Arctigenin from Arctium lappa L. inhibits chikungunya virus by affecting its entry and replication.

BACKGROUND: Dengue and chikungunya, caused by dengue virus (DENV) and chikungunya virus (CHIKV) respectively, are the most common arthropod-borne viral diseases worldwide, for which there are no FDA-approved antivirals or effective vaccines. Arctigenin, a phenylpropanoid lignan from the seeds of Arctium lappa L. is known for its anti-inflammatory, anti-cancer, antibacterial, and immunomodulatory properties. Arctigenin's antimicrobial and immunomodulatory capabilities make it a promising candidate for investigating its potential as an anti-DENV and anti-CHIKV agent. PURPOSE: The aim of the study was to explore the anti-DENV and anti-CHIKV effects of arctigenin and identify the possible mechanisms of action. METHODS: The anti-DENV or anti-CHIKV effects of arctigenin was assessed using various in vitro and in silico approaches. Vero CCL-81 cells were infected with DENV or CHIKV and treated with arctigenin at different concentrations, temperature, and time points to ascertain the effect of the compound on virus entry or replication. In silico molecular docking was performed to identify the interactions of the compound with viral proteins. RESULTS: Arctigenin had no effects on DENV. Various time- and temperature-dependent assays revealed that arctigenin significantly reduced CHIKV RNA copy number and infectious virus particles and affected viral entry. Entry bypass assay revealed that arctigenin inhibited the initial steps of viral replication. In silico docking results revealed the high binding affinity of the compound with the E1 protein and the nsp3 macrodomain of CHIKV. CONCLUSION: This study demonstrates the in-vitro anti-CHIKV potential of arctigenin and suggests that the compound might affect CHIKV entry and replication. Further preclinical and clinical studies are needed to identify its safety and efficacy as an anti-CHIKV drug.

Repurposed drugs in combinations exert additive anti-chikungunya virus activity: an in-vitro study.

Chikungunya virus (CHIKV) infection causes chikungunya, a viral disease that currently has no specific antiviral treatment. Several repurposed drug candidates have been investigated for the treatment of the disease. In order to improve the efficacy of the known drugs, combining drugs for treatment is a promising approach. The current study was undertaken to explore the antiviral activity of a combination of repurposed drugs that were reported to have anti-CHIKV activity. We explored the effect of different combinations of six effective drugs (2-fluoroadenine, emetine, lomibuvir, enalaprilat, metyrapone and resveratrol) at their non-toxic concentrations against CHIKV under post infection treatment conditions in Vero cells. Focus-forming unit assay, real time RT-PCR, immunofluorescence assay, and western blot were used to determine the virus titre. The results revealed that the combination of 2-fluoroadenine with either metyrapone or emetine or enalaprilat exerted inhibitory activity against CHIKV under post-infection treatment conditions. The effect of these drug combinations was additive in nature compared to the effect of the individual drugs. The results suggest an additive anti-viral effect of these drug combinations against CHIKV. The findings could serve as an outline for the development of an innovative therapeutic approach in the future to treat CHIKV-infected patients.

Understanding the resurgence of chikungunya virus during 2020-2021 in Pune, India, based on genomic analyses: A seven year study.

A surge in chikungunya was observed during 2020-21 in Pune district of Maharashtra, India. Whole genome sequencing and phylogenetic analysis of 21 samples/sequences revealed them as Indian ocean lineage of East Central South African genotype. Two distinct sequence clusters were found to circulate during 2020-21; one with E1:K211E and E2:V264A mutations while the other had E1:I317V mutation along with E1:K211E and E2: V264A mutations. The former, the predominant cluster (n = 18), clustered with chikungunya virus (CHIKV) strains of pre 2014 period while the latter (n = 3) clustered with 2016-2018 period Indian strains. Though E1: A226V was not detected in any of the 21 sequences, several unique mutations were detected in the strains which might have played key roles in the enhanced virus transmission during the period. The study highlights parallel evolution, introduction from the neighboring regions and cocirculation of two sequence clusters of CHIKV in Pune. The complete genome data can be useful to determine how the circulating strains differ from candidate vaccines and might help to predict the protective efficacy of chikungunya vaccine candidates.

Seroprevalence of dengue virus infection in Pune City in India, 2019: A decadal change.

BACKGROUND: The burden of dengue infection needs to be monitored along with tracking of the changes in dengue virus (DENV) transmission intensity for vaccine introduction decisions. METHODS: The seroprevalence of dengue was investigated in Pune City in India, in early 2019 using 1654 sera from apparently healthy human participants enrolled randomly through multistage cluster sampling. We used 797 retrospective human sera from late 2009 for comparison. All sera were assessed for the presence of dengue-specific IgG antibodies. A subset (n = 230) was tested for serotype-specific plaque reduction-neutralizing antibodies against all four serotypes. RESULTS: The dengue IgG seroprevalence of 62.9% (95% CI 59.4-66.1) in 2009 increased to 88.4% (95% CI 86.8-89.8) in 2019. Age-stratified dengue seroprevalence revealed a gradual increase in IgG seropositivity from 70.1% in 0-9 years to 85.0% in 10-19 years. The annual probability of dengue infection estimated as a force of infection was 4.1 (95% CI 3.8-4.5) in 2009, which increased to 10.9 (95% CI 10.2-11.6) in 2019. Analysis of dengue serotype-specific neutralizing antibodies revealed DENV-3 as the dominant serotype. The age of exposure to at least one dengue serotype was reduced in 2019 over 2009. CONCLUSIONS: There was a significant increase in the intensity of dengue virus transmission in Pune City over the decade. Since over 85% of the participants above nine years of age had exposure to DENV by 2019, dengue vaccine introduction can be considered. Moreover, such repeated serosurveys in different regions might inform about the readiness of the population for dengue vaccination.

Corrigendum: Drug repurposing approach against chikungunya virus: an in vitro and in silico study.

[This corrects the article DOI: 10.3389/fcimb.2023.1132538.].

Drug repurposing approach against chikungunya virus: an in vitro and in silico study.

The chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes mosquitoes. There are no licenced antivirals or vaccines for treatment or prevention. Drug repurposing approach has emerged as a novel concept to find alternative uses of therapeutics to battle pathogens. In the present study, anti CHIKV activity of fourteen FDA-approved drugs was investigated by in vitro and in silico approaches. Focus-forming unit assay, immunofluorescence test, and quantitative RT-PCR assay were used to assess the in vitro inhibitory effect of these drugs against CHIKV in Vero CCL-81 cells. The findings showed that nine compounds, viz., temsirolimus, 2-fluoroadenine, doxorubicin, felbinac, emetine, lomibuvir, enalaprilat, metyrapone and resveratrol exhibit anti chikungunya activity. Furthermore, in silico molecular docking studies performed by targeting CHIKV structural and non-structural proteins revealed that these drugs can bind to structural protein targets such as envelope protein, and capsid, and non-structural proteins NSP2, NSP3 and NSP4 (RdRp). Findings from in vitro and in silico studies reveal that these drugs can suppress the infection and replication of CHIKV and further in vivo studies followed by clinical trials are warranted.

Effect of Sauropus androgynus L. Merr. on dengue virus-2: An in vitro and in silico study.

ETHNOPHARMACOLOGICAL RELEVANCE: Sauropus androgynus L. Merr. (Euphorbiaceae) commonly known as "multigreen" and "multivitamin" is consumed as a vegetable and used in traditional medicine to relieve fever. AIM OF THE STUDY: This in vitro study is aimed to explore the activities of the lipophilic fraction of the leaves of S. androgynus (LFSA) against dengue (DENV), chikungunya (CHIKV) viruses and malaria (P. falciparum strain 3D7) parasite. MATERIALS AND METHODS: The LFSA was analyzed by using GC-FID and GC-MS. The antiviral activity of LFSA was studied using the Vero CCL-81 cell line. The cytotoxicity assay was performed using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Focus forming unit (FFU), cell-based immunofluorescence (IFA) assays, and quantitative RT-PCR, were used to determine and confirm antiviral activity against DENV and CHIKV. The antiparasitic activity of LFSA was carried out against P. falciparum strain 3D7 grown in fresh O+ human erythrocytes culture. RESULTS: Twelve compounds were identified in LFSA using GC/MS. The most abundant compound was squalene (36.9%), followed by vitamin E (12.5%) and linolenic acid (10.2%). Significant reduction in DENV titre was observed under pre- and post-infection treatment conditions at a concentration of 31.25 µg/ml, but no anti-malarial and anti-CHIKV activity was observed. The Autodock-Vina-based in-silico docking study revealed that ß-sitosterol could form a strong interaction with the DENV E glycoprotein. CONCLUSION: Our findings suggest that LFSA can inhibit DENV infection and might act as a potent prophylactic/therapeutic agent against DENV-2. In-silico results suggested that ß-sitosterol may block the viral entry by inhibiting the fusion process.

A Transcriptomics-Based Bioinformatics Approach for Identification and In Vitro Screening of FDA-Approved Drugs for Repurposing against Dengue Virus-2.

The rising incidence of dengue virus (DENV) infections in the tropical and sub-tropical regions of the world emphasizes the need to identify effective therapeutic drugs against the disease. Repurposing of drugs has emerged as a novel concept to combat pathogens. In this study, we employed a transcriptomics-based bioinformatics approach for drug identification against DENV. Gene expression omnibus datasets from patients with different grades of dengue disease severity and healthy controls were used to identify differentially expressed genes in dengue cases, which were then applied to the query tool of Connectivity Map to identify the inverse gene-disease-drug relationship. A total of sixteen identified drugs were investigated for their prophylactic, virucidal, and therapeutic effects against DENV. Focus-forming unit assay and quantitative RT-PCR were used to evaluate the antiviral activity. Results revealed that five compounds, viz., resveratrol, doxorubicin, lomibuvir, elvitegravir, and enalaprilat, have significant anti-DENV activity. Further, molecular docking studies showed that these drugs can interact with a variety of protein targets of DENV, including the glycoprotein, the NS5 RdRp, NS2B-NS3 protease, and NS5 methyltransferase The in vitro and in silico results, therefore, reveal that these drugs have the ability to decrease DENV-2 production, suggesting that these drugs or their derivatives could be attempted as therapeutic agents against DENV infections.

In Vitro Antiviral Activity of Potential Medicinal Plant Extracts Against Dengue and Chikungunya Viruses.

Dengue and chikungunya are two important mosquito-borne infections which are known to occur extensively in tropical and subtropical areas. Presently, there is no treatment for these viral diseases. In vitro antiviral screening of 25 extracts prepared from the plants of Vitex negundo, Plumeria alba, Ancistrocladus heyneanus, Bacopa monnieri, Anacardium occidentale, Cucurbita maxima, Simarouba glauca, and Embelia ribes using different solvents and four purified compounds (anacardic acid, chloroquinone, glaucarubinone, and methyl gallate) were carried out for their anti-dengue virus (DENV) and anti-chikungunya virus (CHIKV) activities. Maximum nontoxic concentrations of the chloroform, methanol, ethyl acetate, petroleum ether, dichloromethane, and hydroalcoholic extracts of eight plants were used. The antiviral activity was assessed by focus-forming unit assay, quantitative real-time RT-PCR, and immunofluorescence assays. Extracts from Plumeria alba, Ancistrocladus heyneanus, Bacopa monnieri, and Cucurbita maxima showed both anti-DENV and CHIKV activity while extract from Vitex negundo showed only anti-DENV activity. Among the purified compounds, anacardic acid, chloroquinone and methyl gallate showed anti-dengue activity while only methyl gallate had anti-chikungunya activity. The present study had identified the plant extracts with anti-dengue and anti-chikungunya activities, and these extracts can be further characterized for finding effective phytopharmaceutical drugs against dengue and chikungunya.

First case of Zika virus infection during an outbreak of chikungunya in a rural region of Maharashtra state, India.

BACKGROUND: In July 2021, an outbreak of chikungunya virus (CHIKV) was reported in a rural region of Maharashtra state, India. METHODS: Serum samples of symptomatic cases (n=33) were screened for dengue virus (DENV), CHIKV and Zika virus (ZIKV) by molecular and serological assays. RESULTS: The first case of ZIKV infection from Maharashtra was detected and confirmed by molecular and serological assays. Complete genome sequencing revealed that the ZIKV sequence belongs to the Asian genotype and had a closer homology with pre-epidemic strains present before 2007. CONCLUSIONS: ZIKV surveillance needs to be strengthened in the regions experiencing dengue and chikungunya outbreaks.

Targeted in vitro gene silencing of E2 and nsP1 genes of chikungunya virus by biocompatible zeolitic imidazolate framework.

Chikungunya fever caused by the mosquito-transmitted chikungunya virus (CHIKV) is a major public health concern in tropical, sub-tropical and temperate climatic regions. The lack of any licensed vaccine or antiviral agents against CHIKV warrants the development of effective antiviral therapies. Small interfering RNA (siRNA) mediated gene silencing of CHIKV structural and non-structural genes serves as a potential antiviral strategy. The therapeutic efficiency of siRNA can be improved by using an efficient delivery system. Metal-organic framework biocomposits have demonstrated an exceptional capability in protecting and efficiently delivering nucleic acids into cells. In the present study, carbonated ZIF called ZIF-C has been utilized to deliver siRNAs targeted against E2 and nsP1 genes of CHIKV to achieve a reduction in viral replication and infectivity. Cellular transfection studies of E2 and nsP1 genes targeting free siRNAs and ZIF-C encapsulated siRNAs in CHIKV infected Vero CCL-81 cells were performed. Our results reveal a significant reduction of infectious virus titre, viral RNA levels and percent of infected cells in cultures transfected with ZIF-C encapsulated siRNA compared to cells transfected with free siRNA. The results suggest that delivery of siRNA through ZIF-C enhances the antiviral activity of CHIKV E2 and nsP1 genes directed siRNAs.

TNFA and IL10 Polymorphisms and IL-6 and IL-10 Levels Influence Disease Severity in Influenza A(H1N1)pdm09 Virus Infected Patients.

Cytokines are key modulators of immune response, and dysregulated production of proinflammatory and anti-inflammatory cytokines contributes to the pathogenesis of influenza A(H1N1)pdm09 virus infection. Cytokine production is impacted by single nucleotide polymorphisms (SNPs) in the genes coding for them. In the present study, SNPs in the IL6, TNFA, IFNG, IL17A, IL10, and TGFB were investigated for their association with disease severity and fatality in influenza A(H1N1)pdm09-affected patients with mild disease (n = 293) and severe disease (n = 86). Among those with severe disease, 41 patients had fatal outcomes. In a subset of the patients, levels of IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17 were assayed in the plasma for their association with severe disease. The frequency of TNFA rs1800629 G/A allele was significantly higher in severe cases and survived severe cases group compared to that of those with mild infection (OR with 95% for mild vs. severe cases 2.95 (1.52-5.73); mild vs. survived severe cases 4.02 (1.84-8.82)). IL10 rs1800896-rs1800872 G-C haplotype was significantly lower (OR with 95% 0.34 (0.12-0.95)), while IL10 rs1800896-rs1800872 G-A haplotype was significantly higher (OR with 95% 12.11 (2.23-76.96)) in fatal cases group compared to that of the mild group. IL-6 and IL-10 levels were significantly higher in fatal cases compared to that of survived severe cases. IL-6 levels had greater discriminatory power than IL-10 to predict progression to fatal outcome in influenza A(H1N1)pdm09 virus-infected patients. To conclude, the present study reports the association of TNFA and IL10 SNPs with severe disease in Influenza A(H1N1)pdm09 virus-infected subjects. Furthermore, IL-6 levels can be a potential biomarker for predicting fatal outcomes in Influenza A(H1N1)pdm09 virus infected subjects.

Predictors of Dengue-Related Mortality and Disease Severity in a Tertiary Care Center in North India.

BACKGROUND: There is lack of reliable predictors of disease severity and mortality in dengue. The present study was carried out to identify these predictors during the 2015 outbreak in India. METHODS: This prospective observational study included confirmed adult dengue patients hospitalized between August and November 2015 in a tertiary care centre in New Delhi, India. Appropriate statistical tests were used to compare clinicolaboratory characteristics, derive predictors of severe disease and mortality, and compute a predictive score for mortality. Serotyping was done. RESULTS: Data of 369 patients were analyzed (mean age, 30.9 years; 67% males). Of these, 198 (54%) patients had dengue fever, 125 (34%) had dengue hemorrhagic fever (grade 1 or 2), and 46 (12%) developed dengue shock syndrome (DSS). Twenty-two (6%) patients died. Late presentation to the hospital (≥5 days after onset) and dyspnea at rest were identified as independent predictors of severe disease. Age ≥24 years, dyspnea at rest and altered sensorium were identified as independent predictors of mortality. A clinical risk score was developed (12*age + 14*sensorium + 10*dyspnea), which, if ≥22, predicted mortality with a high sensitivity (81.8%) and specificity (79.2%). The predominant serotypes in Delhi (2015) were dengue virus DENV2 and DENV4. CONCLUSION: Age ≥24 years, dyspnea at rest, and altered sensorium were identified as independent predictors of mortality. Platelet counts did not determine outcome in dengue patients. Timely referral/access to healthcare is important. The clinical risk score for mortality prediction that was developed in this study can be used in all healthcare settings, after validation in larger cohorts.

Detection of dengue-4 virus in pune, western india after an absence of 30 years--its association with two severe cases.

BACKGROUND: Difference in severity of dengue outbreaks has been related to virus serotype, genotype and clades within genotypes. Till the 1980 s, India and Sri Lanka reported low number of dengue hemorrhagic fever (DHF) cases despite circulation of all four serotypes of dengue virus (DENV). Since the 1990 s the occurrence of DHF has increased. The increase has been attributed to changes in virus lineage especially with regard to DENV-2 and DENV-3. DENV-1 has been associated with dengue fever (DF) outbreaks and DENV-4 reports have been rare. The emergence of DENV-4 was reported recently in 2003 in Delhi and in 2007 in Hyderabad. The last report of DENV-4 from Maharashtra was in 1975 from Amalner. RESULTS: We report on the detection of DENV-4 in Pune, Maharashtra after an absence of almost 30 years. Two cases were detected in 2009-10, serotyped by multiplex reverse transcriptase polymerase chain reaction (RT-PCR). Both the cases were recorded as severe dengue (Category 3) requiring intensive care unit (ICU) level of treatment. Depending on the hemagglutination inhibiting (HI) antibody titres the 2009 case was characterized as a primary infection and the 2010 case as a secondary infection. Both the cases presented plasma leakage and neither showed any kind of haemorrhage. The 2009 case survived while the 2010 case was fatal. An isolate was obtained from the 2009 case. Based on envelope (E) gene sequence analysis, the virus belonged to genotype I of DENV-4, and clustered with isolates from India and Sri Lanka and was distant from the isolates from Thailand. The nucleotide and amino acid diversity of the E gene of the Indian isolates increased from 1996 to 2007 to 2009 in context of the E gene sequences of other isolates belonging to genotype I. CONCLUSION: The increasing diversity in the circulating DENV-4 calls for close monitoring of the DENV-4 serotype.