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.

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.

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.

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.

Synthesis of novel rhodamine type Anthrone Spiro-lactam (ASL) analogues and evaluation of antiviral activity against dengue and chikungunya viruses.

A series of Rhodamine type Anthrone-Spirolactam (ASL) derivatives Benzylimin-Anthrone-Spirolactam (ASL-1 to ASL-10) and Benzamide-Anthrone-Spirolactam (ASL-11 and ASL-12) were synthesized via a simple condensation reaction between Anthrone Spiro-lactamine (2) and various aromatic aldehyde and acyl chlorides respectively. Since rhodamine-based compounds were reported to have antiviral activity, the ASL derivatives were examined for in vitro antiviral activity against dengue and chikungunya viruses. Among all the analogues, ASL-3, ASL-6, ASL-7, ASL-8, ASL-9 and ASL-10 were the most potent against dengue virus (DENV) and exerted around one log reduction in virus titre under post-treatment conditions. At the same time ASL-3 was effective under co-treatment conditions. Two analogues ASL-6 and ASL-12 exerted anti-chikungunya virus (CHIKV) activity under post-treatment conditions. In silico docking studies revealed that the ASL derivatives interacted with the proteins of DENV and CHIKV. Together, the results suggest the anti-DENV and CHIKV activity of ASL derivatives which may be exploited further for therapeutic purposes.

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.].

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.

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.

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.

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.

ArVirInd-a database of arboviral antigenic proteins from the Indian subcontinent.

Background: Studies on antigenic proteins for arboviruses are important for providing diagnostics and vaccine development. India and its neighboring countries have a huge burden of arboviral diseases. Data mining for country-specific sequences from existing bioinformatics databases is cumbersome and time-consuming. This necessitated the development of a database of antigenic proteins from arboviruses isolated from the countries of the Indian subcontinent. Methods: Arboviral antigenic protein sequences were obtained from the NCBI and other databases. In silico antigenic characterization was performed (Epitope predictions) and data was incorporated into the database. The front end was designed and developed using HTML, CSS, and PHP. For the backend of the database, we have used MySQL. Results: A database, named ArVirInd, is created as a repository of information on curated antigenic proteins. This enlists sequences by country and year of outbreak or origin of the viral strain. For each entry, antigenic information is provided along with functional sites, etc. Researchers can search this database by virus/protein name, country, and year of collection (or in combination) as well as peptide search for epitopes. It is available publicly via the Internet at http://www.arvirind.co.in. ArVirInd will be useful in the study of immune informatics, diagnostics, and vaccinology for arboviruses.

Studies on the antiviral activity of chebulinic acid against dengue and chikungunya viruses and in silico investigation of its mechanism of inhibition.

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.

Decadal Change in Seroprevalence of Chikungunya Virus Infection in Pune City, India.

Chikungunya virus (CHIKV) is an arthropod-borne virus capable of causing large outbreaks. We aimed to determine the decadal change in the extent of chikungunya virus infection from 2009 to 2019. We implemented a prospective cross-sectional survey in Pune City using a 30-cluster approach with probability-proportion-to-size (PPS) sampling, with blood samples collected from 1654 participants in early 2019. The study also included an additional 799 blood samples from an earlier serosurvey in late 2009. The samples were tested by an in-house anti-CHIKV IgG ELISA assay. The overall seroprevalence in 2019 was 53.2% (95% CI 50.7−55.6) as against 8.5% (95% CI 6.5−10.4) in 2009. A fivefold increase in seroprevalence was observed in a decade (p < 0.00001). The seroprevalence increased significantly with age; however, it did not differ between genders. Modeling of age-stratified seroprevalence data from 2019 coincided with a recent outbreak in 2016 followed by the low-level circulation. The mean estimated force of infection during the outbreak was 35.8% (95% CI 2.9−41.2), and it was 1.2% after the outbreak. To conclude, the study reports a fivefold increase in the seroprevalence of chikungunya infection over a decade in Pune City. The modeling approach considering intermittent outbreaks with continuous low-level circulation was a better fit and coincided with a recent outbreak reported in 2016. Community engagement and effective vector control measures are needed to avert future chikungunya outbreaks.

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.