Unique epitopes recognized by antibodies induced in Chikungunya virus-infected non-human primates: implications for the study of immunopathology and vaccine development.

Chikungunya virus (CHIKV) is an Alphavirus that causes chronic and incapacitating arthralgia in humans. Although patient cohort studies have shown the production of CHIKV specific antibodies, the fine specificity of the antibody response against CHIKV is not completely defined. The macaque model of CHIKV infection was established due to limitations of clinical specimens. More importantly, its close relation to humans will allow the study of chronic infection and further identify important CHIKV targets. In this study, serum samples from CHIKV-infected macaques collected at different time-points post infection were used to characterize the antibody production pattern and kinetics. Results revealed that anti-CHIKV antibodies were neutralizing and the E2 glycoprotein, Capsid, nsP1, nsP3 and nsP4 proteins were targets of the anti-CHIKV antibody response in macaques. Furthermore, linear B-cell epitopes recognized by these anti-CHIKV antibodies were identified, and mapped to their structural localization. This characterizes the specificity of anti-CHIKV antibody response in macaques and further demonstrates the importance of the different regions in CHIKV-encoded proteins in the adaptive immune response. Information from this study provides critical knowledge that will aid in the understanding of CHIKV infection and immunity, vaccine design, and pre-clinical efficacy studies.

Diversity of Phlebotomus perniciosus in Provence, southeastern France: Detection of two putative new phlebovirus sequences.

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.

Longitudinal analysis of the human antibody response to Chikungunya virus infection: implications for serodiagnosis and vaccine development.

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.

Chikungunya virus infection of corneal grafts.

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 Réunion during the 2005­2006 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-PCR­positive 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.

Phenotypic and genotypic characterization of dengue virus isolates differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome.

Dengue viruses (DENV) cause 50-100 million cases of acute febrile disease every year, including 500,000 reported cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Viral factors have been proposed to influence the severity of the disease, but markers of virulence have never been identified on DENV. Three DENV serotype-1 isolates from the 2007 epidemic in Cambodia that are derived from patients experiencing the various clinical forms of dengue were characterized both phenotypically and genetically. Phenotypic characteristics in vitro, based on replication kinetics in different cell lines and apoptosis response, grouped isolates from DF and DHF patients together, whereas the virus isolate from a DSS patient showed unique features: a lower level of replication in mammalian cells and extensive apoptosis in mosquito cells. Genomic comparison of viruses revealed six unique amino acid residues in the membrane, envelope, and in non-structural genes in the virus isolated from the DSS patient.

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.

Seroepidemiology of nine zoonoses in Viljujsk, Republic of Sakha (Northeastern Siberia, Russian Federation).

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.

Increase in dengue fever imported from Côte d’Ivoire and West Africa to France.

Dengue is usually not considered a significant health problem in Africa because severe forms of dengue illness are rarely reported. In the absence of local surveillance data, the investigation of dengue cases imported to France contributed to document the circulation of dengue virus in this area. From 1 July 2006 to 31 December 2008, a total of 148 dengue cases imported to metropolitan France were reported through the mandatory notification system. Arthralgia and signs of severity (haemorrhage, thrombocytopenia) were less frequent in patients returning from West African countries. DENV-3 was isolated in two patients from Côte d’Ivoire in 2008. The number and proportion of patients returning from Côte d’Ivoire to France increased significantly in 2008 compared with the previous 18-month period. In parallel, the marginal increase in air travel does not explain the high increase observed in imported dengue cases to France. Our data illustrate increased dengue circulation and the emergence of DENV-3 in this area, with public health implications for epidemiological surveillance and case management locally.

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.

Imported Mayaro virus infection in the Netherlands.

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.

Active infection of human blood monocytes by Chikungunya virus triggers an innate immune response.

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-alpha 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.

Persistent chronic inflammation and infection by Chikungunya arthritogenic alphavirus in spite of a robust host immune response.

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 10(10) 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-alpha 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.

Destructive arthritis in a patient with chikungunya virus infection with persistent specific IgM antibodies.

BACKGROUND: Chikungunya fever is an emerging arboviral disease characterized by an algo-eruptive syndrome, inflammatory polyarthralgias, or tenosynovitis that can last for months to years. Up to now, the pathophysiology of the chronic stage is poorly understood. CASE PRESENTATION: We report the first case of CHIKV infection with chronic associated rheumatism in a patient who developed progressive erosive arthritis with expression of inflammatory mediators and persistence of specific IgM antibodies over 24 months following infection. CONCLUSIONS: Understanding the specific features of chikungunya virus as well as how the virus interacts with its host are essential for the prevention, treatment or cure of chikungunya disease.

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.

Aedes albopictus mosquito: the main vector of the 2007 Chikungunya outbreak in Gabon.

The primary vector at the origin of the 2007 outbreak in Libreville, Gabon is identified as Aedes albopictus, trapped around the nearby French military camp. The Chikungunya virus was isolated from mosquitoes and found to be identical to the A226V circulating human strain. This is the first field study showing the role of the recently arrived species Aedes albopictus in Chikungunya virus transmission in Central Africa, and it demonstrates this species' role in modifying the epidemiological presentation of Chikungunya in Gabon.

Persisting mixed cryoglobulinemia in Chikungunya infection.

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 degrees C until analysis, became positive after a 2-hour incubation of the sera at 37 degrees 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.

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.

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.

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.