Charitha Lakshini Goonasekara

Dengue fever, a mosquito-borne viral infectious disease caused by the dengue virus, is a significant global health concern, especially in tropical and subtropical regions. Despite preventive efforts, Sri Lanka faces recurring dengue outbreaks, with the Western province being the most affected. Current treatments primarily focus on supportive care, as specific antiviral therapies remain elusive. This review presents an overview of dengue, its clinical presentations, the dengue burden in Sri Lanka, and the potential of Sri Lankan medicinal plants used in traditional medicine for treating dengue. Several plants, such as Munronia pinnata, Azardirachta indica, Cissampelos pareira L., Carica papaya, Zingiber officinale, Curcuma longa, and Bambusa vulgaris, show antiviral properties against dengue. The utilization of these medicinal plants in dengue treatment could offer a promising avenue for further research and drug development.

Background: The natural antibody responses to B-cell epitopes from dengue structural proteins were assessed using immune sera from people having well-defined past dengue infections with one of the four serotypes.Method: Based on an immune-computational analysis previously conducted, nineteen epitopes from the envelope (E) and eight epitopes from pre-membrane (prM), which were more than 50% conserved across all the four DENV serotypes, were selected. Peptides to represent these B-cell epitopes were obtained from commercially available arrays, and were subjected to enzyme linked immunosorbent assay with sera obtained from dengue seropositive healthy volunteers (DENV1 n = 12: DENV2 n = 12: DENV3 n = 12 and DENV4 n = 12), and 10 dengue seronegative healthy volunteers from Sri Lanka. The cut-off value for the positive antibody response was set by taking the mean response of a peptide to the negative sera plus three standard deviations. The peptides (N = 7) showing the broad immune responses were used to generate antibodies in three mice (Balb/c) batches. The mice antisera were then subjected to microneutralization assays against all the four DENV serotypes. An EC₅₀viral neutralization ≥ 40 times the serum dilution was considered as neutralizing.Results: Five of the E-peptide and two prM peptides were recognised by most individuls exposed to infections with each of the four serotypes, showing a serotype cross-reactive broad antibody response. The mice immune sera against the peptides representing the five E protein epitopes neutralized all the four DENV serotypes. Two of these five epitopes are from the Domain II, whereas one of them includes the whole bc-loop region.Conclusion: The antibody responses of highly conserved epitopes across the serotypes, were broadly responsive with sera of all four DENV serotypes collected from individuals infected with only one DENV serotype. Weakly conserved epitopes showed rather specific antibody responses dominated by one or few serotypes.

Although immune responses to the Japanese Encephalitis virus (JEV), and the dengue viruses (DENV) have a potential to modulate the immune responses to each other, this has been poorly investigated. Therefore, we developed an ELISA to identify JEV specific, DENV non cross-reactive antibody responses by identifying JEV specific, highly conserved regions of the virus and proceeded to investigate if the presence of JEV specific antibodies associate with dengue disease severity. 22 JEV specific peptides were identified from highly conserved regions of the virus and the immunogenicity and specificity of these peptides were assessed in individuals who were non-immune to JEV and DENV (JEV^-DENV^-, N = 30), those who were only immune to the JEV and not DENV (JEV⁺DENV^-, N = 30), those who were only immune to DENV(JEV^-DENV⁺, N = 30) and in those who were immune to both viruses (JEV⁺DENV⁺, N = 30). 7/22 peptides were found to be highly immunogenic and specific and these 7 peptides were used as a pool to further evaluate JEV-specific responses. All 30/30 JEV⁺DENV^- and 30/30 JEV⁺DENV⁺ individuals, and only 3/30 (10%) JEV^-DENV⁺ individuals responded to this pool. We further evaluated this pool of 7 peptides in patients following primary and secondary dengue infection during the convalescent period and found that the JEV-specific peptides, were unlikely to cross react with DENV IgG antibodies. We further compared this in-house ELISA developed with the peptide pool with an existing commercial JEV IgG assay to identify JEV-specific IgG following vaccination, and our in-house ELISA was found to be more sensitive. We then proceeded to investigate if the presence of JEV-specific antibodies were associated with dengue disease severity, and we found that those who had past severe dengue (n = 175) were significantly more likely (p<0.0001) to have JEV-specific antibodies than those with past non-severe dengue (n = 175) (OR 5.3, 95% CI 3.3 to 8.3). As our data show that this assay is highly sensitive and specific for detection of JEV-specific antibody responses, it would be an important tool to determine how JEV seropositivity modulate dengue immunity and disease severity when undertaking dengue vaccine trials.

Background: Although immune responses to the Japanese Encephalitis virus (JEV), and the dengue viruses (DENV) have a potential to modulate the immune responses to each other, this has been poorly investigated. Therefore, we developed an ELISA to identify JEV specific, DENV non cross-reactive antibody responses by identifying JEV specific, highly conserved regions of the virus and proceeded to investigate if the presence of JEV specific antibodies associate with dengue disease severity. Methodology/Principal findings: 20 JEV specific peptides were identified from highly conserved regions of the virus and the immunogenicity and specificity of these peptides were assessed in individuals who were non-immune to JEV and DENV (JEV ^- DENV ^- , N=30), those who were only immune to the JEV and not DENV (JEV ⁺ DENV ^- , N=30), those who were only immune to DENV(JEV ^- DENV ⁺ , N=30) and in those who were immune to both viruses (JEV ⁺ DENV ⁺ , N=30). 7/20 peptides were found to be highly immunogenic and specific and these 7 peptides were used as a pool to further evaluate JEV-specific responses. All 30/30 JEV ⁺ DENV ^- and 30/30 JEV ⁺ DENV ⁺ individuals, and only 3/30 (10%) JEV ^- DENV ⁺ individuals responded to this pool. We further evaluated this pool of 7 peptides in patients following primary and secondary dengue infection during the convalescent period and found that the JEV-specific peptides, were unlikely to cross react with DENV IgG antibodies. We further compared this in-house ELISA developed with the peptide pool with an existing commercial JEV IgG assay to identify JEV-specific IgG following vaccination, and our in-house ELISA was found to be more sensitive. We then proceeded to investigate if the presence of JEV-specific antibodies were associated with dengue disease severity, and we found that those who had past severe dengue (n=175) were significantly more likely (p<0.0001) to have JEV-specific antibodies than those with past non-severe dengue (n=175) (OR 5.3, 95% CI 3.3 to 8.3). Conclusions/Significance: As our data show that this assay is highly sensitive and specific for detection of JEV-specific antibody responses, it would be an important tool to determine how JEV seropositivity modulate dengue immunity and disease severity when undertaking dengue vaccine trials. Author summary: Both Japanese Encephalitis virus (JEV), and the dengue viruses (DENV) co-circulate in the same geographical region and have a potential to modulate the immune responses to each other. However, due to the difficulty in identifying antibody responses specific to either virus due to the highly cross-reactive nature of virus-specific antibodies, this has been poorly investigated. Therefore, we developed an ELISA to identify JEV-specific, DENV non cross-reactive antibody responses by identifying JEV-specific, highly conserved regions of the virus and proceeded to investigate if the presence of JEV-specific antibodies associates with dengue disease severity. 20 JEV-specific peptides were identified from highly conserved regions of the virus and the immunogenicity and specificity of these peptides were assessed. We found that seven peptides were highly immunogenic and specific to the JEV and we further evaluated the usefulness of an ELISA developed using these pools of peptides. We found that our in-house ELISA was found to be significantly more sensitive some of the existing commercial assays. As this assay appears to be highly sensitive and specific for detection of JEV-specific antibody responses, it would be an important tool to determine how JEV seropositivity modulate dengue immunity and disease severity when undertaking dengue vaccine trials.

Background: Due to the similarity between the dengue (DENV) and the Japanese encephalitis virus (JEV) there is potential for immune cross-reaction. We sought to identify T cell epitopes that are specific to JEV and do not cross react with DENV. Methodology: 20mer peptides were synthesized from regions which showed >90% conservation. Using IFNγ cultured ELISpot assays, we investigated JEV-specific T cell responses in DENV⁻ and JEV⁻ non-immune individuals (DENV⁻JEV⁻ = 21), JEV seronegative and had not received the JE vaccine, but who were DENV seropositive (DENV⁺JEV⁻ = 22), JEV⁺(seropositive for JEV and had received the JE vaccine), but seronegative for DENV (DENV⁻JEV⁺ = 23). We further assessed the responses to these peptides by undertaking ex vivo IFNγ assays and flow cytometry. Results: None of DENV⁻JEV⁻ individuals responded to any of the 20 JEV-specific peptides. High frequency of responses was seen to 6/20 peptides by individuals who were JEV⁺ but DENV⁻, where over 75% of the individuals responded to at least one peptide. P34 was the most immunogenic peptide, recognized by 20/23 (86.9%) individuals who were DENV⁻JEV⁺, followed by peptide 3 and peptide 7 recognized by 19/23 (82.6%). Peptide 34 from the NS2a region, showed Ex vivo responses to these peptides were less frequent, with only 40% of individuals responding to peptide 34 and 16–28% to other peptides, probably as 5/6 peptides were recognized by CD4+ T cells. Discussion: We identified six highly conserved, T cell epitopes which are highly specific for JEV, in the Sri Lankan population. Since both JEV and DENV co-circulate in the same regions and since both JE and dengue vaccines are likely to be co-administered in the same geographical regions in future, these JEV-specific T cell epitopes would be useful to study JEV-specific T cell responses, in order to further understand how DENV and JEV-specific cellular immune responses influence each other.

Plants have been utilized as medicines to treat various ailments since ancient times. Formulations made by plant materials have been used in traditional, complementary, and alternative medicine and remain widespread in both developing and developed countries. In developing countries, traditional medicines are widely practiced due to its accessibility and affordability, while in developed countries, complementary and alternative medicine are widely popular due to the adverse effects of chemical drugs. Tragia involucrata Linn. (family: Euphorbiaceae) is a highly used medicinal plant used in both Sri Lankan and Indian traditional medical systems. Since this plant is a weed, it is being extensively destroyed due to the lack of knowledge regarding the medicinal value of this plant. Hence, the objective of this study was to collect data on the medicinal value of this plant by correlating its scientifically validated biological activities with its ethnopharmacological uses. An attempt was made to gather as much information available regarding the ethnopharmacological uses and scientifically validated biological activities of Tragia involucrata through authentic traditional texts, scientific journals, and other authentic texts regarding medicinal plants. Thus, the review provides an insight to the capability of Tragia involucrata to be used as a monoherbal formulation for diseases pertaining to multiple systems of the body. With all the scientifically validated biological activities and the ethnopharmacological uses, Tragia involucrata may qualify as a potent candidate to be developed into a phytomedicine to be utilized as both a preventive and as a therapeutic agent.

Cancer is a global burden. In low- and middle-income countries around 70% of deaths are due to cancer. For a number of years natural products have been a good source of agents for combatting cancer and plants have played a huge role in anti-cancer product development. For many centuries, indigenous cultures around the world have used traditional herbal medicine to treat a myriad of diseases including cancer. In Sri Lanka, a number of plants have been reported to have anti-cancer properties and some of the commonly used plants are described in this review with an account of their compounds and modes of action. Only a small number of the plants in Sri Lanka have been tested for their bioactivity and more research is required to determine their medicinal activity with the aim of developing novel drugs to fight this disease.

This study aims to characterize the antigenicity of the Capsid (C) protein and the human antibody responses to C protein from the four dengue virus (DENV) serotypes. Parker hydrophilicity prediction, Emini surface accessibility prediction and Karplus & Schulz flexibility predictions were used to bioinformatically characterize antigenicity. The human antibody response to C protein was assessed by ELISA using immune sera and an array of overlapping DENV2 C peptides. DENV2 C protein peptides P1 (located on C protein at 2–18 a.a), P11 (79–95 a.a) and P12 (86–101 a.a) were recognized by most individuals exposed to infections with only one of the 4 DENV serotypes as well as people exposed to infections with two serotypes. These conserved peptide epitopes are located on the amino (1–40 a.a) and carboxy (70–100 a.a) terminal regions of C protein, which were predicted to be antigenic using different bioinformatic tools. DENV2 C peptide P6 (39–56 a.a) was recognized by all individuals exposed to DENV2 infections, some individuals exposed to DENV4 infections and none of the individuals exposed to DENV1 or 3 infections. Thus, unlike C peptides P1, P11 and P12, which contain epitopes, recognized by DENV serotype cross-reactive antibodies, DENV2 peptide P6 contains an epitope that is preferentially recognized by antibodies in people exposed to this serotype compared to other serotypes. We discuss our results in the context of the known structure of C protein and recent work on the human B-cell response to DENV infection.

B-cell epitopes on the envelope (E) and premembrane (prM) proteins of dengue virus (DENV) were predicted using bioinformatics tools, BepiPred, Ellipro, and SVMTriP. Predicted epitopes, 32 and 17 for E and prM proteins, respectively, were then characterized for their level of conservations. The epitopes, EP4/E (48–55), epitope number 4 of E protein at amino acids 48–55, EP9/E (165–182), EP11/E (218–233), EP20/E (322–349), EP21/E (326–353), EP23/E (356–365), and EP25/E (380–386), showed a high intraserotype conservancy with very low pan-serotype conservancy, demonstrating a potential target as serotype specific diagnostic markers. EP3 (30–41) located in domain-I and EP26/E (393–409), EP27/E (416–435), EP28/E (417–430) located in the stem region of E protein, and EP8/prM (93–112) from the prM protein have a pan-serotype conservancy higher than 70%. These epitopes indicate a potential use as universal vaccine candidates, subjected to verification of their potential in viral neutralization. EP2/E (16–21), EP5/E (62–123), EP6/E (63–89), EP19/E (310–329), and EP24/E (371–402), which have more than 50% pan-serotype conservancies, were found on E protein regions that are important in host cell attachment. Previous studies further show evidence for some of these epitopes to generate cross-reactive neutralizing antibodies, indicating their importance in antiviral strategies for DENV. This study suggests that bioinformatic approaches are attractive first line of screening for identification of linear B-cell epitopes.