Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.
Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age. Gene stability was inferred from obtained quantification data through three widely cited evaluation algorithms i.e. BestKeeper, geNorm, and NormFinder. No single gene showed a satisfactory degree of stability throughout all developmental stages. Therefore, we propose combinations of PGK and ILK for early embryos; RPL32 and RPS17 for late embryos, all four larval instars, and pupae samples; eEF1-γ with STK for adult males; eEF1-γ with RPS17 for non-blood fed females; and eEF1-γ with eIF2α for both blood-fed females and cell culture. The results from this study should be able to provide a more informed selection of normalizing genes during qPCR in Ae.albopictus.
Chikungunya virus (CHIKV) infection is a persistent problem worldwide due to efficient adaptation of the viral vectors, Aedes aegypti and Aedes albopictus mosquitoes. Therefore, the absence of effective anti-CHIKV drugs to combat chikungunya outbreaks often leads to a significant impact on public health care. In this study, we investigated the antiviral activity of drugs that are used to alleviate infection symptoms, namely, the non-steroidal anti-inflammatory drugs (NSAIDs), on the premise that active compounds with potential antiviral and anti-inflammatory activities could be directly subjected for human use to treat CHIKV infections. Amongst the various NSAID compounds, Mefenamic acid (MEFE) and Meclofenamic acid (MECLO) showed considerable antiviral activity against viral replication individually or in combination with the common antiviral drug, Ribavirin (RIBA). The 50% effective concentration (EC50) was estimated to be 13 μM for MEFE, 18 μM for MECLO and 10 μM for RIBA, while MEFE + RIBA (1:1) exhibited an EC50 of 3 μM, and MECLO + RIBA (1:1) was 5 μM. Because MEFE is commercially available and its synthesis is easier compared with MECLO, MEFE was selected for further in vivo antiviral activity analysis. Treatment with MEFE + RIBA resulted in a significant reduction of hypertrophic effects by CHIKV on the mouse liver and spleen. Viral titre quantification in the blood of CHIKV-infected mice through the plaque formation assay revealed that treatment with MEFE + RIBA exhibited a 6.5-fold reduction compared with untreated controls. In conclusion, our study demonstrated that MEFE in combination with RIBA exhibited significant anti-CHIKV activity by impairing viral replication in vitro and in vivo. Indeed, this finding may lead to an even broader application of these combinatorial treatments against other viral infections.
Introduction: Dengue virus (DENV), Zika virus (ZIKV) and Chikungunya virus (CHIKV) are Arboviruses that are transmitted by the same vector, Aedes aegypti. Dengue has become a global problem since the Second World War and is common in more than 110 countries. In Malaysia, dengue is a major disease burden as total economic costs to the country as a result of dengue is close to RM1.05 billion in 2010 and estimated to rise to 1.3 billion by 2020. Apart from Dengue, Zika and Chikungunya are the other important mosquito borne diseases in Malaysia. The aim of this study was to develop a multiplex real-time assay for simultaneous detection of DENV, ZIKV and CHIKV in clinical specimens. Methods: The published singleplex protocols were used with key modifications to implement a triplex assay. A one-step multiplex real-time RT-PCR assay was developed that can simultaneously detect RNA of DENV, ZIKV and CHIKV with good performance for a routine diagnostic use. The assay was evaluated for inter- and intra-reproducibility by mean CT value. The diagnostic sensitivity was tested with 135 archived samples which had been defined positive or negative by routine singleplex assays. Whole blood, plasma and urines were used in this study. Results: Intra- and inter-reproducibility and sensitivity varied from 0.10% to 4.73% and from 0.45% to 5.98% for each virus respectively. The specificity of detection was 100%. The multiplex real-time RT-PCR assay showed concordance with test results performed by routine singleplex assays. No cross reaction was observed for any of the clinical samples. Conclusion: The development of a rapid, sensitive and specific molecular assay for DENV, ZIKV and CHIKV infections will produce a greater diagnostic capacity in our laboratory. This multiplex approach is cost effective and robust with the concurrent detection of 3 viruses of public health concern.
The Asian tiger mosquito, Aedes albopictus (Ae. albopictus), is an important vector that transmits arboviruses such as dengue (DENV), Zika (ZIKV) and Chikungunya virus (CHIKV). Long noncoding RNAs (lncRNAs) are known to regulate various biological processes. Knowledge on Ae. albopictus lncRNAs and their functional role in virus-host interactions are still limited. Here, we identified and characterized the lncRNAs in the genome of an arbovirus vector, Ae. albopictus, and evaluated their potential involvement in DENV and ZIKV infection. We used 148 public datasets, and identified a total of 10, 867 novel lncRNA transcripts, of which 5,809, 4,139, and 919 were intergenic, intronic and antisense respectively. The Ae. albopictus lncRNAs shared many characteristics with other species such as short length, low GC content, and low sequence conservation. RNA-sequencing of Ae. albopictus cells infected with DENV and ZIKV showed that the expression of lncRNAs was altered upon virus infection. Target prediction analysis revealed that Ae. albopictus lncRNAs may regulate the expression of genes involved in immunity and other metabolic and cellular processes. To verify the role of lncRNAs in virus infection, we generated mutations in lncRNA loci using CRISPR-Cas9, and discovered that two lncRNA loci mutations, namely XLOC_029733 (novel lncRNA transcript id: lncRNA_27639.2) and LOC115270134 (known lncRNA transcript id: XR_003899061.1) resulted in enhancement of DENV and ZIKV replication. The results presented here provide an important foundation for future studies of lncRNAs and their relationship with virus infection in Ae. albopictus.
This study is focused towards developing a global consensus sequence of nonstructural protein 2 (NSP2), a protease of Chikungunya Virus (CHIKV) and predict immunogenic promiscuous T-cell epitopes based on various bioinformatics tools. To date, no epitope data is available for the Chikungunya virus in the IEDB database. In this study, 100 available nucleotide sequences of NSP2-CHIKV belonging to different strains were downloaded from the National Centre for Biotechnology Information (NCBI) database. The nucleotide sequences were subjected to translated sequencing using the EXPASY tool followed by protein alignment using the CLC workbench and a global consensus sequence for the respective protein was developed. IEDB tool was used to predict HLA-I and HLA-II binding promiscuous epitopes from the consensus sequence of NSP2-CHIKV. Thirty-four B-cell based epitopes are predicted and the promiscuous epitope is VVDTTGSTKPDPGD at position 341-354. Twenty-six MHC-I short peptide epitopes are predicted to bind with HLA-A. The promiscuous epitopes predicted to bind with HLA-A*01:01 are VTAIVSSLHY, SLSESATMVY, FSKPLVYY, QPTDHVVGEY at positions 317-326, 84-93, 535-544 and 15-24 with percentile ranks 0.17, 0.39, 0.51 and 0.81, respectively. Twenty-four MHC-II short peptide epitopes are predicted for HLA-DRB. The promiscuous epitope predicted to bind with HLA-DRB*01:01 is VVGEYLVLSPQTVLRS from 20-35 with a lowest percentile rank of 0.01. These predicted epitopes can be effective targets towards development of vaccine against CHIKV. Epitopes predicted in this study displayed good binding affinity, antigenicity and promiscuity for the HLA classes. These predicted epitopes can prove to be translationally important towards the development of CHIKV.
Chikungunya virus (CHIKV) is maintained in the sylvatic cycle in West Africa and is transmitted by Aedes mosquito species to monkeys. In 2006, four verified CHIKV isolates were obtained during a survey of arboviruses in monkeys (Macaca fascicularis) in Pahang state, Peninsular Malaysia. RNA was extracted from the CHIKV isolates and used in reverse transcription polymerase chain reactions (RT-PCR) to amplify PCR fragments for sequencing. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the whole viral sequence. A total of 11,238 base pairs (bp) corresponding to open reading frames (ORFs) from our isolates and 47 other registered isolates in the National Center for Biotechnology Information (NCBI) were used to elucidate sequences, amino acids, and phylogenetic relationships and to estimate divergence times by using MEGA 7.0 and the Bayesian Markov chain Monte Carlo method. Phylogenetic analysis revealed that all CHIKV isolates could be classified into the Asian genotype and clustered with Bagan Panchor clades, which are associated with the chikungunya outbreak reported in 2006, with sequence and amino acid similarities of 99.9% and 99.7%, respectively. Minor amino acid differences were found between human and non-human primate isolates. Amino acid analysis showed a unique amino acid at position 221 in the nsP1region, at which a glycine (G) was found only in monkey isolates, whereas arginine (R) was found at the same position only in human isolates. The time to the most recent common ancestor (MRCA) estimation indicated that CHIKV probably started to diverge from human to non-human primates in approximately 2004 in Malaysia. The results suggested that CHIKV in non-human primates probably resulted from the spillover of the virus from humans. The study will be helpful in understanding the movement and evolution of CHIKV in Malaysia and globally.
Carica papaya (papaya) fruits are available throughout the world and it is well accepted as food or as a quasi-drug. Aqueous papaya leaves extract have been used as treatment for dengue fever. This prompted us to carry out the docking study on these nine selected ligands (phyto-constituents of papaya) which are carpaine, dehydrocarpaine I and II, cardenolide, p-coumaric acid, chlorogenic acid, caricaxanthin, violaxanthin and zeaxanthin. These phytoconstituents were evaluated on the docking behaviour of dengue serotype 3 RNA-dependent RNA polymerase (RdRp); influenza A (H1N9) virus neuraminidase (NA); chikungunya virus glycoprotein (E3-E2-E1) and chikungunya virus non-structural protein2 (nsP2) protease using Discovery Studio Version 3.1. In addition, molecular physicochemical, drug-likeness, ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) and TOPKAT (Toxicity Prediction by Komputer Assisted Technology) analyses were done. The molecular physicochemical analysis revealed that cardenolide and p-coumaric acid (2 ligands) complied with Lipinski’s rule of five. Dehydrocarpaine II, cardenolide, caricaxanthin, violaxanthin and zeaxanthin all the five ligands were predicted to have plasma protein binding (PPB) effect. Docking studies and binding free energy calculations revealed that p-coumaric acid exhibited very least binding energy irrespective of its target protein. Hence, the results of this present study exhibited the potential of these nine ligands as antiviral agent.
OBJECTIVES: We conducted an epidemiological review of the chikungunya fever situation in Singapore and described the measures taken to prevent the chikungunya virus from becoming entrenched in the tropical city-state.
METHODS: All laboratory-confirmed cases and outbreak investigation reports maintained by the Communicable Diseases Division, Ministry of Health, and Aedes mosquito surveillance data obtained by the National Environment Agency during the period 2006 and 2009 were reviewed and analysed.
RESULTS: Sporadic cases were imported into Singapore until the first local transmission occurred in an urban area where Aedes aegypti was the predominant vector. Subsequent introduction of a mutant viral strain (A226V) in early 2008 resulted in the rapid spread to suburban and rural areas where Aedes albopictus was the primary vector. 1072 cases including 812 (75.7%) indigenous cases were reported. The main sources of importation were India and Malaysia. Foreign contract workers were identified as high-risk for indigenous infections.
CONCLUSIONS: The disease was successfully brought under control through aggressive vector control measures directed at A. albopictus. Although the incidence has sharply declined since January 2009, a high degree of vigilance is maintained to prevent a recurrence of epidemic transmission which can occur even with a well-established nationwide mosquito control programme.
The Chikungunya virus (CHIKV) is an arthropod borne virus. In the last 50 years, it has been the cause of numerous outbreaks in tropical and temperate regions, worldwide. There is limited understanding regarding the underlying molecular mechanisms involved in CHIKV replication and how the virus interacts with its host. In the present study, comparative proteomics was used to identify secreted host proteins that changed in abundance in response to early CHIKV infection. Two-dimensional gel electrophoresis was used to analyse and compare the secretome profiles of WRL-68 cells infected with CHIKV against mock control WRL-68 cells. The analysis identified 25 regulated proteins in CHIKV infected cells. STRING network analysis was then used to predict biological processes that may be affected by these proteins. The processes predicted to be affected include signal transduction, cellular component and extracellular matrix (ECM) organization, regulation of cytokine stimulus and immune response. These results provide an initial view of CHIKV may affect the secretome of infected cells during early infection. The results presented here will compliment earlier results from the study of late host response. However, functional characterization will be necessary to further enhance our understanding of the roles played by these proteins in the early stages of CHIKV infection in humans.
Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have led to significant impact on health care in the developing world. Here, we evaluated the antiviral effects of tetracycline (TETRA) derivatives and other common antiviral agents against CHIKV. Our results showed that within the TETRA derivatives group, Doxycycline (DOXY) exhibited the highest inhibitory effect against CHIKV replication in Vero cells. On the other hand, in the antiviral group Ribavirin (RIBA) showed higher inhibitory effects against CHIKV replication compared to Aciclovir (ACIC). Interestingly, RIBA inhibitory effects were also higher than all but DOXY within the TETRA derivatives group. Docking studies of DOXY to viral cysteine protease and E2 envelope protein showed non-competitive interaction with docking energy of -6.6±0.1 and -6.4±0.1 kcal/mol respectively. The 50% effective concentration (EC50) of DOXY and RIBA was determined to be 10.95±2.12 μM and 15.51±1.62 μM respectively, while DOXY+RIBA (1:1 combination) showed an EC50 of 4.52±1.42 μM. When compared, DOXY showed higher inhibition of viral infectivity and entry than RIBA. In contrast however, RIBA showed higher inhibition against viral replication in target cells compared to DOXY. Assays using mice as animal models revealed that DOXY+RIBA effectively inhibited CHIKV replication and attenuated its infectivity in vivo. Further experimental and clinical studies are warranted to investigate their potential application for clinical intervention of CHIKV disease.
This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.
The past decade has seen the re-emergence of Chikungunya virus (CHIKV) as a major global health threat, affecting millions around the world. Although fatal infections are rare among infected patients, the occurrence of long-lasting polyarthralgia has a significant impact on patients' quality of lives and ability to work. These issues were the stimuli for this study to determine the potential of baicalin, a bioflavonoid, as the novel antiviral compound against CHIKV. It was found that baicalin was well tolerated by Vero, BHK-21 and HEK 293T cells with maximal nontoxic doses >600 μM, ≈ 350 μM and ≈110 μM, respectively. Antiviral assays indicated that baicalin was the most effective inhibitor when tested for its direct virucidal activity with EC50 ≈ 7 μM, followed by inhibition of virus entry into the host cell, attachment of virus particle to cellular receptors and finally intracellular replication of viral RNA genome. In silico analysis using molecular docking demonstrated close interactions between baicalin and CHIKV envelope protein with considerably strong binding affinity of -9.7 kcal/mol. qRT-PCR analysis revealed that baicalin had the greatest effect on the synthesis of viral negative stand RNA with EC50 ≈ 0.4 μM followed by the inhibition of synthesis of positive-strand genomic (EC50 ≈ 13 μM) and subgenomic RNAs (EC50 ≈ 14 μM). These readings indicate that the compound efficiently inhibits replicase complexes formation but is a less potent inhibitor of existing replicase complexes. Coherent with this hypothesis, the use of recombinant CHIKV replicons harboring Renilla luciferase marker showed that replication of corresponding replicon RNAs was only slightly downregulated at higher doses of baicalin, with EC50 > 100 μM. Immunofluorescence and western blotting experiments demonstrated dose-dependent inhibition of expression of different viral proteins. It was also observed that levels of important protein markers for cellular autophagy (LC3) and apoptosis (Bax) were reduced in baicalin treatment groups as compared with untreated virus infected controls. In summary, given its low toxicity and high efficacy against CHIKV, baicalin has great potential to be developed as the novel antiviral compound for CHIKV. In vivo studies to evaluate its activity in a more complexed system represent a necessary step for future analysis.
BACKGROUND: Chikungunya virus (CHIKV) of the Central/East African genotype has caused large outbreaks worldwide in recent years. In Malaysia, limited CHIKV outbreaks of the endemic Asian and imported Central/East African genotypes were reported in 1998 and 2006. Since April 2008, an unprecedented nationwide outbreak has affected Malaysia.
OBJECTIVE: To study the molecular epidemiology of the current Malaysian CHIKV outbreak, and to evaluate cross-neutralisation activity of serum from infected patients against isolates of Asian and Central/East African genotypes.
STUDY DESIGN: Serum samples were collected from 83 patients presenting in 2008, and tested with PCR for the E1 gene, virus isolation, and for IgM. Phylogenetic analysis was performed on partial E1 gene sequences of 837bp length. Convalescent serum from the current outbreak and Bagan Panchor outbreak (Asian genotype, 2006) were tested for cross-neutralising activity against representative strains from each outbreak.
RESULTS: CHIKV was confirmed in 34 patients (41.0%). The current outbreak strain has the A226V mutation in the E1 structural protein, and grouped with Central/East African isolates from recent global outbreaks. Serum cross-neutralisation activity against both Central/East African and Asian genotypes was observed at titres from 40 to 1280.
CONCLUSIONS: The CHIKV strain causing the largest Malaysian outbreak is of the Central/East African genotype. The presence of the A226V mutation, which enhances transmissibility of CHIKV by Aedes albopictus, may explain the extensive spread especially in rural areas. Serum cross-neutralisation of different genotypes may aid potential vaccines and limit the effect of future outbreaks.
Chikungunya virus (CHIKV) and dengue virus (DENV) co-circulate in areas endemic with the Aedes mosquito vectors. Both viruses cause similar illnesses which may be difficult to distinguish clinically. CHIKV is also associated with persistent arthralgia.
In 1998, Malaysia experienced its first chikungunya virus (CHIKV) outbreak in the suburban areas followed by another two in 2006 (rural areas) and 2008 (urban areas), respectively. Nevertheless, there is still a lack of documented data regarding the magnitude of CHIKV exposure in the Malaysian population. The aim of this study was to determine the extent of chikungunya virus infection in healthy Malaysian adults residing in outbreak-free locations.
Chikungunya fever (CHIKF) is a global infectious disease which can affect a wide range of age groups. The pathological and immunological response upon Chikungunya virus (CHIKV) infection have been reported over the last few years. However, the clinical profile and immune response upon CHIKV infection in children remain largely unknown. In this study, we analyzed the clinical and immunological response, focusing on the cytokine/chemokine profile in a CHIKV-infected pediatric cohort from Sarawak, Malaysia. Unique immune mediators triggered upon CHIKV infection were identified through meta-analysis of the immune signatures between this pediatric group and cohorts from previous outbreaks. The data generated from this study revealed that a broad spectrum of cytokines/chemokines is up-regulated in a sub-group of virus-infected children stratified according to their viremic status during hospitalization. Furthermore, different immune mediator profiles (the levels of pro-inflammatory cytokines, chemokines and growth and other factors) were observed between children and adults. This study gives an important insight to understand the immune response of CHIKV infection in children and would aid in the development of better prognostics and clinical management for children.
A serum survey of several characteristic groups of humans in urban, rural, and forested areas of Peninsular Malaysia for evidence of infection with three alphaviruses (Sindbis, getah, and chikungunya) was made on 4384 specimens collected between 1965 and 1969. Analysis of the serological results indicated that 1) persons residing in predominantly rural and forested areas have higher frequencies of specific alphavirus antibody of all three viruses than persons residing in urban areas, 2) human infection with chikungunya virus appears to be at a low level of activity but is widespread, although more common and recent in the northern part of the country, and 3) Sindbis and getah viruses probably do not represent a threat to the public health, but chikungunya virus remains a potential menance and may be responsible for future epidemics transmitted by A. aegypti and A. albopictus mosquitoes.
Plaque reduction neutralization tests, using five group A arboviruses (chikungunya, Ross River, Getah, Bebaru and Sindbis), were done on sera from human populations in 44 Southeast Asia and Pacific island localities. Specificity of the plaque neutralization test was determined by examining convalescent sera from patients with known alphavirus infections. Chikungunya-specific neutralizing antibodies were demonstrated in sera of persons living in South Vietnam, Northern Malaysia, Indonesia (Kalimantan and Sulawesi), as well as Luzon, Marinduque, Cebu and Mindanao islands in the Philippines. Evidence of Ross River virus infection was found among populations living in West New Guinea and Papua New Guinea mainland, the Bismark Archipelago, Rossel Island and the Solomon Islands. There appeared to be no geographic overlap in the distribution of chikungunya and Ross River viruses, with the separation in their distribution corresponding with Weber's line in the Pacific. Sindbis neutralizing antibodies were found in 7 of 21 populations sampled, but in general the prevalence of infection was low. Four sera, from Vietnam, Malaysia and Mindanao gave monospecific reactions with Getah virus. No evidence of specific Bebaru virus infection was detected. The epidemiology of these five alphaviruses in Southeast Asia and the Pacific islands is discussed.
Chikungunya fever (CHIKF) is currently distributed in Africa and in South and Southeast Asia; outbreaks have occurred periodically in the region over the past 50 years. After a large outbreak had occurred in countries in the western Indian Ocean region in 2005, several countries reported cases of CHIKF from travelers who had visited affected areas. In Japan, there have been only 15 cases of CHIKF patients so far, according to the National Institute of Infectious Diseases. Therefore, to evaluate the clinical and radiological features associated with the disease, we describe 6 imported cases of CHIKF. All of the patients had had prolonged arthralgia on admission to our hospital, and diagnosis was confirmed with specific antibodies by using an IgM-capture enzyme-linked immunoassay and a plaque reduction neutralizing antibody assay. Magnetic resonance imaging (MRI) of one patient revealed erosive arthritis and tenosynovitis during the convalescence stage. Clinicians should be aware of the late consequences of infection by the chikungunya virus (CHIKV) and recognize the possible association of subacute and chronic arthritis features. In addition, competent vectors of CHIKV, Aedes aegypti, can now be found in many temperate areas of the eastern and western hemispheres, including Japan. This fact raises concern that the virus could be introduced and become established in these areas. This necessitates an increased awareness of the disease, because imported cases are likely to contribute to the spread of CHIKV infection wherever the competent mosquito vectors are distributed.