variants isolated from humans and from the vector, and also on viral protease expression, for functional studies and antiviral candidate drug selection. The laboratory also collaborates with clinical teams in Reunion and metropolitan France working on humoral and cellular immune responses and on the different clinical forms of the disease. The Epidemiology and Public Health Department of IMTSSA conducted an epidemiological study of all gendarmes working in Reunion at the end of the epidemic (June 2006). This study, done in partnership with the tropical virology laboratory and CNRS, is helping to complete the clinical description of the epidemic, in an unbiased population. In 2007, it will form the basis for a prospective cohort study in which these patients will be monitored for several years to better document the chronic phase of the disease in a population with excellent healthcare access. Finally, the department has provided the civil authorities with advice and support in disease-control operations in Reunion. Communication played an important role in the management of this crisis, showing how crucial it now is for healthcare professionals to develop relevant skills. The Army Health Service in Maarseilles was never isolated from its university partners, as witnessed by clinical collaboration between Laveran HIA and CHU Nord (a Marseilles teaching hospital) and by virological cooperation between the IMTSSA and Etablissement français du sang (EFS) laboratories. This experience is highly encouraging with respect to the creation in Marseilles of a healthcare research network (RTRS) devoted to tropical and emerging infectious diseases.

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 ΔNS2B-NS3pro protease was purified as a functional protein with a purity greater than 90% suitable for crystallization attempts.

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.

International audience; After West Nile virus (WNV) reappeared in Camargue in September 2000, a multi species surveillance system was first set up in three districts in 2001. It was then extended to the entire French Mediterranean costal areas at the end of 2003. The integrated surveillance system was jointly coordinated by the ministry of health and the ministry of agriculture. It combined a multidisciplinary approach, involving; human, equine, avian and entomological surveillance. Between 2003 and 2006, WNV was detected in three occurrences; in the district of Var in summer 2003 with 7 human cases and 4 equine cases; in Camargue in September 2004 with 32 equine cases and 13 birds’ seroconversions, and finally in the district of Pyrénées-Orientales at the end of September 2006 with 5 equine cases. The virus circulation was limited in time and space. Control measures were implemented in the West Nile outbreak districts, integrating local antivectorial activities, prevention individual measures (against mosquito-bites) and restriction on blood donations.; Après le retour du virus West Nile (VWN) en Camargue en septembre 2000, une surveillance multi espèces a été mise en oeuvre en 2001 dans les trois départements camarguais pour être étendue fin 2003 et ultérieurement à l’ensemble du pourtour méditerranéen. Ce dispositif de surveillance du VWN VWN, coordonné par les ministères de la santé et de l’agriculture, combine une approche pluridisciplinaire avec des volets ; humain, équin, aviaire et entomologique. De 2003 à 2006, le VWN a été détecté à trois reprises ; dans le département du Var en été 2003 avec 7 cas humains et 4 cas équins, à nouveau en Camargue en septembre 2004 avec 32 cas équins et 13 séroconversions aviaires et dans le département des Pyrénées-Orientales fin septembre 2006 avec 5 cas équins. La circulation virale est restée localisée dans le temps et dans l’espace. La souche virale a été identifiée en 2004 chez les oiseaux. Des mesures de contrôle ont été mises en oeuvre dans les départements touchés par le VWN, intégrant des actions ciblées de démoustication, des mesures de prévention individuelle contre les piqûres de moustiques et des restrictions de dons du sang.

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.

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).

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.

The resurgence of Chikungunya virus is described during an urban epidemic in Kinshasa Democratic Republic of the Congo, after 39 years without any isolation of the virus. Chikungunya virus was isolated in sera from nine patients with clinical symptoms. A 1,200 bp long partial sequence of the E1/3'UTR genomic region was determined for each isolate. All sequences clustered in the central African lineage. They constitute Chikungunya virus reference sequences for the Democratic Republic of the Congo.

West Nile virus (WNV) is a common arbovirosis, transmitted by mosquitoes mainly Culex. WNV is commonly responsible for equine epizootics and epidemics in humans in sub-Saharan Africa. It has been occasionally described in Southern Europe and in some Mediterranean countries. Since 1994, WNV clinical aspects seem to change with an increase of central neurological involvement and a higher mortality, especially among people older than 50 years. In 1999, WNV reached New York, being responsible for severe clinical manifestations. It spread all over North America in less than four years Four (only four states of the United States are free). As a consequence of an equine epizootic in Camargue in 2000, French medical authorities settled a survey of the WNV circulation in the South of France. During 2003, this efficient action led to identify 6 human cases in a formerly WNV-free French Mediterranean coast, but not in Camargue.

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.