Evaluation of analgesic and prophylactic activity of curcumin against chikungunya-infected acute/chronic arthralgic mice.

Chikungunya virus (CHIKV) infection, a global public health problem, might lead to acute/chronic polyarthritis causing long-term morbidity among infected patients. But, except nonsteroidal anti-inflammatory drugs (NSAIDs) with gastrointestinal, cardiovascular, and immune-related side-effects, no Food and Drug Administration (FDA)-approved analgesic drug is available till date for the treatment of CHIKV-induced arthritis. Curcumin, a plant product with minimal toxicity has been FDA-approved as a Generally Recognized As Safe drug. This study aimed to determine the analgesic and prophylactic effect of curcumin, if any, among CHIKV-induced arthralgic mice. Arthritic pain was evaluated by von Frey assay, locomotory behavior by open-field test, and feet swelling by calipers. Cartilage integrity and proteoglycan loss were evaluated by Safranin O staining followed by Osteoarthritis Research Society International (OARSI), Standardized Microscopic Arthritis Scoring of Histological sections (SMASH) score, and type II collagen loss by immunohistochemistry. Mice were administered high (HD), mid (MD), and low (LD) curcumin doses, before (PT: pretreatment), during (CT: cotreatment) and after (Post-T: posttreatment) CHIKV-infection. Curcumin treatment using PTHD (2000 mg/kg), CTHD , and Post-TMD (1000 mg/kg) significantly alleviated CHIKV-induced arthritic pain by improving pain-threshold, locomotory behavior and reducing feet swelling of infected mice. Also, decreased proteoglycan loss and cartilage erosion with lower OARSI, SMASH scores were observed among these three subgroups compared to infected ones. Compared to infected ones, one- to twofold increased intensity of type II collagen in knee medial femoral condyle and medial tibial plateau regions of these subgroups was observed by immunohistochemical staining. Thus, this study highlighted both the analgesic (CT, Post-T), and prophylactic (PT) activity of curcumin in alleviating CHIKV-induced acute/chronic arthritis within mouse model.

Impact of apoptotic biomarkers for prognosis of dengue disease severity among eastern Indian patients.

Dengue virus (DENV) induced severe manifestations is a precursor for fatality among infected patients. Previous autopsy examinations of severe dengue (SD) patients reported presence of apoptotic cells in liver, brain, intestinal and lung tissues. Thus, serum-level of major apoptotic proteins of dengue patients was evaluated in the current study, along with their biochemical parameters. Patients were categorized according to World Health Organization (WHO)-defined classification. DENV-infection was screened among 165 symptomatic patients by quantitative reverse transcription polymerase chain reaction, antidengue IgM, and IgG ELISA. Serum levels of apoptotic (Capase-3,7,8, Bcl-2 and FasL) and hepatic-markers, lipid profile, hematological parameters of 78 dengue-positive patients were determined by sandwich-ELISA/immunoturbidimetry/auto-analyzer. Significantly higher levels of caspase-3,7,8 and FasL was detected among SD patients compared to those without warning (WOW) signs. Amongst biochemical parameters, bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase serum concentrations significantly increased among severe patients. Principal component analysis followed by hierarchical clustering differentiated severe and with warning dengue patient groups from those WOW using caspase-3,7,8 and FasL biomarkers-thus clearly distinguishing severe-dengue group. Correlation analyses also established strong positive correlation between caspase-3,7,8 and FasL. Thus, serum level of caspase-3,7,8 and FasL during early stage of infection could be used as biomarkers for WHO-defined dengue disease severity.

Differential genotypic signatures of Toll-like receptor polymorphisms among dengue-chikungunya mono- and co-infected Eastern Indian patients.

Dengue (DENV) and chikungunya (CHIKV) viral infections trigger high patient morbidity and mortality. Mono-/co-infection of these viruses activates innate immune response, triggering Toll-like receptor (TLR) pathways. The present study investigated the differential role of TLR3, 7 and 8 single-nucleotide polymorphisms (SNPs) between mono- and co-infected Eastern Indian patients. Interaction of TLR polymorphic variants with signal peptidase complex (SPC18) was explored which might affect immune signalling against DENV/CHIKV infections. Out of 550 febrile symptomatic patients, 128 DENV-CHIKV co-infected samples were genotyped for eight SNPs of TLR3 (rs3775290-chr4:186083063), TLR7 (rs179008-chrX:12885540, rs5741880-chrX:12869297, rs179010-chrX:12884766, rs3853839-chrX:12889539) and TLR8 (rs5744080-chrX:12919685, rs3764879-chrX:12906578, rs3764880-chrX:12906707) by PCR-RFLP along with 157 healthy individuals. Statistical analysis established genotypic association of TLR SNPs with DENV-CHIKV co-infection, and difference between mono- and co-infected patients and their role in determining high viral load (HVL) during competitive viral replication among co-infected patients. In silico protein-protein docking evaluated interactive effect of TLR variants with SPC18. The findings revealed patients with CC genotypes of TLR7 and 8 SNPs were significantly susceptible towards co-infection, whereas specific genotypes of TLR7 and 8 imparted protection against co-infection. Differential analysis between mono-/co-infected patients revealed distinct genotypic distribution of TLR3, 7 and 8 SNPs. Co-infected patients with TT-rs179010 exhibited DENV-HVL, whereas CHIKV-HVL was detected among patients with other genotypes. Molecular docking of TLR7-rs179008 Q variant and TLR8-rs3764880 V variant with SPC18 generated better free binding energy. This study underlined the importance of TLR7 and 8 SNPs towards mono-/co-infection of DENV/CHIKV, with certain genotypes associated with co-infection susceptibility. Moreover, it suggested a probable role of specific genotypes of TLR7 and 8 polymorphisms imparting high dengue/chikungunya viral load among co-infected patients.

Increased CRP, anti-CCP antibody, IL-2R, COMP levels in prognosis of post-chikungunya chronic arthritis and protective role of their specific genotypes against arthritic manifestation.

Chikungunya infection leads to acute/chronic polyarthritis/polyarthralgia causing long-term morbidity among patients. Prognosis of post-chikungunya chronic arthritis (PCA) is of utmost necessity for proper disease management. Arthritic and hepatic biomarkers were evaluated among chikungunya patients without arthritis, with acute arthritis and with post-chikungunya chronic arthritis in the study. Serum levels of arthritic [CRP (C-reactive protein), anti cyclic-citrullinated-peptide (anti-CCP) antibody, soluble interleukin-2 receptor (sIL-2R), cartilage oligomeric matrix protein (COMP)] and hepatic [ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), albumin and bilirubin] biomarkers of 167 chikungunya positive patients were determined by sandwich-ELISA/immunoturbidimetry/auto-analyser. 167 chikungunya-patients and 102 healthy controls were genotyped to understand role of CRP-rs3093059/rs3091244, IL-2R-rs743777 and COMP-rs144778694 polymorphisms towards chikungunya virus (CHIKV) infectivity and arthralgic manifestation. CRP, anti-CCP antibody, IL-2R and COMP levels significantly increased among PCA patients. Concentrations of AST, ALT, AST/ALT-ratio, bilirubin and ALP increased among arthritic chikungunya patients. Principal component analysis differentiated PCA groups from acute (AA) and non-arthritic groups. Patients with IL-2R-rs743777-GA, G-allele and COMP-rs144778694-GA genotypes were susceptible to chikungunya infection. Moreover, patients with CRP-rs3093059-CT, rs3091244-TT, IL-2R-rs743777-GA and COMP-rs144778694-AA genotypes were significantly protected from arthralgia, whereas, COMP-rs144778694-GA genotype was susceptible towards it. Patients with certain genotypes of CRP, IL-2R and COMP demonstrated significantly higher biomarker serum-levels among patients suffering from AA with/without PCA. Thus, both serum biomarker levels and polymorphic genotypes of infected patients play decisive role in development of PCA.

In silico and in vitro evaluation of silibinin: a promising anti-Chikungunya agent.

Chikungunya virus (CHIKV) infection and subsequent high patient morbidity is a global threat. The present study aimed to identify the potent antiviral agent against Chikungunya virus, with minimum in vitro cytotoxicity. CHIKV nsP4 3D structure was determined using the I-TASSER server followed by its refinement and pocket determination. Furthermore, high-throughput molecular docking was employed to identify candidate CHIKV nsP4 inhibitors in a library containing 214 compounds. The top ranked compound was evaluated further with various assays, including cytotoxicity, antiviral activity, time of drug addition, viral entry attachment, and microneutralization assays. High-throughput computational screening indicated silibinin to have the best interaction with CHIKV nsP4 protein, immature and mature glycoproteins with highest negative free binding energy, - 5.24 to - 5.86 kcal/mol, and the lowest inhibitory constant, 50.47 to 143.2 µM. Further in vitro analysis demonstrated silibinin could exhibit statistically significant (p < 0.05) dose-dependent anti-CHIKV activity within 12.5-100-µM concentrations with CC50 as 50.90 µM. In total, 50 µM silibinin interfered with both CHIKV attachment (75%) and entry (82%) to Vero cells. Time of addition assay revealed silibinin interfered with late phase of the CHIKV replication cycle. Microneutralization assay revealed that silibinin could inhibit clearing of 50% Vero cell monolayer caused by CHIKV-induced CPE at a minimum dose of 25 µM. These data indicated silibinin to be a promising candidate drug against CHIKV infection.

Association of C-reactive protein polymorphisms with serum-CRP concentration and viral load among dengue-chikungunya mono/co-infected patients.

India being endemic to Dengue (DENV) and chikungunya (CHIKV) infections faces high patient-mortality and morbidity with overlapping clinical features. C-reactive protein (CRP) acts as early defence system in response to these infections. This study investigated role of CRP single-nucleotide polymorphism (SNP) genotypes and protein levels towards DENV/CHIKV mono and co-infection among eastern Indian patients. 128 DENV-CHIKV co-infected, 206 DENV and 167 CHIKV mono-infected patients were subjected to genotyping of two CRP SNPs by PCR-RFLP along with 102 healthy individuals. CRP levels were determined by immunoturbidimetry. Statistical correlation of CRP genotypes with CRP concentration, DENV-CHIKV mono/co-infection and viral load was performed. Patients with rs3093059-CT and rs3091244-TT were more susceptible to DENV-CHIKV co-infection, whereas, rs3091244-CT might have imparted protection against CHIKV mono-infection. DENV-HVL was more prevalent within rs3093059-TT and rs3091244-CT co-infected patients, whereas, CHIKV-HVL among rs3091244-CC. Acute phase co-infected patients had significantly higher CRP level compared to mono-infections. Both mono and co-infected patients with aches/pain exhibited 2-3-fold higher CRP levels compared to those without. rs3093059-CT and rs3091244-CT co-infected patients had higher CRP concentration compared to rs3093059-TT and rs3091244-CC, respectively. Co-infected patients with WHO-defined warning signs had higher anti-dengue IgG/IgM ratio and serum CRP level compared to those without warning signs. Thus, patient's CRP genotype might play significant role in determining serum-CRP concentration, viral load and DENV-CHIKV mono/co-infection.

Re-emergence of Chikungunya virus infection in Eastern India.

Chikungunya fever is a major public health issue in India. Re-emergence of chikungunya virus (CHIKV) in West Bengal was detected after 32 years in 2006. After 2010, this infection was in apparent decline, but in 2016 a massive outbreak affected the country. Present study was carried out to understand CHIKV infection dynamics during recent outbreaks in West Bengal, Eastern India and its implication on disease manifestations. Blood was collected from 641 symptomatic patients. Patients' sera were serologically diagnosed to detect presence of anti-chikungunya-IgM antibodies. Viral RNA was extracted; presence of CHIKV genome and its respective viral load was determined by real time quantitative reverse transcription-PCR (real-time qRT-PCR). Statistical analysis was performed using EPI INFO software. CHIKV infection was detected in 24.64% of symptomatic patients. Middle-aged patients (31-40 years) were predominantly affected; clinically, both arthralgia and joint-swelling were significantly prevalent among CHIKV-infected patients. Myalgia, joint-swelling, and arthralgic manifestation were found in significantly higher frequency among patients with high chikungunya viral load (> 10,000 copies/ml). Thus, this study clearly indicated the re-emergence of CHIKV in Eastern India. Significant presence of myalgia, joint swelling, and arthralgia among chikungunya patients with high viral load implied association of disease severity with viral load; requiring vigilance for proper management of infected patients as this disease is highly morbid in nature. However, in addition to chikungunya virus, other viral, bacterial, and protozoal infections also occur during post-monsoon season in India, having overlapping symptoms. Hence, continuous monitoring of these infections is required for better clinical management of patients.