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.

Serum ferritin level as a prognostic biomarker for predicting dengue disease severity: A systematic review and meta-analysis.

Serum ferritin levels serves as biomarkers in many inflammatory and infectious diseases. This current systematic review and meta-analysis evaluated whether serum ferritin levels are associated with severe dengue and its utility as a biomarker of disease severity. Literature searches were conducted in PubMed, Scopus, ScienceDirect, the Cochrane library, and Google Scholar. A total of 18 studies examining the serum ferritin levels in dengue cases in the context of disease severity (nine studies having dengue classification as non-severe vs. severe dengue cases, and nine studies having dengue classification as dengue without warning signs (DwoWS), dengue with warning signs (DwWS), and severe dengue cases) were included and the quality of the studies was assessed using the Quality in Prognostic Factor Studies tool. The meta-analysis was performed using STATA software to calculate the effect size as a standardized mean difference (SMD) or Hedges 'g' for the continuous outcome. Higher serum ferritin levels were found in severe dengue cases compared to non-severe cases [SMD (Hedges 'g') 4.05 (95% C.I. 2.09-6.00), (I2  = 98.8%)]. In the second group, DwWS cases showed high serum ferritin levels compared to DwoWS [SMD 2.01 (95% C.I. 0.92-3.10), (I2  = 97.89%)], and severe dengue cases showed higher levels of serum ferritin compared to DwWS [SMD 2.66 (95% C.I. 1.72-4.48), (I2  = 98.78%)] and DwoWS cases [SMD 6.65 (95% C.I. 1.72-11.59), (I2  = 99.78%]. Subgroup analysis for the country of study (India vs. others), ferritin testing methods, and ferritin measurement day revealed testing method as a significant contributor to heterogeneity. To conclude, the present study suggests serum ferritin as a prognostic marker for dengue disease severity. Multi-centric studies involving a large number of dengue patients with a uniform case definition accounting for all the confounding variables might help in determining a universal cut-off value to discriminate between non-severe and severe dengue.

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.

Anti-Dengue Activity of Lipophilic Fraction of Ocimum basilicum L. Stem.

Ocimum basilicum L. is used to cure many types of fever in traditional medicine. This study aims to explore the antiviral activity of the lipophilic fraction of the stem of O. basilicum (LFOB) against dengue virus (DENV) and chikungunya virus (CHIKV). The LFOB was analyzed using GC-FID and GC-MS. The antiviral activity of LFOB 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). In vitro antiviral activity and FFU assay were used to determine and confirm antiviral activity against DENV and CHIKV. Twenty-six compounds were identified in LFOB using GC/MS. The most abundant compounds were ß-sitosterol (22.9%), stigmasterol (18.7%), and campesterol (12.9%). Significant reduction in DENV titre was observed under pre- and post-infection treatment conditions at a concentration of 3.125 µg/mL, but no anti-CHIKV activity was observed. Our earlier and the present AutoDock-Vina-based in silico docking study revealed that ß-sitosterol and stigmasterol could form strong interactions with the DENV E glycoprotein and DENV RdRp domain, respectively. Our findings suggest that LFOB can inhibit DENV infection and might act as a potent prophylactic/therapeutic agent against DENV-2. In silico results suggested that ß-sitosterol and stigmasterol may block the viral entry by inhibiting the fusion process and viral replication respectively.

In vitro and in vivo studies reveal α-Mangostin, a xanthonoid from Garcinia mangostana, as a promising natural antiviral compound against chikungunya virus.

BACKGROUND: Chikungunya virus (CHIKV), a serious health problem in several tropical countries, is the causative agent of chikungunya fever. Approved antiviral therapies or vaccines for the treatment or prevention of CHIKV infections are not available. As diverse natural phenolic compounds have been shown to possess antiviral activities, we explored the antiviral activity of α-Mangostin, a xanthanoid, against CHIKV infection. METHODS: The in vitro prophylactic and therapeutic effects of α-Mangostin on CHIKV replication in Vero E6 cells were investigated by administering it under pre, post and cotreatment conditions. The antiviral activity was determined by foci forming unit assay, quantitative RT-PCR and cell-based immune-fluorescence assay. The molecular mechanism of inhibitory action was further proposed using in silico molecular docking studies. RESULTS: In vitro studies revealed that 8 µM α-Mangostin completely inhibited CHIKV infectivity under the cotreatment condition. CHIKV replication was also inhibited in virus-infected mice. This is the first in vivo study which clearly showed that α-Mangostin is effective in vivo by significantly reducing virus replication in serum and muscles. Molecular docking indicated that α-Mangostin can efficiently interact with the E2-E1 heterodimeric glycoprotein and the ADP-ribose binding cavity of the nsP3 macrodomain. CONCLUSIONS: The findings suggest that α-Mangostin can inhibit CHIKV infection and replication through possible interaction with multiple CHIKV target proteins and might act as a prophylactic/therapeutic agent against CHIKV.

Oligonucleotide-Based Approaches to Inhibit Dengue Virus Replication.

Dengue fever is one of the most common viral infections affecting humans. It is an expanding public health problem, particularly in tropical and subtropical regions. No effective vaccine or antiviral therapies against Dengue virus (DENV) infection are available. Therefore, there is a strong need to develop safe and effective therapeutic strategies that can reduce the burden and duration of hospitalizations due to this life-threatening disease. Oligonucleotide-based strategies are considered as an attractive means of inhibiting viral replication since oligonucleotides can be designed to interact with any viral RNA, provided its sequence is known. The resultant targeted destruction of viral RNA interferes with viral replication without inducing any adverse effects on cellular processes. In this review, we elaborate the ribozymes, RNA interference, CRISPR, aptamer and morpholino strategies for the inhibition of DENV replication and discuss the challenges involved in utilizing such approaches.

In Vitro Antiviral Activity of α-Mangostin against Dengue Virus Serotype-2 (DENV-2).

Dengue virus (DENV), a member of the family Flaviviridae, is a threat for global health as it infects more than 100 million people yearly. Approved antiviral therapies or vaccines for the treatment or prevention of DENV infections are not available. In the present study, natural compounds were screened for their antiviral activity against DENV by in vitro cell line-based assay. α-Mangostin, a xanthanoid, was observed to exert antiviral activity against DENV-2 under pre-, co- and post-treatment testing conditions. The antiviral activity was determined by foci forming unit (FFU) assay, quantitative RT-PCR and cell-based immunofluorescence assay (IFA). A complete inhibition of DENV-2 was observed at 8 µM under the co-treatment condition. The possible inhibitory mechanism of α-Mangostin was also determined by docking studies. The molecular docking experiments indicate that α-Mangostin can interact with multiple DENV protein targets such as the NS5 methyltransferase, NS2B-NS3 protease and the glycoprotein E. The in vitro and in silico findings suggest that α-Mangostin possesses the ability to suppress DENV-2 production at different stages of its replication cycle and might act as a prophylactic/therapeutic agent against DENV-2.

Seroprevalence and incidence of primary dengue infections among children in a rural region of Maharashtra, Western India.

BACKGROUND: Dengue infections have become a huge threat to public health systems in developing countries. Data on seroprevalence and incidence of dengue infections are lacking from rural regions of India. The objective of present study was to investigate the seroprevalence and incidence of dengue infection utilizing repeated serosurveys from a rural region of Maharashtra, Western India. METHODS: In the present study, 819 children between ages 5 to 15 years from 21 villages in Pune District of Maharashtra, India were sampled in 2014 and 2016. The sera were tested for the presence of dengue specific IgG using an indirect IgG ELISA kit. RESULTS: Overall seroprevalence of dengue was 15.3% (95% confidence intervals (CI) 12.9-17.8%) in 2014 and 20.5% (95% CI 17.8-23.4%) in 2016. Among the 694 children who were seronegative at baseline (2014), 78 seroconverted. Overall incidence rate of primary dengue was 54.2 infections/1000 children years (95% CI 43.0-67.3). Incidence of primary dengue infection was higher in children from urbanized villages compared to rural villages (Incidence rate ratio (IRR) 2.6 (95% CI 1.3-5.2)). In rural villages, incidence of primary dengue infection was higher in children aged 10 years or above as compared to those aged below 10 years (IRR 9.75 (95% CI 1.21-77.9). CONCLUSIONS: The study provides the incidence rates of primary dengue infections from a rural region of India. More multi centric studies investigating the incidence of dengue will provide accurate estimate of incidence of dengue and help formulate well directed policies. The results also suggest that urbanization and transitions in demographic settings might favour dengue outbreaks in rural regions and these regions need to be targeted for vector control measures.

Higher levels of dengue-virus-specific IgG and IgA during pre-defervescence associated with primary dengue hemorrhagic fever.

Dengue hemorrhagic fever (DHF), although predominantly associated with secondary infections, has also been reported in primary infections. An enhanced immune response including antibodies and cytokines is implicated in the pathogenesis of secondary DHF. However, the factors operating in primary DHF are poorly understood. To understand the role of the antibody response, the relative levels of different antibody isotypes during the acute phase of infection in primary and secondary dengue infections were determined. Levels of DENV-specific IgM, IgG, IgA and IgE were measured in the serum samples of 200 dengue patients and 20 dengue-naïve individuals. Samples were collected within 15 days of onset of illness. The DENV-specific IgM levels were significantly higher in DF cases compared to DHF, which was more evident in secondary infections and in post-defervescence samples. The levels of IgG, IgA and IgE were higher in DHF cases, with greater significance in primary infections. A higher level of IgG in DHF cases was evident in pre-defervescence samples, whilst the IgE level was higher in pre- and post-defervescence samples. There was a significant correlation of IgG titres with platelet counts, with higher titres associated with lower platelet counts. It is speculated that IgG, IgA and IgE produced in response to primary infections may contribute to pathogenesis, whilst IgM produced in response to secondary infections may protect against progression to severe disease.

Pertussis Vaccines Scarcely Provide Protection against Bordetella parapertussis Infection in Children-A Systematic Review and Meta-Analysis.

BACKGROUND: Pertussis, or whooping cough, is a global public health concern. Pertussis vaccines have demonstrated good protection against Bordetella pertussis infections, but their effectiveness against Bordetella parapertussis remains debated due to conflicting study outcomes. METHODS: A systematic review and meta-analysis were conducted to assess the effectiveness of pertussis vaccines in protecting children against B. parapertussis infection. A comprehensive search of PubMed, Web of Science, and Scopus databases was conducted, and randomized controlled trials (RCTs) and observational studies that met inclusion criteria were included in the analysis. RESULTS: The meta-analysis, involving 46,533 participants, revealed no significant protective effect of pertussis vaccination against B. parapertussis infection (risk ratio: 1.10, 95% confidence interval: 0.83 to 1.44). Subgroup analyses by vaccine type and study design revealed no significant protection. The dearth of recent data and a limited pool of eligible studies, particularly RCTs, underscore a critical gap that warrants future research in the domain. CONCLUSIONS: These findings offer crucial insights into the lack of effectiveness of pertussis vaccines against B. parapertussis. Given the rising incidence of cases and outbreaks, coupled with the lack of cross-protection by the existing vaccines, there is an urgent need to develop vaccines that include specific antigens to protect against B. parapertussis.

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.

Explorations on the antiviral potential of zinc and magnesium salts against chikungunya virus: implications for therapeutics.

Background: Chikungunya virus (CHIKV), which causes chikungunya fever, is an arbovirus of public health concern with no approved antiviral therapies. A significant proportion of patients develop chronic arthritis after an infection. Zinc and magnesium salts help the immune system respond effectively against viral infections. This study explored the antiviral potential of zinc sulphate, zinc acetate, and magnesium sulphate against CHIKV infection. Methods: The highest non-toxic concentration of the salts (100 µM) was used to assess the prophylactic, virucidal, and therapeutic anti-CHIKV activities. Dose-dependent antiviral effects were investigated to find out the 50% inhibitory concentration of the salts. Entry bypass assay was conducted to find out whether the salts affect virus entry or post entry stages. Virus output in all these experiments was estimated using a focus-forming unit assay, real-time RT-PCR, and immunofluorescence assay. Results: Different time- and temperature-dependent assays revealed the therapeutic antiviral activity of zinc and magnesium salts against CHIKV. A minimum exposure of 4 hours and treatment initiation within 1 to 2 hours of infection are required for inhibition of CHIKV. Entry assays revealed that zinc salt affected virus-entry. Entry bypass assays suggested that both salts affected post-entry stages of CHIKV. In infected C57BL6 mice orally fed with zinc and magnesium salts, a reduction in viral RNA copy number was observed. Conclusion: The study results suggest zinc salts exert anti-CHIKV activity at entry and post entry stages of the virus life cycle, while magnesium salt affect CHIKV at post entry stages. Overall, the study highlights the significant antiviral potential of zinc sulphate, zinc acetate, and magnesium sulphate against CHIKV, which can be exploited in designing potential therapeutic strategies for early treatment of chikungunya patients, thereby reducing the virus-associated persistent arthritis.

Cellular Immune Responses Against γ-Inactivated Antigen in the Recovered Cases of Kyasanur Forest Disease.

Kyasanur Forest Disease Virus (KFDV) is a tick-borne flavivirus that causes life-threatening hemorrhagic fever in humans with case fatality rates of 3-5%. Relatively little is known about the mechanism of its pathogenesis or host immune responses to KFDV infection. Here, we investigated KFDV-specific cellular immune responses in the recovered cases of Kyasanur Forest Disease (KFD). Peripheral blood mononuclear cells of the recovered KFD cases and healthy controls were exposed to γ-inactivated KFDV antigen ex vivo. The proliferation index was determined using an enzyme-linked immunosorbent assay-based lymphoproliferative assay. The frequencies of CD4+ and CD8+ T cells expressing intracellular interferon (IFN)-γ in response to stimulation with γ-inactivated KFDV antigen were determined using flow cytometry. A significant increase in lymphoproliferation and a high frequency of CD4+ and CD8+ T cells secreting IFN-γ against γ-inactivated KFDV antigen were found in the recovered KFD group compared to the healthy control group. In conclusion, the study indicated the generation of cellular immune responses in individuals who recovered from KFD and can be used as indicators of cellular immunity in KFD vaccine studies.

Severe disease during both primary and secondary dengue virus infections in pediatric populations.

Dengue is a global epidemic causing over 100 million cases annually. The clinical symptoms range from mild fever to severe hemorrhage and shock, including some fatalities. The current paradigm is that these severe dengue cases occur mostly during secondary infections due to antibody-dependent enhancement after infection with a different dengue virus serotype. India has the highest dengue burden worldwide, but little is known about disease severity and its association with primary and secondary dengue infections. To address this issue, we examined 619 children with febrile dengue-confirmed infection from three hospitals in different regions of India. We classified primary and secondary infections based on IgM:IgG ratios using a dengue-specific enzyme-linked immunosorbent assay according to the World Health Organization guidelines. We found that primary dengue infections accounted for more than half of total clinical cases (344 of 619), severe dengue cases (112 of 202) and fatalities (5 of 7). Consistent with the classification based on binding antibody data, dengue neutralizing antibody titers were also significantly lower in primary infections compared to secondary infections (P ≤ 0.0001). Our findings question the currently widely held belief that severe dengue is associated predominantly with secondary infections and emphasizes the importance of developing vaccines or treatments to protect dengue-naive populations.

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.

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.