A Combination of Doxycycline and Ribavirin Alleviated Chikungunya Infection

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.     

Differential Proteome Analysis of Chikungunya Virus Infection on Host Cells

Background

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach.

Methodology and Principal Findings

The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE). Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP) and cell cycle regulation.

Conclusion/Significance

This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1) regulation (in favour of virus survival, replication and transmission). While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.     

Development of a Hamster Model for Chikungunya Virus Infection and Pathogenesis

Chikungunya virus is transmitted by mosquitoes and causes severe, debilitating infectious arthritis in humans. The need for an animal model to study the disease process and evaluate potential treatments is imminent as the virus continues its spread into novel geographic locations. Golden hamsters (Mesocricetus auratus) are often used as outbred laboratory animal models for arboviral diseases. Here we demonstrate that hamsters inoculated with chikungunya virus developed viremia and histopathologic lesions in their limbs and joints similar to those seen in human patients. The virus disseminated rapidly and was found in every major organ, including brain, within a few days of infection. Hamsters did not manifest overt clinical signs, and the virus was generally cleared within 4 days, followed by a strong neutralizing antibody response. These results indicate that hamsters are highly susceptible to chikungunya virus infection and develop myositis and tenosynovitis similar to human patients followed by a complete recovery. This animal model may be useful for testing antiviral drugs and vaccines.     

Chikungunya Virus Infection of Cell Monolayers by Cell-to-Cell and Extracellular Transmission

Both cell-to-cell and extracellular transmission of chikungunya virus infection was demonstrated in BHK21/C13 cell monolayers. The mode of virus infection may depend on the cell line. Virus transmission in L-929 and guinea pig lung cell lines was extracellular.     

Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection

In December of 2013, chikungunya virus (CHIKV), an alphavirus in the family Togaviridae, was introduced to the island of Saint Martin in the Caribbean, resulting in the first autochthonous cases reported in the Americas. As of January 2015, local and imported CHIKV has been reported in 50 American countries with over 1.1 million suspected cases. CHIKV causes a severe arthralgic disease for which there are no approved vaccines or therapeutics. Furthermore, the lack of a commercially available, sensitive, and affordable diagnostic assay limits surveillance and control efforts. To address this issue, we utilized an insect-specific alphavirus, Eilat virus (EILV), to develop a diagnostic antigen that does not require biosafety containment facilities to produce. We demonstrated that EILV/CHIKV replicates to high titers in insect cells and can be applied directly in enzyme-linked immunosorbent assays without inactivation, resulting in highly sensitive detection of recent and past CHIKV infection, and outperforming traditional antigen preparations.     

Native Wolbachia from Aedes albopictus Blocks Chikungunya Virus Infection In Cellulo

Wolbachia, a widespread endosymbiont of terrestrial arthropods, can protect its host against viral and parasitic infections, a phenotype called "pathogen blocking". However, in some cases Wolbachia may have no effect or even enhance pathogen infection, depending on the host-Wolbachia-pathogen combination. The tiger mosquito Aedes albopictus is naturally infected by two strains of Wolbachia, wAlbA and wAlbB, and is a competent vector for different arboviruses such as dengue virus (DENV) and Chikungunya virus (CHIKV). Interestingly, it was shown in some cases that Ae. albopictus native Wolbachia strains are able to inhibit DENV transmission by limiting viral replication in salivary glands, but no such impact was measured on CHIKV replication in vivo. To better understand the Wolbachia/CHIKV/Ae. albopictus interaction, we generated a cellular model using Ae. albopictus derived C6/36 cells that we infected with the wAlbB strain. Our results indicate that CHIKV infection is negatively impacted at both RNA replication and virus assembly/secretion steps in presence of wAlbB. Using FISH, we observed CHIKV and wAlbB in the same mosquito cells, indicating that the virus is still able to enter the cell in the presence of the bacterium. Further work is needed to decipher molecular pathways involved in Wolbachia-CHIKV interaction at the cellular level, but this cellular model can be a useful tool to study the mechanism behind virus blocking phenotype induced by Wolbachia. More broadly, this underlines that despite Wolbachia antiviral potential other complex interactions occur in vivo to determine mosquito vector competence in Ae. albopictus.     

Transformation During Mixed Pneumococcal Infection of Mice

The recent demonstration by others of transformation during peritoneal infection of mice by two genetically distinct pneumococcal strains supports the notion that transformation may be significant in pneumococcal infection in nature. These studies confirm the occurrence of transformation during mixed infection of mice and define some conditions for its occurrence and its significance. Mice were inoculated with deoxyribonucleic acid (DNA) donor (small type III capsule, low virulence, streptomycin-susceptible) and recipient (noncapsulated, low virulence, streptomycin-resistant) pneumococci, and the bacteremia in mice that died was evaluated. Transformants (large type III capsule, virulent, streptomycin-resistant) were isolated from up to 80% of mice that died from mixed peritoneal infection. Transformation occurred in mice that received donor and recipient 6 hr apart; hence, active DNA was released and competence developed during growth in vivo. Transformation was detected only with progressive infection by both strains, and then transformants were few in the blood and apparently were not responsible for the death of the animals. In doubly infected mice treated with streptomycin, transformation was enhanced; transformants numerically dominated the bacteremia and seemed to cause the death of the mice. Transformation was also demonstrated for the first time during infection of the respiratory tract.     

Cross-Inhibition of Chikungunya Virus Fusion and Infection by Alphavirus E1 Domain III Proteins

Alphaviruses are small enveloped RNA viruses that include important emerging human pathogens, such as chikungunya virus (CHIKV). These viruses infect cells via a low-pH-triggered membrane fusion reaction, making this step a potential target for antiviral therapies. The E1 fusion protein inserts into the target membrane, trimerizes, and refolds to a hairpin-like conformation in which the combination of E1 domain III (DIII) and the stem region (DIII-stem) pack against a core trimer composed of E1 domains I and II (DI/II). Addition of exogenous DIII proteins from Semliki Forest virus (SFV) has been shown to inhibit E1 hairpin formation and SFV fusion and infection. Here we produced and characterized DIII and DI/II proteins from CHIKV and SFV. Unlike SFV DIII, both core trimer binding and fusion inhibition by CHIKV DIII required the stem region. CHIKV DIII-stem and SFV DIII-stem showed efficient cross-inhibition of SFV, Sindbis virus, and CHIKV infections. We developed a fluorescence anisotropy-based assay for the binding of SFV DIII-stem to the core trimer and used it to demonstrate the relatively high affinity of this interaction (K_d [dissociation constant], ∼85 nM) and the importance of the stem region. Together, our results support the conserved nature of the key contacts of DIII-stem in the alphavirus E1 homotrimer and describe a sensitive and quantitative in vitro assay for this step in fusion protein refolding.     

基孔肯雅热

基孔肯雅热(Chikungunya fever,CHIK)是由基孔肯雅病毒(Chikungunya virus,CHIKV)病毒引起的,伊蚊叮咬传播的,以发热、关节疼痛等为主要特征的自限性传染性疾病。1952年在坦桑尼亚发生的暴发流行中首次分离CHIKV[1-2],亚洲地区于     

Cytokines in Acute Chikungunya

Introduction

Acute chikungunya (CHIKV) is predominantly an acute onset of excruciatingly painful, self-limiting musculoskeletal (MSK) arbovirus illness and this was further reported by us during the 2006 Indian epidemic [Chopra et al. Epidemiol Infect 2012]. Selected serum cytokines profile in subjects within one month of onset of illness is being presented.

Methods

Out of 509 clinical CHIKV cases (43% population) identified during a rural population survey, 225 subjects consented blood investigations. 132 examined within 30 days of febrile onset are the study cohort. Anti-CHIKV IgM and IgG antibodies tested by immunochromatography and indirect immunofluorescence respectively. Interferons (IFN)-α, -β and -γ, Interferon Gamma-Induced Protein-10 (CXCL-10/IP-10), Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-13 (IL-13), Monocyte Chemoattractant Protein-1 (MCP-1), Interleukin–4 (IL-4) and Interleukin–10 (IL-10) performed by ELISA. Samples collected from neighboring community a year prior to the epidemic used as healthy controls.

Results

Seropositivity for anti-CHIKV IgM and IgG was 65% and 52% respectively. IFN-α, IFN-β, IFN-γ, CXCL10/IP-10 and IL-1β showed intense response in early acute phase. Cytokines (particularly TNF-α, MCP-1, IL-4, IL-6 and IL-10) was maximum in extended symptomatic phase and remained elevated in recovered subjects. Higher (p<0.05) IFN and IL-4 seen in patients seropositive for anti-CHIKV IgG. Elderly cases (≥65 years) showed elevated cytokines (except IFN) and anti-CHIKV antibodies near similar to younger subjects. Significant correlations (p<0.05) found between cytokines and clinical features (fatigue, low back ache, myalgia) and anti-CHIKV antibodies.

Conclusion

An intense cytokine milieu was evident in the early and immediate persistent symptomatic phase and in recovered subjects. Early persistent IgM and lower IgG to anti-CHKV and intense Th2 cytokine phenotype seem to be associated with delay in resolution of MSK symptoms. Intriguingly, maximum TNF-α, IL-6 and IL-13 with low anti-CHIKV IgM response found in subjects recovered from CHIKV within one month of illness.