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Chronic rheumatological manifestations similar to those of rheumatoid arthritis (RA) are described after chikungunya virus infection. We aimed to compare the relevance of joint counts and symptoms to clinical outcomes in RA and chronic chikungunya disease. Forty patients with chronic chikungunya arthralgia and 40 patients with RA were enrolled in a cross-sectional study. The association of tenderness and swelling, clinically assessed in 28 joints, and patient evaluations of pain and musculoskeletal stiffness with modified Health Assessment Questionnaire (HAQ) and quality of life (QoL) assessments were investigated. Tender and swollen joint counts, pain and stiffness scores were all associated with the HAQ disability index in RA (all r > 0.55, p ≤ 0.0002), but only stiffness was significantly associated with disability in chikungunya (r = 0.38, p = 0.02). Joint counts, pain and stiffness were also associated with most QoL domains in RA patients. In contrast, in chikungunya disease, tender joint counts were associated only with one QoL domain and swollen joints for none, while pain and stiffness were associated with several domains. Our results confirm the relevance of joint counts in RA, but suggest that in chronic chikungunya disease, joint counts have more limited value. Stiffness and pain score may be more important to quantify chikungunya arthritis impact.

Mosquito saliva facilitates blood feeding through the anti-haemostatic, anti-inflammatory and immunomodulatory properties of its proteins. However, the potential contribution of non-coding RNAs to host manipulation is still poorly understood. We analysed small RNAs from Aedes aegypti saliva and salivary glands and show here that chikungunya virus-infection triggers both the siRNA and piRNA antiviral pathways with limited effects on miRNA expression profiles. Saliva appears enriched in specific miRNA subsets and its miRNA content is well conserved among mosquitoes and ticks, clearly pointing to a non-random sorting and occurrence. Finally, we provide evidence that miRNAs from Ae. aegypti saliva may target human immune and inflammatory pathways, as indicated by prediction analysis and searching for experimentally validated targets of identical human miRNAs. Overall, we believe these observations convincingly support a scenario where both proteins and miRNAs from mosquito saliva are injected into vertebrates during blood feeding and contribute to the complex vector–host–pathogen interactions.

The mosquito Aedes albopictus is an invasive species first detected in Europe in Albania in 1979, and now established in 28 European countries. Temperature is a limiting factor in mosquito activities and in the transmission of associated arboviruses namely chikungunya (CHIKV) and dengue (DENV). Since 2007, local transmissions of CHIKV and DENV have been reported in mainland Europe, mainly in South Europe. Thus, the critical question is how far north transmission could occur. In this context, the Albanian infestation by Ae. albopictus is of interest because the species is present up to 1200 m of altitude; this allows using altitude as a proxy for latitude. Here we show that Ae. albopictus can transmit CHIKV at 28 °C as well as 20 °C, however, the transmission of DENV is only observed at 28 °C. We conclude that if temperature is the key environmental factor limiting transmission, then transmission of CHIKV, but not DENV is feasible in much of Europe.

Chikungunya virus (CHIKV) is the causative agent of Chikungunya fever, an acute febrile and arthritogenic illness with no effective treatments available. The development of effective therapeutic strategies could be significantly accelerated with detailed knowledge of the molecular components behind CHIKV replication. However, drug discovery is hindered by our incomplete understanding of their main components. The RNA-dependent RNA-polymerase (nsP4-CHIKV) is considered the key enzyme of the CHIKV replication complex and a suitable target for antiviral therapy. Herein, the nsP4-CHIKV was extensively characterized through experimental and computational biophysical methods. In the search for new molecules against CHIKV, a compound designated LabMol-309 was identified as a strong ligand of the nsp4-CHIKV and mapped to bind to its active site. The antiviral activity of LabMol-309 was evaluated in cellular-based assays using a CHIKV replicon system and a reporter virus. In conclusion, this study highlights the biophysical features of nsP4-CHIKV and identifies a new compound as a promising antiviral agent against CHIKV infection.

Significant attempts are being made worldwide in an attempt to develop a tool that, with a simple analysis, is capable of distinguishing between different arboviruses. Herein, we employ molecular fluorescence spectroscopy as a sensitive and specific rapid tool, with simple methodology response, capable of identifying spectral variations between serum samples with or without the dengue or chikungunya viruses. Towards this, excitation emission matrices (EEM) of clinical samples from patients with dengue or chikungunya, in addition to uninfected controls, were separated into a training or test set and analysed using multi-way classification models such as n-PLSDA, PARAFAC-LDA and PARAFAC-QDA. Results were evaluated based on calculations of accuracy, sensitivity, specificity and F score; the most efficient model was identified to be PARAFAC-QDA, whereby 100% was obtained for all figures of merit. QDA was able to predict all samples in the test set based on the scores present in the factors selected by PARAFAC. The loadings obtained by PARAFAC can be used in future studies to prove the direct or indirect relationship of spectral changes caused by the presence of these viruses. This study demonstrates that molecular fluorescence spectroscopy has a greater capacity to detect spectral variations related to the presence of such viruses when compared to more conventional techniques.

Aedes aegypti is the main vector of arboviral diseases such as dengue, chikungunya and Zika. A key feature for disease transmission modeling and vector control planning is adult mosquito dispersal. We studied Ae aegypti adult dispersal by conducting a mark-capture study of naturally occurring Ae. aegypti from discarded containers found along a canal that divided two residential communities in Donna, Texas, USA. Stable isotopes were used to enrich containers with either 13C or 15N. Adult mosquitoes were collected outdoors in the yards of households throughout the communities with BG Sentinel 2 traps during a 12-week period. Marked mosquito pools with stable isotopes were used to estimate the mean distance travelled using three different approaches (Net, Strip or Circular) and the probability of detecting an isotopically marked adult at different distances from the larval habitat of origin. We consistently observed, using the three approaches that male (Net: 220 m, Strip: 255 m, Circular: 250 m) Ae. aegypti dispersed further in comparison to gravid (Net: 135 m, Strip: 176 m, Circular: 189 m) and unfed females (Net: 192 m, Strip: 213 m, Circular: 198 m). We also observed that marked male capture probability slightly increased with distance, while, for both unfed and gravid females, such probability decreased with distance. Using a unique study design documenting adult dispersal from natural larval habitat, our results suggest that Ae. aegypti adults disperse longer distances than previously reported. These results may help guide local vector control authorities in their fight against Ae. aegypti and the diseases it transmits, suggesting coverage of 200 m for the use of insecticides and innovative vector control tools.

The mosquito Aedes aegypti is the primary vector of diseases such as dengue, Zika, chikungunya, and yellow fever. Improving control techniques requires a better understanding of the mosquito’s life cycle, including spatial population dynamics in endemic regions. One of the most promising techniques consists of introducing genetically modified male mosquitoes. Several models proposed to describe this technique present mathematical issues or rely on numerous parameters, making their application challenging to real-world situations. We propose a model describing the spatial population dynamics of the Aedes aegypti in the presence of genetically modified males. This model presents some mathematical improvements compared to the literature allowing deeper mathematical analysis. Moreover, this model relies on few parameters, which we show how to obtain or estimate from the literature. Through numerical simulations, we investigate the impacts of environmental heterogeneity, the periodicity of genetically modified male releases, and released genetically modified males quantity on the population dynamics of Aedes aegypti. The main results point to that the successful application of this vector control technique relies on releasing more than a critical amount of modified males with a frequency exceeding a specific critical value.

Chikungunya virus (CHIKV), a highly infectious and rapidly spread viral pathogen, is classified as a pathogenic agent at the biosafety level 3. Operation of live authentic CHIKV needs a specific laboratory with the P3 or above containment, which greatly confines the CHIKV-associated studies. To establish an evaluation system of CHIKV that can be utilized in a BSL2 laboratory, we constructed a pseudovirus (PsV) system of CHIKV containing double reporter genes (ZsGreen1 and luciferase). The fluorescent ZsGreen1 is a convenient and cheap reporter for monitoring the efficiency of transfection and titration of PsV. The enzyme luciferase is a sensitive reporter for the application of PsV to neutralization assay or drug screening. The CHIKV PsV produced in this study, with a titer of up to 3.16 × 106 TU/ml, was confirmed by Western blotting and transmission electronic microscopy (TEM). Finally, we developed a microneutralization assay with the CHIKV PsV produced in this study, which was successfully applied to evaluate neutralizing activities of convalescent sera from CHIKV-infected patients. In summary, we have established a convenient and sensitive double-reporter CHIKV pseudovirus system, which provides a safe and effective platform for screening anti-CHIKV drugs and evaluating vaccines against CHIKV.

Chebulinic acid (CA), originally isolated from the flower extract of the plant Terminalia chebula, has been shown to inhibit infection of herpes simplex virus-2 (HSV-2), suggestively by inhibiting the host entry step of viral infection. Like HSV-2, the dengue virus (DENV) and chikungunya virus (CHIKV) also use receptor glycosaminoglycans (GAG) to gain host entry, therefore, the activity of CA was tested against these viruses. Co-treatment of 8 µM CA with DENV-2 caused 2 log decrease in the virus titer (4.0 log10FFU/mL) at 120 h post infection, compared to virus control (5.95 log10FFU/mL). In contrast, no inhibitory effect of CA was observed against CHIKV infection under any condition. The mechanism of action of CA was investigated in silico by employing DENV-2 and CHIKV envelope glycoproteins. During docking, CA demonstrated equivalent binding at multiple sites on DENV-2 envelope protein, including GAG binding site, which have previously been reported to play a crucial role in host attachment and fusion, indicating blocking of these sites. However, CA did not show binding to the GAG binding site on envelope protein-2 of CHIKV. The in vitro and in silico findings suggest that CA possesses the ability to inhibit DENV-2 infection at the entry stage of its infection cycle and may be developed as a potential therapeutic agent against it.

Reverse transcription polymerase chain reaction (RT-PCR) is the gold standard for the molecular diagnosis of many infectious diseases, including RNA viruses, but is generally limited to settings with access to trained personnel and laboratory resources. We have previously reported a fundamentally simpler thermal cycling platform called Adaptive PCR, which dynamically controls thermal cycling conditions during each cycle by optically monitoring the annealing and melting of mirror-image L-DNA surrogates of the PCR primers and targets. In this report, we integrate optically-controlled reverse transcription and single-channel monitoring of L-DNAs to develop a multiplexed Adaptive RT-PCR instrument and assay for the detection of Zika, dengue, and chikungunya virus RNA with high target specific and low limits of detection. The assay is demonstrated to detect as low as 5 copies/reaction of Zika or chikungunya RNA and 50 copies/reaction of dengue RNA. The multiplexed Adaptive RT-PCR instrument is robust and has many of the features required to implement diagnostic assays for RNA viruses in settings that lack traditional laboratory resources.

Brazil, which is hyperendemic for dengue virus (DENV), has had recent Zika (ZIKV) and (CHIKV) Chikungunya virus outbreaks. Since March 2016, CHIKV is the arbovirus infection most frequently diagnosed in Rio de Janeiro. In the analysis of 1835 syndromic patients, screened by real time RT-PCR, 56.4% of the cases were attributed to CHIKV, 29.6% to ZIKV, and 14.1% to DENV-4. Sequence analyses of CHIKV from sixteen samples revealed that the East-Central-South-African (ECSA) genotype of CHIKV has been circulating in Brazil since 2013 [95% bayesian credible interval (BCI): 03/2012-10/2013], almost a year before it was detected by arbovirus surveillance program. Brazilian cases are related to Central African Republic sequences from 1980’s. To the best of our knowledge, given the available sequence published here and elsewhere, the ECSA genotype was likely introduced to Rio de Janeiro early on 2014 (02/2014; BCI: 07/2013-08/2014) through a single event, after primary circulation in the Bahia state at the Northestern Brazil in the previous year. The observation that the ECSA genotype of CHIKV was circulating undetected underscores the need for improvements in molecular methods for viral surveillance.

Chikungunya fever, caused by chikungunya virus (CHIKV) and transmitted to humans by infected Aedes mosquitoes, has posed a global threat in several countries in 2015. Recent outbreaks in La Réunion, Italy and China are related with a new variant of CHIKV with shorter extrinsic incubation period in contaminated mosquitoes, but the role of this new variant on the spread of chikungunya fever is unclear. We develop a mathematical model that incorporates the virus mutation dynamics in the transmission of CHIKV among mosquitoes and humans. Our numerical simulations show that a substantial virus mutation rate combined with high virus transmission probabilities from mosquito to human, could result in sustainable chikungunya fever outbreaks. Further, we apply Markov Chain Monte Carlo sampling method to fit our model to the 2007 chikungunya fever outbreak data in North-Eastern Italy where the mutant strain was detected. We conclude that the basic reproduction number might be underestimated without considering the mutation dynamics, and our estimation shows that the basic reproduction number of the 2007 Italy outbreak was $${\pmb{\mathscr{R}}}_{{\bf{0}}}$$ R 0 = 2.035[95%Cl: 1.9424 - 2.1366]. Sensitivity analysis shows that the transmission rate of the mutant strain from mosquitoes to human is more influential on $${\pmb{\mathscr{R}}}_{{\bf{0}}}$$ R 0 than the shortened extrinsic incubation period. We conclude that the virus mutation dynamics could play an important role in the transmission of CHIKV, and there is a crucial need to better understand the mutation mechanism.

Reverse genetics is an important tool in the elucidation of viral replication and the development of countermeasures; however, these methods are impeded by laborious and inefficient replicon delivery methods. This paper demonstrates the use of a baculovirus to facilitate the efficient delivery of autonomous CHIKV replicons into mosquito and mammalian cells in vitro as well as adult mosquitoes in vivo. The efficacy of this approach was verified via co-localization among an eGFP reporter, nsP1, and dsRNA as well as through the inhibition of an RNA-dependent RNA polymerase (RdRp) null mutation (DDAA) in nsP4, or the treatment of a known antiviral compound (6-azauridine). We also investigated the correlation between CHIKV replicon-launched eGFP expression and the effectiveness of CHIKV replicon variants in inducing IFN-β expression in human cell lines. This delivery method based on a single vector is applicable to mosquito and mammalian cells in seeking to decipher the mechanisms underlying CHIKV replication, elucidate virus–host interactions, and develop antivirals. This study presents an effective alternative to overcome many of the technological issues related to the study and utilization of autonomous arbovirus replicons.

We describe a novel vaccine platform that can generate protective immunity to chikungunya virus (CHIKV) in C57BL/6J mice after a single immunization by employing an infectious RNA (iRNA), which upon introduction into a host cell launches an infectious attenuated virus. We and others have previously reported that an engineered deletion of 183 nucleotides in the nsP3 gene attenuates chikungunya virus (CHIKV) and reduces in vivo viral replication and viremia after challenge in mice, macaques and man. Here, we demonstrated that in vitro transfection of iRNA carrying the nsP3 deletion generated infectious viruses, and after intramuscular injection, the iRNA induced robust antibody responses in mice. The iRNA was superior at eliciting binding and neutralizing antibody responses as compared to a DNA vaccine encoding the same RNA (iDNA) or a non-propagating RNA replicon (RREP) lacking the capsid encoding gene. Subsequent challenge with a high dose of CHIKV demonstrated that the antibody responses induced by this vaccine candidate protected animals from viremia. The iRNA approach constitutes a novel vaccine platform with the potential to impact the spread of CHIKV. Moreover, we believe that this approach is likely applicable also to other positive-strand viruses.

The molecular detection system has evolved over last two decades and is rapidly replacing the conventional confirmatory techniques in diagnostic virology. However the major limitation in implementation of available molecular detection assays is the non availability of field deployable nucleic acid isolation platform coupled with gene amplification technique. The rapid and early molecular detection is crucial for employing effective measure against many viral infections. The re-emergence of chikungunya virus (CHIKV) has led to epidemics since 2004 in several parts of the world including India. The main association of CHIKV with severe arthritis and long-lasting arthralgia and closely mimics symptoms of Dengue and Zika virus infection requiring laboratory confirmation. In this study, a simple magnetic bead based ribonucleic acid extraction method was optimized, which was coupled with isothermal polymerase spiral reaction (PSR) technique for early and rapid detection. Subsequently, the polymerase spiral reaction reagents were converted to dry down format that led to a rapid user friendly field compatible sample processing to answer method for rapid and onsite detection of Chikungunya virus. Both the methods were evaluated with a panel of clinical samples. The sensitivity of the assays were compared with available commercial viral RNA extraction platform and qRT-PCR. The in-house nucleic acid extraction system based on magnetic bead followed by dry down RT-Polymerase Spiral Reaction assay was found to be highly sensitive with 10 copies of RNA as limit of detection in CHIKV clinical specimens. With respect to other closely related viruses no cross reactivity was observed. This novel methodology has the potential to revolutionize the diagnosis of infectious agents in resource limited settings around the world.

Chikungunya and Zika are arboviruses transmitted by the mosquito Aedes aegypti. Mosquito fecundity and egg viability are important parameters of vectorial capacity. Here we aim to understand, comparatively, the effects of Chikungunya virus (CHIKV) and Zika virus (ZIKV) infections on the fecundity and fertility of young and old Aedes aegypti females. Using artificial infection blood feeding experiments we observed that both CHIKV and ZIKV do not alter the number of eggs laid when compared to uninfected females, although the egg fertility significantly decreases in both young and old CHIKV-infected females. There is an upward trend of null females (infertile females) from 2.1% in young to 6.8% in old ZIKV-infected females. Together, our data revealed that CHIKV and ZIKV affects differently Ae. aegypti physiology, that may be related to different viral spread in nature.

New epidemics of infectious diseases can emerge any time, as illustrated by the emergence of chikungunya virus (CHIKV) and Zika virus (ZIKV) in Latin America. During new epidemics, public health officials face difficult decisions regarding spatial targeting of interventions to optimally allocate limited resources. We used a large-scale, data-driven, agent-based simulation model (ABM) to explore CHIKV mitigation strategies, including strategies based on previous DENV outbreaks. Our model represents CHIKV transmission in a realistic population of Colombia with 45 million individuals in 10.6 million households, schools, and workplaces. Our model uses high-resolution probability maps for the occurrence of the Ae. aegypti mosquito vector to estimate mosquito density in Colombia. We found that vector control in all 521 municipalities with mosquito populations led to 402,940 fewer clinical cases of CHIKV compared to a baseline scenario without intervention. We also explored using data about previous dengue virus (DENV) epidemics to inform CHIKV mitigation strategies. Compared to the baseline scenario, 314,437 fewer cases occurred when we simulated vector control only in 301 municipalities that had previously reported DENV, illustrating the value of available data from previous outbreaks. When varying the implementation parameters for vector control, we found that faster implementation and scale-up of vector control led to the greatest proportionate reduction in cases. Using available data for epidemic simulations can strengthen decision making against new epidemic threats.

International audience; Arboviral diseases such as chikungunya, dengue, and Zika viruses have been threatening the European countries since the introduction in 1979 of the major vector Aedes albopictus. In 2017, more than three hundred of CHIKV autochthonous cases were reported in Italy, highlighting the urgent need for a risk assessment of arboviral diseases in European countries. In this study, the vector competence for three major arboviruses were analyzed in eight Ae. albopictus populations from europe. Here we show that Southern european Ae. albopictus were susceptible to CHIKV, DENV-1 and ZIKV with the highest vector competence for CHIKV. Based on vector competence data and vector distribution, a prediction risk map for CHIKV was generated stressing the fear of CHIKV and to a lesser extent, of other arboviruses for Europe, calling us for new public health strategies. Recent epidemics involving several arboviral diseases such as chikungunya (CHIKV), dengue (DENV) and Zika (ZIKV) have received global attention as major public health issues 1,2. The Aedes spp. mosquitoes, main vectors of these arboviruses, have extended their distribution owing to human activities (trade and travels) and climate change; they are no longer restricted to tropical regions and have initiated the invasion of European regions 3-6. Although Europe was not considered prone to arboviral diseases, the presence of newly introduced competent mosquitoes coupled to a growing number of imported cases 7 led to local transmissions of chikungunya and den-gue fever in Croatia 8,9 , France 10-14 , and Italy 15. The 2017 CHIKV outbreak in Italy was attributed to few incident cases in Anzio in June, subsequently spreading to Guardavalle Marina later and to Rome in October. A total of 337 infections were reported, 61 of which occurred in the capital city of Rome 16. This outbreak exemplifies that the presence of Ae. albopictus in Italy favors the occurrence of CHIKV epidemics 16. Mosquitoes are able to transmit arboviruses by acquiring a viremic blood meal from an infected host. Along with blood, the ingested virus enters in the mosquito midgut and infects the epithelial cells that may in turn disseminate the virus to the mosquito internal tissues or organs. After the extrinsic incubation period (EIP) 17-19 , the virus may reach the salivary glands where the viral cycle in the vector ends up with the virus transmitted to the host by the mosquito saliva 20. The virus efficiency to cross each anatomical barrier (midgut and salivary glands) depends on several genetic or biological factors regulating mosquito antiviral immunity 21. Geographic populations of a same mosquito species may not share the same immunological background, leading to varying susceptibilities to transmit arboviruses 22. A vector competence analysis of European Ae. albopictus populations is critical for assessing the risk of arbovi-ral diseases outbreaks as the establishment of competent mosquitoes can become the breeding ground for various arboviruses. To this aim, we collected eight European Ae. albopictus populations from Croatia, Greece, Italy, Montenegro, and Switzerland, and experimentally infected them with three arboviruses, CHIKV (Alphavirus, Togaviridae), and DENV and ZIKV (Flavivirus, Flaviviridae). Based on data obtained, we elaborated a vector competence data-driven prediction for CHIKV transmission using computational modeling to assist in evaluating the current risk of arboviral diseases transmission in Southern Europe.

Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Goes, Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Goes, Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Biologia. Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Goes, Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Bioqu?mica M?dica Leopoldo de Meis. Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Universidade do Estado do Par?. Departamento de Morfologia e Ci?ncias Fisiol?gicas. Centro de Ci?ncias Biol?gicas e da Sa?de. Bel?m, PA, Brazil. Universidade Federal do Rio de Janeiro. N?cleo Multidisciplinar de Pesquisas. Polo Avan?ado de Xer?m. Duque de Caxias, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Goes, Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro. N?cleo Multidisciplinar de Pesquisas. Polo Avan?ado de Xer?m. Duque de Caxias, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Biologia. Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Bioqu?mica M?dica Leopoldo de Meis. Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Goes, Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil. Universidade Federal do Rio de Janeiro. Instituto de Bioqu?mica M?dica Leopoldo de Meis. Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil Universidade Federal do Rio de Janeiro. Instituto de Bioqu?mica M?dica Leopoldo de Meis. Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Goes, Centro de Ci?ncias da Sa?de. Rio de Janeiro, RJ, Brazil. The global situation of diseases transmitted by arthropod-borne viruses such as Dengue (DENV), Yellow Fever (YFV), Chikungunya (CHIKV) and Zika (ZIKV) viruses is alarming and treatment of human infection by these arboviruses faces several challenges. The discovery of broad-spectrum antiviral molecules, able to inactivate different groups of viruses, is an interesting approach. The viral envelope is a common structure among arboviruses, being a potential target for antivirals. Porphyrins are amphipathic molecules able to interact with membranes and absorb light, being widely used in photodynamic therapy. Previously, we showed that heme, Co-protoporphyrin IX (CoPPIX) and Sn-protoporphyrin IX (SnPPIX) directly inactivate DENV and YFV infectious particles. Here we demonstrate that the antiviral activity of these porphyrins can be broadened to CHIKV, ZIKV, Mayaro virus, Sindbis virus and Vesicular Stomatitis virus. Porphyrin treatment causes viral envelope protein loss, affecting viral morphology, adsorption and entry into target cells. Also, light-stimulation enhanced the SnPPIX activity against all tested arboviruses. In summary, CoPPIX and SnPPIX were shown to be efficient broad-spectrum compounds to inactivate medically and veterinary important viruses.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. The outbreak of CHIKV infection has been seen in many tropical and subtropical regions of the biosphere. Current reports evidenced that after outbreaks in 2005–06, the fitness of this virus propagating in Aedes albopictus enhanced due to the epistatic mutational changes in its envelope protein. In our study, we evaluated the prevalence of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) in CHIKV proteome. IDPs/IDPRs are known as members of a ‘Dark Proteome’ that defined as a set of polypeptide segments or whole protein without unique three-dimensional structure within the cellular milieu but with significant biological functions, such as cell cycle regulation, control of signaling pathways, and maintenance of viral proteomes. However, the intrinsically disordered aspects of CHIKV proteome and roles of IDPs/IDPRs in the pathogenic mechanism of this important virus have not been evaluated as of yet. There are no existing reports on the analysis of intrinsic disorder status of CHIKV. To fulfil this goal, we have analyzed the abundance and functionality of IDPs/IDPRs in CHIKV proteins, involved in the replication and maturation. It is likely that these IDPs/IDPRs can serve as novel targets for disorder based drug design.

Mayaro virus (MAYV) causes an acute febrile illness similar to that produced by chikungunya virus (CHIKV), an evolutionary relative in the Semliki Forest virus complex of alphaviruses. MAYV emergence is typically sporadic, but recent isolations and outbreaks indicate that the virus remains a public health concern. Given the close phylogenetic and antigenic relationship between CHIKV and MAYV, and widespread distribution of CHIKV, we hypothesized that prior CHIKV immunity may affect MAYV pathogenesis and/or influence its emergence potential. We pre-exposed immunocompetent C57BL/6 and immunocompromised A129 or IFNAR mice to wild-type CHIKV, two CHIKV vaccines, or a live-attenuated MAYV vaccine, and challenged with MAYV. We observed strong cross-protection against MAYV for mice pre-exposed to wild-type CHIKV, and moderately but significantly reduced cross-protection from CHIKV-vaccinated animals. Immunity to other alphavirus or flavivirus controls provided no protection against MAYV disease or viremia. Mechanistic studies suggested that neutralizing antibodies alone can mediate this protection, with T-cells having no significant effect on diminishing disease. Finally, human sera obtained from naturally acquired CHIKV infection cross-neutralized MAYV at high titers in vitro. Altogether, our data suggest that CHIKV infection can confer cross-protective effects against MAYV, and the resultant reduction in viremia may limit the emergence potential of MAYV.

Chikungunya virus (CHIKV) is considered a public health problem due to its rapid spread and high morbidity. This study aimed to determine the genetic diversity and phylogenetic relationships of CHIKVs in Colombia. A descriptive and retrospective study was carried out using sera of patients infected with Chikungunya during the outbreak in Colombia. The whole genomes of CHIKV (n = 16) were sequenced with an Illumina Hi-seq 2500 and were assembled using the Iterative Virus Assembler software. A Bayesian inference phylogenetic analysis was carried out with 157 strains of worldwide origin. The Colombian CHIKV sequences were grouped in the Asian genotype; however, three independent phylogenetic subclades were observed, probably the result of three separate introductions from Panama, Nicaragua, and St. Barts. Each subclade showed several different non-synonymous mutations (nsP2-A153V; nsp2-Y543H; nsp2-G720A; nsP3-L458P; Capside R78Q), that may have functional consequences for CHIKV biology and pathogenesis. These same mutations may affect the efficacy of potential CHIKV vaccines.

Zika virus infection and dengue and chikungunya fevers are emerging viral diseases that have become public health threats. Their aetiologic agents are transmitted by the bite of genus Aedes mosquitoes. Without effective therapies or vaccines, vector control is the main strategy for preventing the spread of these diseases. Increased insecticide resistance calls for biorational actions focused on control of the target vector population. The chitin required for larval survival structures is a good target for biorational control. Chitin synthases A and B (CHS) are enzymes in the chitin synthesis pathway. Double-stranded RNA (dsRNA)-mediated gene silencing (RNAi) achieves specific knockdown of target proteins. Our goal in this work, a new proposed RNAi-based bioinsecticide, was developed as a potential strategy for mosquito population control. DsRNA molecules that target five different regions in the CHSA and B transcript sequences were produced in vitro and in vivo through expression in E. coli HT115 and tested by direct addition to larval breeding water. Mature and immature larvae treated with dsRNA targeting CHS catalytic sites showed significantly decreased viability associated with a reduction in CHS transcript levels. The few larval and adult survivors displayed an altered morphology and chitin content. In association with diflubenzuron, this bioinsecticide exhibited insecticidal adjuvant properties.

The lack of vaccines and antiviral treatment, along with the increasing number of cases of Zika virus (ZIKV) and Chikungunya virus (CHIKV) infections, emphasize the need for searching for new therapeutic strategies. In this context, the marine brown seaweed Canistrocarpus cervicornis has been proved to hold great antiviral potential. Hence, the aim of this work was to evaluate the anti-ZIKV and anti-CHIKV activity of a marine dolastane isolated from brown seaweed C. cervicornis and its crude extract. Vero cells were used in antiviral assays, submitted to ZIKV and CHIKV, and treated with different concentrations of C. cervicornis extract or dolastane. The crude extract of C. cervicornis showed inhibitory activities for both ZIKV and CHIKV, with EC50 values of 3.3 μg/mL and 3.1 μg/mL, respectively. However, the isolated dolastane showed a more significant and promising inhibitory effect (EC50 = 0.95 µM for ZIKV and 1.3 µM for CHIKV) when compared to both the crude extract and ribavirin, which was used as control. Also, the dolastane showed a very potent virucidal activity against CHIKV and was able to inhibit around 90% of the virus infectivity at 10 μM. For the ZIKV, the effects were somewhat lower, although interesting, at approximately 64% in this same concentration. Further, we observed that both the extract and the dolastane were able to inhibit the replication of ZIKV and CHIKV at different times of addition post-infection, remaining efficient even if added after 8 hours post-infection, but declining soon after. A synergistic effect using sub-doses of the extract and isolates was associated with ribavirin, inhibiting above 80% replication even at the lowest concentrations. Therefore, this work has unveiled the anti-ZIKV and CHIKV potential of C. cervicornis crude extract and an isolated dolastane, which, in turn, can be used as a preventive or therapeutic strategy in the future.

The Chikungunya (CHIK) virus is epidemic in Brazil, with 170,000 cases in the first half of 2016. More than 60% of patients present relapsing and remitting chronic arthralgia with debilitating pain lasting years. There are no specific therapeutic agents to treat and rehabilitee infected persons with CHIK. Persistent pain can lead to incapacitation, requiring long-term pharmacological treatment. Advances in non-pharmacological treatments are necessary to promote pain relief without side effects and to restore functionality. Clinical trials indicate transcranial direct current stimulation (tDCS) can treat a broad range of chronic pain disorders, including diffuse neuromuscular pain and arthralgia. Here, we demonstrate that the tDCS across the primary motor cortex significantly reduces pain in the chronic phase of CHIK. High-resolution computational model was created to analyze the cortical electric field generated during tDCS and a diffuse and clustered brain current flow including M1 ipsilateral and contralateral, left DLPFC, nucleus accumbens, and cingulate was found. Our findings suggest tDCS could be an effective, inexpensive and deployable therapy to areas lacking resources with a significant number of patients with chronic CHIK persistent pain.

Development of platforms for a reliable, rapid, sensitive and selective detection of chikungunya virus (CHIGV) is the need of the hour in developing countries. To the best of our knowledge, there are no reports available for the electrochemical detection of CHIGVDNA. Therefore, we aim at developing a biosensor based on molybdenum disulphide nanosheets (MoS2 NSs) for the point-of-care diagnosis of CHIGV. Briefly, MoS2 NSs were synthesized by chemical route and characterized using scanning electron microscopy, transmission electron microscopy, UV-Vis spectroscopy, Raman spectroscopy and X-Ray Diffraction. MoS2 NSs were then subjected to physical adsorption onto the screen printed gold electrodes (SPGEs) and then employed for the detection of CHIGV DNA using electrochemical voltammetric techniques. Herein, the role of MoS2 NSs is to provide biocompatibility to the biological recognition element on the surface of the screen printed electrodes. The detection strategy employed herein is the ability of methylene blue to interact differentially with the guanine bases of the single and double-stranded DNA which leads to change in the magnitude of the voltammetric signal. The proposed genosensor exhibited a wide linear range of 0.1 nM to 100 µM towards the chikungunya virus DNA.

Aedes aegypti is the primary urban mosquito vector of viruses causing dengue, Zika and chikungunya fevers –for which vaccines and effective pharmaceuticals are still lacking. Current strategies to suppress arbovirus outbreaks include removal of larval-breeding sites and insecticide treatment of larval and adult populations. Insecticidal control of Ae. aegypti is challenging, due to a recent rapid global increase in knockdown-resistance (kdr) to pyrethroid insecticides. Widespread, heavy use of pyrethroid space-sprays has created an immense selection pressure for kdr, which is primarily under the control of the voltage-gated sodium channel gene (vgsc). To date, eleven replacements in vgsc have been discovered, published and shown to be associated with pyrethroid resistance to varying degrees. In Mexico, F1,534C and V1,016I have co-evolved in the last 16 years across Ae. aegypti populations. Recently, a novel replacement V410L was identified in Brazil and its effect on vgsc was confirmed by electrophysiology. Herein, we screened V410L in 25 Ae. aegypti historical collections from Mexico, the first heterozygote appeared in 2002 and frequencies have increased in the last 16 years alongside V1,016I and F1,534C. Knowledge of the specific vgsc replacements and their interaction to confer resistance is essential to predict and to develop strategies for resistance management.

Outbreaks of arbovirus infections vectored by invasive Aedes albopictus have already occurred and are predicted to become increasingly frequent in Southern Europe. We present a probabilistic model to assess risk of arbovirus outbreaks based on incident cases worldwide, on the probability of arrival of infected travelers, and on the abundance of the vector species. Our results show a significant risk of Chikungunya outbreak in Rome from mid June to October in simulations with high human biting rates (i.e. when ≥50% of the population is bitten every day). The outbreak risk is predicted to be highest for Chikungunya and null for Zika. Simulated increase of incident cases in selected endemic countries has no major impact on the outbreak risk. The model correctly estimated the number of imported cases and can be easily adapted to other urban areas where Ae. albopictus is the only potential vector present.

Chikungunya virus (CHIKV) is a mosquito-borne virus, which has created an alarming threat in the world due to unavailability of vaccine and antiviral compounds. The CHIKV nsP2 contains ATPase, RTPase, helicase and protease activities, whereas, nsP1 is a viral capping enzyme. In alphaviruses, the four non-structural proteins form the replication complex in the cytoplasm and this study characterizes the interaction between CHIKV nsP1 and nsP2. It was observed that, both the proteins co-localize in the cytoplasm and interact in the CHIKV infected cells by confocal microscopy and immunoprecipitation assay. Further, it was demonstrated through mutational analysis that, the amino acids 1-95 of nsP2 and 170-288 of nsP1 are responsible for their direct interaction. Additionally, it was noticed that, the ATPase activity of nsP2 is enhanced in the presence of nsP1, indicating the functional significance of this interaction. In silico analysis showed close (≤1.7 Å) polar interaction (hydrogen bond) between Glu4, Arg7, 96, 225 of nsP2 with Lys256, 206, Val367 and Phe312 of nsP1 respectively. Hence, this investigation provides molecular characterization of CHIKV nsP1-nsP2 interaction which might be a useful target for rational designing of antiviral drugs.

Chikungunya virus (CHIKV), a mosquito-borne pathogen, consists of three genotypes: East/Central/South African (ECSA), West African (WA), and Asian. Although a current rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity to ECSA-genotype viruses, it showed poor performance against the Asian-genotype virus that is spreading in the American continents. To understand the basis for the low performance of this IC test against Asian-genotype virus, we re-examined the anti-CHIKV monoclonal antibodies (mAbs) used in the assay for their interaction with E1-antigen of the three CHIKV genotypes. We found that the reactivity of one mAb for Asian-genotype virus was lower than that for ECSA virus. Comparison of E1 amino acid sequences revealed that the ECSA virus used to generate these mAbs possesses glutamic acid (E) at position 350, in contrast to WA and Asian, which possess aspartic acid (D) at this position. Site-directed mutagenesis confirmed that the mutation altered mAb reactivity, since E-to-D substitution at position 350 in ECSA reduced recognition by the mAb, while D-to-E substitution at this position in Asian and WA increased affinity for the mAb. Taken together, these results indicate that residue 350 of the CHIKV 6K-E1 is a key element affecting the performance of this IC assay.

Arboviruses that replicate in mosquitoes activate innate immune response within mosquitoes. Regulatory non-coding microRNAs (miRNA) are known to be modulated in mosquitoes during chikungunya infection. However, information about targets of these miRNAs is scant. The present study was aimed to identify and analyze targets of miRNAs that are regulated during chikungunya virus (CHIKV) replication in Aedes aegypti cells and in the mosquito. Employing next-generation sequencing technologies, we identified a total of 126 miRNAs from the Ae. aegypti cell line Aag2. Of these, 13 miRNAs were found to be regulated during CHIKV infection. Putative targets of three of the most significantly regulated miRNAs- miR-100, miR-2b and miR-989 were also analyzed using quantitative PCRs, in cell lines and in mosquitoes, to validate whether they were the targets of the miRNAs. Our study expanded the list of miRNAs known in Ae. aegypti and predicted targets for the significantly regulated miRNAs. Further analysis of some of these targets revealed that ubiquitin-related modifier is a target of miRNA miR-2b and plays a significant role in chikungunya replication.

It has been observed that replication of Chikungunya virus (CHIKV) in C6/36 Aedes albopictus cells has little effect on virus evolution. To characterize evolutionary patterns associated with CHIKV replication in mosquito cells, we performed serial passages of the LR2006 strain in Ae. albopictus cells (75 and 30 passages in C6/36 and U4.4 respectively) and Ae. aegypti cells (100 passages in AA-A20 and in AE) and studied genotypic changes accompanying adaptation during this evolutionary process. Quantitative analysis revealed cell specific patterns: low mutation rates in C6/36 cells except when a CHIKV strain pre-adapted to mammalian was used and typical features of adaptation to cell culture conditions with a high number of fixed mutations in AE and AA-A20 cells probably due to the weak permissiveness of these latter cell lines. Altogether, these results suggested that both cell line and viral strain influence rates of viral evolution. In contrast, characteristics and distribution of mutations were qualitatively very similar in all mosquito cells with a high level of parallel evolution including 4 deletion mutations. Serial passage in mammalian cells of viruses pre-adapted to mosquito cells revealed disappearance of almost all shared mutations suggesting that many of these mutational patterns are vector-specific.

The arrival and rapid spread of the mosquito-borne viral disease Chikungunya across the Americas is one of the most significant public health developments of recent years, preceding and mirroring the subsequent spread of Zika. Globalization in trade and travel can lead to the importation of these viruses, but climatic conditions strongly affect the efficiency of transmission in local settings. In order to direct preparedness for future outbreaks, it is necessary to anticipate global regions that could become suitable for Chikungunya transmission. Here, we present global correlative niche models for autochthonous Chikungunya transmission. These models were used as the basis for projections under the representative concentration pathway (RCP) 4.5 and 8.5 climate change scenarios. In a further step, hazard maps, which account for population densities, were produced. The baseline models successfully delineate current areas of active Chikungunya transmission. Projections under the RCP 4.5 and 8.5 scenarios suggest the likelihood of expansion of transmission-suitable areas in many parts of the world, including China, sub-Saharan Africa, South America, the United States and continental Europe. The models presented here can be used to inform public health preparedness planning in a highly interconnected world.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes febrile disease, muscle and joint pain, which can become chronic in some individuals. The non-structural protein 3 (nsP3) plays essential roles during infection, but a complete understanding of its function is lacking. Here we used a microscopy-based approach to image CHIKV nsP3 inside human cells. The SNAP system consists of a self-labelling enzyme tag, which catalyses the covalent linking of exogenously supplemented synthetic ligands. Genetic insertion of this tag resulted in viable replicons and specific labelling while preserving the effect of nsP3 on stress granule responses and co-localisation with GTPase Activating Protein (SH3 domain) Binding Proteins (G3BPs). With sub-diffraction, three-dimensional, optical imaging, we visualised nsP3-positive structures with variable density and morphology, including high-density rod-like structures, large spherical granules, and small, low-density structures. Next, we confirmed the utility of the SNAP-tag for studying protein turnover by pulse-chase labelling. We also revealed an association of nsP3 with cellular lipid droplets and examined the spatial relationships between nsP3 and the non-structural protein 1 (nsP1). Together, our study provides a sensitive, specific, and versatile system for fundamental research into the individual functions of a viral non-structural protein during infection with a medically important arthropod-borne virus (arbovirus).

Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.

Chikungunya and dengue are arboviral infections with overlapping clinical symptoms. A subset of chikungunya infection occurs also as co-infections with dengue, resulting in complications during diagnosis and patient management. The present study was undertaken to identify the global metabolome of patient sera infected with chikungunya as mono infections and with dengue as co-infections. Using nuclear magnetic resonance (NMR) spectroscopy, the metabolome of sera of three disease conditions, namely, chikungunya and dengue as mono-infections and when co-infected were ascertained and compared with healthy individuals. Further, the cohorts were analyzed on the basis of age, onset of fever and joint involvement. Here we show that many metabolites in the serum are significantly differentially regulated during chikungunya mono-infection as well as during chikungunya co-infection with dengue. We observed that glycine, serine, threonine, galactose and pyrimidine metabolisms are the most perturbed pathways in both mono and co-infection conditions. The affected pathways in our study correlate well with the clinical manifestation like fever, inflammation, energy deprivation and joint pain during the infections. These results may serve as a starting point for validations and identification of distinct biomolecules that could be exploited as biomarker candidates thereby helping in better patient management.

Monocyte chemoattractant protein-1 (MCP-1/CCL2)-mediated migration of monocytes is essential for immunological surveillance of tissues. During chikungunya virus (CHIKV) infection however, excessive production of MCP-1 has been linked to disease pathogenesis. High MCP-1 serum levels are detected during the viremic phase of CHIKV infection and correlate with the virus titre. In vitro CHIKV infection was also shown to stimulate MCP-1 production in whole blood; yet the role and the mechanism of MCP-1 production upon infection of human peripheral blood mononuclear cells remain unknown. Here we found that active CHIKV infection stimulated production of MCP-1 in monocytes. Importantly however, we found that communication with other leukocytes is crucial to yield MCP-1 by monocytes upon CHIKV infection. Indeed, blocking interferon-α/β receptor or the JAK1/JAK2 signalling downstream of the receptor abolished CHIKV-mediated MCP-1 production. Additionally, we show that despite the apparent correlation between IFN type I, CHIKV replication and MCP-1, modulating the levels of the chemokine did not influence CHIKV infection. In summary, our data disclose the complexity of MCP-1 regulation upon CHIKV infection and point to a crucial role of IFNβ in the chemokine secretion. We propose that balance between these soluble factors is imperative for an appropriate host response to CHIKV infection.

Most arthropod-borne viruses (arboviruses), perpetuated by alternation between a vertebrate host and an insect vector, are likely to emerge through minor genetic changes enabling the virus to adapt to new hosts. In the past decade, chikungunya virus (CHIKV; Alphavirus, Togaviridae) has emerged on La Réunion Island following the selection of a unique substitution in the CHIKV E1 envelope glycoprotein (E1-A226V) of an East-Central-South African (ECSA) genotype conferring a higher transmission rate by the mosquito Aedes albopictus. Assumed to have occurred independently on at least four separate occasions, this evolutionary convergence was suspected to be responsible for CHIKV worldwide expansion. However, assumptions on CHIKV emergence were mainly based on viral genetic changes and the role of the mosquito population quasispecies remained unexplored. Here we show that the nature of the vector population is pivotal in selecting the epidemic CHIKV. We demonstrate using microsatellites mosquito genotyping that Ae. albopictus populations are genetically differentiated, contributing to explain their differential ability to select the E1-226V mutation. Aedes albopictus, newly introduced in Congo coinciding with the first CHIKV outbreak, was not able to select the substitution E1-A226V nor to preferentially transmit a CHIKV clone harboring the E1-226V as did Ae. albopictus from La Réunion.

Chikungunya, a re-emerging arbovirus transmitted to humans by Aedes aegypti and Ae. albopictus mosquitoes, causes debilitating disease characterized by an acute febrile phase and chronic joint pain. Chikungunya has recently spread to the island of St. Martin and subsequently throughout the Americas. The disease is now affecting 42 countries and territories throughout the Americas. While chikungunya is mainly a tropical disease, the recent introduction and subsequent spread of Ae. albopictus into temperate regions has increased the threat of chikungunya outbreaks beyond the tropics. Given that there are currently no vaccines or treatments for chikungunya, vector control remains the primary measure to curtail transmission. To investigate the effectiveness of a containment strategy that combines disease surveillance, localized vector control and transmission reduction measures, we developed a model of chikungunya transmission dynamics within a large residential neighborhood, explicitly accounting for human and mosquito movement. Our findings indicate that prompt targeted vector control efforts combined with measures to reduce transmission from symptomatic cases to mosquitoes may be highly effective approaches for controlling outbreaks of chikungunya, provided that sufficient detection of chikungunya cases can be achieved.

Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia and is now considered endemic in countries across Asia and Africa. The tissue tropism of CHIKV infection in humans remains, however, ill-defined. Due to the fact that myositis is commonly observed in most patients infected with CHIKV, we sought to develop a clinically relevant cellular model to better understand the pathogenesis of CHIKV infection. In this study, primary human skeletal muscle myoblasts (HSMM) were established as a novel human primary cell line that is highly permissive to CHIKV infection, with maximal amounts of infectious virions observed at 16 hours post infection. Genome-wide microarray profiling analyses were subsequently performed to identify and map genes that are differentially expressed upon CHIKV infection. Infection of HSMM cells with CHIKV resulted in altered expressions of host genes involved in skeletal- and muscular-associated disorders, innate immune responses, cellular growth and death, host metabolism and virus replication. Together, this study has shown the establishment of a clinically relevant primary human cell model that paves the way for the further analysis of host factors and their involvement in the various stages of CHIKV replication cycle and viral pathogenesis.

Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z’ factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 103 M−1 sec−1 respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that has recently engendered large epidemics around the world. There is no specific antiviral for treatment of patients infected with CHIKV and development of compounds with significant anti-CHIKV activity that can be further developed to a practical therapy is urgently required. Andrographolide is derived from Andrographis paniculata, a herb traditionally used to treat a number of conditions including infections. This study sought to determine the potential of andrographolide as an inhibitor of CHIKV infection. Andrographolide showed good inhibition of CHIKV infection and reduced virus production by approximately 3log10 with a 50% effective concentration (EC50) of 77 μM without cytotoxicity. Time-of-addition and RNA transfection studies showed that andrographolide affected CHIKV replication and the activity of andrographolide was shown to be cell type independent. This study suggests that andrographolide has the potential to be developed further as an anti-CHIKV therapeutic agent.

Outbreaks involving the Asian genotype Chikungunya virus (CHIKV) caused over one million infections in the Americas recently. The outbreak was preceded by a major nationwide outbreak in the Philippines. We examined the phylogenetic and phylogeographic relationships of representative CHIKV isolates obtained from the 2012 Philippines outbreak with other CHIKV isolates collected globally. Asian CHIKV isolated from the Philippines, China, Micronesia and Caribbean regions were found closely related, herein denoted as Cosmopolitan Asian CHIKV (CACV). Three adaptive amino acid substitutions in nsP3 (D483N), E1 (P397L) and E3 (Q19R) were identified among CACV. Acquisition of the nsP3-483N mutation in Compostela Valley followed by E1-397L/E3-19R in Laguna preceded the nationwide spread in the Philippines. The China isolates possessed two of the amino acid substitutions, nsP3-D483N and E1-P397L whereas the Micronesian and Caribbean CHIKV inherited all the three amino acid substitutions. The unique amino acid substitutions observed among the isolates suggest multiple independent virus dissemination events. The possible biological importance of the specific genetic signatures associated with the rapid global of the virus is not known and warrant future in-depth study and epidemiological follow-up. Molecular evidence, however, supports the Philippines outbreak as the possible origin of the CACV.

Chikungunya virus (CHIKV) infection in human is associated with debilitating and persistent arthralgia and arthritis. Currently, there is no specific vaccine or effective antiviral available. Anti-CHIKV Phosphorodiamidate Morpholino Oligomer (CPMO) was evaluated for its antiviral efficacy and cytotoxcity in human cells and neonate murine model. Two CPMOs were designed to block translation initiation of a highly conserved sequence in CHIKV non-structural and structural polyprotein, respectively. Pre-treatment of HeLa cells with CPMO1 signficantly suppressed CHIKV titre, CHIKV E2 protein expression and prevented CHIKV-induced CPE. CPMO1 activity was also CHIKV-specific as shown by the lack of cross-reactivity against SINV or DENV replication. When administered prophylactically in neonate mice, 15 μg/g CPMO1v conferred 100% survival against CHIKV disease. In parallel, these mice demonstrated significant reduction in viremia and viral load in various tissues. Immunohistological examination of skeletal muscles and liver of CPMO1v-treated mice also showed healthy tissue morphology, in contrast to evident manifestation of CHIKV pathogenesis in PBS- or scrambled sCPMO1v-treated groups. Taken together, our findings highlight for the first time that CPMO1v has strong protective effect against CHIKV infection. This warrants future development of morpholino as an alternative antiviral agent to address CHIKV infection in clinical applications.