Extended Preclinical Safety,Efficacy and Stability Testing of a Live-attenuated Chikungunya Vaccine Candidate

We recently described a new, live-attenuated vaccine candidate for chikungunya (CHIK) fever, CHIKV/IRES. This vaccine was shown to be well attenuated, immunogenic and efficacious in protecting against CHIK virus (CHIKV) challenge of mice and nonhuman primates. To further evaluate its preclinical safety, we compared CHIKV/IRES distribution and viral loads in interferon-α/β receptor-incompetent A129 mice to another CHIK vaccine candidate, 181/clone25, which proved highly immunogenic but mildly reactive in human Phase I/II clinical trials. Compared to wild-type CHIK virus, (wt-CHIKV), both vaccines generated lower viral loads in a wide variety of tissues and organs, including the brain and leg muscle, but CHIKV/IRES exhibited marked restrictions in dissemination and viral loads compared to 181/clone25, and was never found outside the blood, spleen and muscle. Unlike wt-CHIKV, which caused disrupted splenic architecture and hepatic lesions, histopathological lesions were not observed in animals infected with either vaccine strain. To examine the stability of attenuation, both vaccines were passaged 5 times intracranially in infant A129 mice, then assessed for changes in virulence by comparing parental and passaged viruses for footpad swelling, weight stability and survival after subcutaneous infection. Whereas strain 181/clone25 p5 underwent a significant increase in virulence as measured by weight loss (from <10% to >30%) and mortality (from 0 to 100%), CHIKV/IRES underwent no detectible change in any measure of virulence (no significant weight loss and no mortality). These data indicate greater nonclinical safety of the CHIKV/IRES vaccine candidate compared to 181/clone25, further supporting its eligibility for human testing.     

Chikungunya disease: infection-associated markers from the acute to the chronic phase of arbovirus-induced arthralgia

At the end of 2005, an outbreak of fever associated with joint pain occurred in La Réunion. The causal agent, chikungunya virus (CHIKV), has been known for 50 years and could thus be readily identified. This arbovirus is present worldwide, particularly in India, but also in Europe, with new variants returning to Africa. In humans, it causes a disease characterized by a typical acute infection, sometimes followed by persistent arthralgia and myalgia lasting months or years. Investigations in the La Réunion cohort and studies in a macaque model of chikungunya implicated monocytes-macrophages in viral persistence. In this Review, we consider the relationship between CHIKV and the immune response and discuss predictive factors for chronic arthralgia and myalgia by providing an overview of current knowledge on chikungunya pathogenesis. Comparisons of data from animal models of the acute and chronic phases of infection, and data from clinical series, provide information about the mechanisms of CHIKV infection-associated inflammation, viral persistence in monocytes-macrophages, and their link to chronic signs.     

Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication

Background

Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection.

Methods

Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection.

Results

Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1.

Conclusion

Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.     

Novel chikungunya vaccine candidate with an IRES-based attenuation and host range alteration mechanism

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that has recently caused devastating urban epidemics of severe and sometimes chronic arthralgia. As with most other mosquito-borne viral diseases, control relies on reducing mosquito populations and their contact with people, which has been ineffective in most locations. Therefore, vaccines remain the best strategy to prevent most vector-borne diseases. Ideally, vaccines for diseases of resource-limited countries should combine low cost and single dose efficacy, yet induce rapid and long-lived immunity with negligible risk of serious adverse reactions. To develop such a vaccine to protect against chikungunya fever, we employed a rational attenuation mechanism that also prevents the infection of mosquito vectors. The internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. Testing in both normal outbred and interferon response-defective mice indicated that the new vaccine candidate is highly attenuated, immunogenic and efficacious after a single dose. Furthermore, it is incapable of replicating in mosquito cells or infecting mosquitoes in vivo. This IRES-based attenuation platform technology may be useful for the predictable attenuation of any alphavirus.     

Proteomic analysis of CHIKV-infected human fibroblast-like synoviocytes: Identification of host factors potentially associated with CHIKV replication and cellular pathogenesis

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes arthralgic fever. Fibroblast-like synoviocytes play a key role in joint damage in inflammatory arthritides and can additionally serve as target cells for CHIKV infection. To gain a better understanding of CHIKV-induced arthralgia, the interaction between CHIKV and synoviocytes was investigated at the protein level. A gel-enhanced liquid chromatography-mass spectrometry (GeLC-MS/MS) approach was used to examine protein expression from primary human fibroblast-like synoviocytes (HFLS) infected with clinical isolates of CHIKV at 12 and 24 hr post infection. Our analysis identified 259 and 241 proteins of known function that were differentially expressed (>1.5 or <−1.5 fold change) following CHIKV infection at 12 and 24 hpi, respectively. These proteins are involved in cellular homeostasis, including cellular trafficking, cytoskeletal organization, immune response, metabolic process, and protein modification. Some of these proteins have previously been reported to participate in arthralgia/arthritis and the death of infected cells. Our results provide information on the CHIKV-induced modulation of cellular proteins of HFLS at an early stage of infection, as well as highlighting biological processes associated with CHIKV infection in the main target cells of the joint.     

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.     

First Reported Chikungunya Fever Outbreak in the Republic of Congo, 2011

Background

Chikungunya is an Aedes -borne disease characterised by febrile arthralgia and responsible for massive outbreaks. We present a prospective clinical cohort study and a retrospective serological study relating to a CHIK outbreak, in the Republic of Congo in 2011.

Methodology and Findings

We analysed 317 suspected cases, of which 308 (97.2%) lived in the city of Brazzaville (66.6% in the South area). Amongst them, 37 (11.7%) were CHIKV+ve patients (i.e., biologically confirmed by a real-time RT-PCR assay), of whom 36 (97.3%) had fever, 22 (66.7%) myalgia and 32 (86.5%) arthralgia. All tested negative for dengue. The distribution of incident cases within Brazzaville districts was compared with CHIKV seroprevalence before the outbreak (34.4% in 517 blood donors), providing evidence for previous circulation of CHIKV. We applied a CHIK clinical score to 126 patients recruited within the two first day of illness (including 28 CHIKV+ves (22.2%)) with sensitivity (78.6%) and specificity (72.4%) values comparing with those of the referent study in Reunion Island. The negative predictive value was high (92%), but the positive predictive value (45%) indicate poor potential contribution to medical practice to identify CHIKV+ve patients in low prevalence outbreaks. However, the score allowed a slightly more accurate follow-up of the evolution of the outbreak than the criterion "fever+arthralgia". The complete sequencing of a Congolase isolate (Brazza_MRS1) demonstrated belonging to the East/Central/South African lineage and was further used for producing a robust genome-scale CHIKV phylogenetic analysis.

Conclusions/Significance

We describe the first Chikungunya outbreak declared in the Republic of Congo. The seroprevalence study conducted amongst blood donors before outbreak provided evidence for previous CHIKV circulation. We suggest that a more systematic survey of the entomological situation and of arbovirus circulation is necessary in Central Africa for better understanding the environmental, microbiological and sociological determinants of emergence.     

Chikungunya Virus-associated Long-term Arthralgia: A 36-month Prospective Longitudinal Study

Background

Arthritogenic alphaviruses, including Chikungunya virus (CHIKV), are responsible for acute fever and arthralgia, but can also lead to chronic symptoms. In 2006, a Chikungunya outbreak occurred in La Réunion Island, during which we constituted a prospective cohort of viremic patients (n = 180) and defined the clinical and biological features of acute infection. Individuals were followed as part of a longitudinal study to investigate in details the long-term outcome of Chikungunya.

Methodology/Principal Findings

Patients were submitted to clinical investigations 4, 6, 14 and 36 months after presentation with acute CHIKV infection. At 36 months, 22 patients with arthralgia and 20 patients without arthralgia were randomly selected from the cohort and consented for blood sampling. During the 3 years following acute infection, 60% of patients had experienced symptoms of arthralgia, with most reporting episodic relapse and recovery periods. Long-term arthralgias were typically polyarthralgia (70%), that were usually symmetrical (90%) and highly incapacitating (77%). They were often associated with local swelling (63%), asthenia (77%) or depression (56%). The age over 35 years and the presence of arthralgia 4 months after the disease onset are risk factors of long-term arthralgia. Patients with long-term arthralgia did not display biological markers typically found in autoimmune or rheumatoid diseases. These data helped define the features of CHIKV-associated chronic arthralgia and permitted an estimation of the economic burden associated with arthralgia.

Conclusions/Significance

This study demonstrates that chronic arthralgia is a frequent complication of acute Chikungunya disease and suggests that it results from a local rather than systemic inflammation.     

Chronic Joint Disease Caused by Persistent Chikungunya Virus Infection Is Controlled by the Adaptive Immune Response

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that causes incapacitating disease in humans characterized by intense joint pain that can persist for weeks, months, or even years. Although there is some evidence of persistent CHIKV infection in humans suffering from chronic rheumatologic disease symptoms, little is known about chronic disease pathogenesis, and no specific therapies exist for acute or chronic CHIKV disease. To investigate mechanisms of chronic CHIKV-induced disease, we utilized a mouse model and defined the duration of CHIKV infection in tissues and the associated histopathological changes. Although CHIKV RNA was readily detectable in a variety of tissues very early after infection, CHIKV RNA persisted specifically in joint-associated tissues for at least 16 weeks. Inoculation of Rag1^−/− mice, which lack T and B cells, resulted in higher viral levels in a variety of tissues, suggesting that adaptive immunity controls the tissue specificity and persistence of CHIKV infection. The presence of CHIKV RNA in tissues of wild-type and Rag1^−/− mice was associated with histopathological evidence of synovitis, arthritis, and tendonitis; thus, CHIKV-induced persistent arthritis is not mediated primarily by adaptive immune responses. Finally, we show that prophylactic administration of CHIKV-specific monoclonal antibodies prevented the establishment of CHIKV persistence, whereas therapeutic administration had tissue-specific efficacy. These findings suggest that chronic musculoskeletal tissue pathology is caused by persistent CHIKV infection and controlled by adaptive immune responses. Our results have significant implications for the development of strategies to mitigate the disease burden associated with CHIKV infection in humans.     

Homology modeling, molecular dynamics, e-pharmacophore mapping and docking study of Chikungunya virus nsP2 protease

To date, no suitable vaccine or specific antiviral drug is available to treat Chikungunya viral (CHIKV) fever. Hence, it is essential to identify drug candidates that could potentially impede CHIKV infection. Here, we present the development of a homology model of nsP2 protein based on the crystal structure of the nsP2 protein of Venezuelan equine encephalitis virus (VEEV). The protein modeled was optimized using molecular dynamics simulation; the junction peptides of a nonstructural protein complex were then docked in order to investigate the possible protein–protein interactions between nsP2 and the proteins cleaved by nsP2. The modeling studies conducted shed light on the binding modes, and the critical interactions with the peptides provide insight into the chemical features needed to inhibit the CHIK virus infection. Energy-optimized pharmacophore mapping was performed using the junction peptides. Based on the results, we propose the pharmacophore features that must be present in an inhibitor of nsP2 protease. The resulting pharmacophore model contained an aromatic ring, a hydrophobic and three hydrogen-bond donor sites. Using these pharmacophore features, we screened a large public library of compounds (Asinex, Maybridge, TOSLab, Binding Database) to find a potential ligand that could inhibit the nsP2 protein. The compounds that yielded a fitness score of more than 1.0 were further subjected to Glide HTVS and Glide XP. Here, we report the best four compounds based on their docking scores; these compounds have IDs of 27943, 21362, ASN 01107557 and ASN 01541696. We propose that these compounds could bind to the active site of nsP2 protease and inhibit this enzyme. Furthermore, the backbone structural scaffolds of these four lead compounds could serve as building blocks when designing drug-like molecules for the treatment of Chikungunya viral fever.     

A mouse model for Chikungunya: young age and inefficient type-I interferon signaling are risk factors for severe disease

Chikungunya virus (CHIKV) is a re-emerging arbovirus responsible for a massive outbreak currently afflicting the Indian Ocean region and India. Infection from CHIKV typically induces a mild disease in humans, characterized by fever, myalgia, arthralgia, and rash. Cases of severe CHIKV infection involving the central nervous system (CNS) have recently been described in neonates as well as in adults with underlying conditions. The pathophysiology of CHIKV infection and the basis for disease severity are unknown. To address these critical issues, we have developed an animal model of CHIKV infection. We show here that whereas wild type (WT) adult mice are resistant to CHIKV infection, WT mouse neonates are susceptible and neonatal disease severity is age-dependent. Adult mice with a partially (IFN-alpha/betaR(+/-)) or totally (IFN-alpha/betaR(-/-)) abrogated type-I IFN pathway develop a mild or severe infection, respectively. In mice with a mild infection, after a burst of viral replication in the liver, CHIKV primarily targets muscle, joint, and skin fibroblasts, a cell and tissue tropism similar to that observed in biopsy samples of CHIKV-infected humans. In case of severe infections, CHIKV also disseminates to other tissues including the CNS, where it specifically targets the choroid plexuses and the leptomeninges. Together, these data indicate that CHIKV-associated symptoms match viral tissue and cell tropisms, and demonstrate that the fibroblast is a predominant target cell of CHIKV. These data also identify the neonatal phase and inefficient type-I IFN signaling as risk factors for severe CHIKV-associated disease. The development of a permissive small animal model will expedite the testing of future vaccines and therapeutic candidates.     

A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals.     

Mosquito-bite infection of humanized mice with chikungunya virus produces systemic disease with long-term effects

Chikungunya virus (CHIKV) is an emerging, mosquito-borne alphavirus responsible for acute to chronic arthralgias and neuropathies. Although it originated in central Africa, recent reports of disease have come from many parts of the world, including the Americas. While limiting human CHIKV cases through mosquito control has been used, it has not been entirely successful. There are currently no licensed vaccines or treatments specific for CHIKV disease, thus more work is needed to develop effective countermeasures. Current animal research on CHIKV is often not representative of human disease. Most models use CHIKV needle inoculation via unnatural routes to create immediate viremia and localized clinical signs; these methods neglect the natural route of transmission (the mosquito vector bite) and the associated human immune response. Since mosquito saliva has been shown to have a profound effect on viral pathogenesis, we evaluated a novel model of infection that included the natural vector, Aedes species mosquitoes, transmitting CHIKV to mice containing components of the human immune system. Humanized mice infected by 3–6 mosquito bites showed signs of systemic infection, with demonstrable viremia (by qRT-PCR and immunofluorescent antibody assay), mild to moderate clinical signs (by observation, histology, and immunohistochemistry), and immune responses consistent with human infection (by flow cytometry and IgM ELISA). This model should give a better understanding of human CHIKV disease and allow for more realistic evaluations of mechanisms of pathogenesis, prophylaxis, and treatments.     

A potent neutralizing IgM mAb targeting the N218 epitope on E2 protein protects against Chikungunya virus pathogenesis

Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore, this study aimed to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model.

To evaluate their antigenicity and neutralization profile, indirect enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA) and a plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated, and reverse genetics techniques were then used to create an infectious CHIKV clone with a single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. The survival rate, CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralize CHIKV replication. Further analyses of 3E7b binding and neutralization of CHIKV single-mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In a pre-binding neutralization assay, 3E7b blocks CHIKV attachment to permissive cells, possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection, 3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively, these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design.     

Recombinant Modified Vaccinia Virus Ankara Expressing Glycoprotein E2 of Chikungunya Virus Protects AG129 Mice against Lethal Challenge

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections.     

Long-Term Arthralgia after Mayaro Virus Infection Correlates with Sustained Pro-inflammatory Cytokine Response

Mayaro virus (MAYV), an alphavirus similar to chikungunya virus (CHIKV), causes an acute debilitating disease which results in the development of long-term arthralgia in more than 50% of infected individuals. Currently, the immune response and its role in the development of MAYV-induced persistent arthralgia remain unknown. In this study, we evaluated the immune response of individuals with confirmed MAYV infection in a one-year longitudinal study carried out in Loreto, Peru. We report that MAYV infection elicits robust immune responses that result in the development of a strong neutralizing antibody response and the secretion of pro-inflammatory immune mediators. The composition of these inflammatory mediators, in some cases, differed to those previously observed for CHIKV. Key mediators such as IL-13, IL-7 and VEGF were strongly induced following MAYV infection and were significantly increased in subjects that eventually developed persistent arthralgia. Although a strong neutralizing antibody response was observed in all subjects, it was not sufficient to prevent the long-term outcomes of MAYV infection. This study provides initial immunologic insight that may eventually contribute to prognostic tools and therapeutic treatments against this emerging pathogen.     

The cell biology of Chikungunya virus infection

Chikungunya virus (CHIKV) infection causes a disease which appears to affect multiple cell types and tissues. The acute phase is manifested by a non-fatal febrile illness, polyarthralgia and maculopapular rashes in adults, but with recurrent arthralgia that may linger for months during convalescence. The issue of cellular and tissue tropism of CHIKV has elicited interest primarily because of this lingering incapacitating chronic joint pain, as well as clear encephalopathy in severe cases among neonates during the re-emergence of the virus in recent epidemics. The principle cell types productively infected by CHIKV are skin fibroblasts, epithelial cells and lymphoid tissues. There is controversy as to whether CHIKV productively infects haematopoietic cells and neurones/glia. CHIKV infection triggers rapid and robust innate immune responses which quickly clears the acute phase infection. However, significant acute as well as chronic infection of less obvious cell types, such as monocytes, neurones/glia or even CNS neural progenitors may conceivably occur. There is therefore a need to ascertain the full range potential of CHIKV tropism, fully understand the cellular responses triggered during the acute the convalescent phases, and explore possible cell types that might be the source of chronic problems associated with CHIKV infection.     

基孔肯雅病毒检测方法及进展

基孔肯雅病毒是引起基孔肯雅热的病原体,主要经伊蚊传播,感染者的症状主要以发热、皮疹和关节疼痛为主。该病毒主要分布在非洲、东南亚等地区,近年来在印度洋地区造成大规模流行。我国主要以输入性病例为主,未发生大规模流行。对基孔肯雅病毒的实验室检测方法及最新研究进展进行了综述。     

Proteomics Profiling of Chikungunya-Infected Aedes albopictus C6/36 Cells Reveal Important Mosquito Cell Factors in Virus Replication

Chikungunya virus (CHIKV) is the only causative agent of CHIKV fever with persistent arthralgia, and in some cases may lead to neurological complications which can be highly fatal, therefore it poses severe health issues in many parts of the world. CHIKV transmission can be mediated via the Aedes albopictus mosquito; however, very little is currently known about the involvement of mosquito cellular factors during CHIKV-infection within the mosquito cells. Unravelling the neglected aspects of mosquito proteome changes in CHIKV-infected mosquito cells may increase our understanding on the differences in the host factors between arthropod and mammalian cells for successful replication of CHIKV. In this study, the CHIKV-infected C6/36 cells with differential cellular proteins expression were profiled using two-dimensional gel electrophoresis (2DE) coupled with the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 2DE analysis on CHIKV-infected C6/36 cells has shown 23 mosquito cellular proteins that are differentially regulated, and which are involved diverse biological pathways, such as protein folding and metabolic processes. Among those identified mosquito proteins, spermatogenesis-associated factor, enolase phosphatase e-1 and chaperonin-60kD have been found to regulate CHIKV infection. Furthermore, siRNA-mediated gene knockdown of these proteins has demonstrated the biological importance of these host proteins that mediate CHIKV infection. These findings have provided an insight to the importance of mosquito host factors in the replication of CHIKV, thus providing a potential channel for developing novel antiviral strategies against CHIKV transmission.     

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 K₂₅₂Q 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.