Group IV Getah Virus in Culex Mosquitoes, Malaysia.

A new Getah virus (GETV) strain, B254, was isolated from Culex fuscocephalus mosquitoes captured at Mount Ophir, Malaysia, in 2012. Phylogenetic analyses revealed that GETV B254 is distinct from the old Malaysia GETV MM2021 strain but closely related to group IV GETV from Russia (LEIV16275Mag), China (YN12031), and Thailand (GETV/SW/Thailand/2017).

Serological evidence of DENV, JEV, and ZIKV among the indigenous people (Orang Asli) of Peninsular Malaysia.

Dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV) are mosquito-borne flavivirus of medical importance in tropical countries such as Malaysia. However, much remains unknown regarding their prevalence among the underserved indigenous people (Orang Asli) living in communities in the forest fringe areas of Peninsular Malaysia. Information on the prevalence of diseases is necessary to elevate the effectiveness of disease control and preventive measures. This study aimed to determine the seroprevalence of the three major flaviviruses among the Orang Asli and investigate the association between demographic factors and seropositivities. Sampling activities were conducted in the Orang Asli villages to obtain serum samples and demographic data from consenting volunteers. The presence of DENV, JEV, and ZIKV immunoglobulin G (IgG) antibodies in the sera were examined using commercial enzyme-linked immunosorbent assay kits. A focus reduction neutralization assay was performed to measure virus-specific neutralizing antibodies. A total of 872 serum samples were obtained from the Orang Asli volunteers. Serological assay results revealed that DENV IgG, JEV IgG, and ZIKV IgG seropositivities among the Orang Asli were at 4.9%, 48.4%, and 13.2%, respectively. Neutralizing antibodies (FRNT50 ≥ 1:40) against JEV and ZIKV were found in 86.7% and 100.0%, respectively, out of the samples tested. Positive serology to all three viruses corresponded significantly to the age of the volunteers with increasing seropositivity in older volunteers. Findings from the study suggest that Orang Asli are at significant risk of contracting JEV and ZIKV infections despite the lack of active transmission of the viruses in the country.

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages.

BACKGROUND: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required. METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay. RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P < 0.001). CONCLUSION: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

Dengue Outbreak during Ongoing Civil War, Taiz, Yemen.

We identified dengue in ≈51% of patients given a clinical diagnosis of suspected dengue in Taiz, Yemen, during 2016. The cosmopolitan genotype of dengue virus type 2 was most common; viruses appeared to have originated in Saudi Arabia. Damage to public health infrastructure during the ongoing civil war might enable dengue to become endemic to Yemen.

Baicalein and baicalin as Zika virus inhibitors.

At present, there is no effective antiviral agent for Zika virus (ZIKV), an arbovirus that is known for its teratogenic effects on newborns. Baicalein and baicalin were found to be capable of downregulating ZIKV replication up to 10 hours postinfection, while prophylactic effects were evident in pre-treated cells. Baicalein exhibited its highest potency during intracellular ZIKV replication, whereas baicalin was most effective against virus entry. Our in silico interaction assays predicted that both compounds exhibited the strongest binding affinities towards ZIKV NS5, while the virus envelope glycoprotein was the least likely target protein. These findings serve as a crucial platform for further in-depth studies to decipher the underlying anti-ZIKV mechanism(s) of each compound.

Operational utility of the reverse-transcription recombinase polymerase amplification for detection of dengue virus.

BACKGROUND: A method for rapid detection of dengue virus using the reverse-transcription recombinase polymerase amplification (RT-RPA) was recently developed, evaluated and made ready for deployment. However, reliance solely on the evaluation performed by experienced researchers in a well-structured and well-equipped reference laboratory may overlook the potential intrinsic problems that may arise during deployment of the assay into new application sites, especially for users unfamiliar with the test. Appropriate assessment of this newly developed assay by users who are unfamiliar with the assay is, therefore, vital. METHODS: An operational utility test to elucidate the efficiency and effectiveness of the dengue RT-RPA assay was conducted among a group of researchers new to the assay. Nineteen volunteer researchers with different research experience were recruited. The participants performed the RT-RPA assay and interpreted the test results according to the protocol provided. Deviation from the protocol was identified and tabulated by trained facilitators. Post-test questionnaires were conducted to determine the user satisfaction and acceptability of the dengue RT-RPA assay. RESULTS: All the participants completed the test and successfully interpreted the results according to the provided instructions, regardless of their research experience. Of the 19 participants, three (15.8%) performed the assay with no deviations and 16 (84.2%) performed the assay with only 1 to 5 deviations. The number of deviations from protocol, however, was not correlated with the user laboratory experience. The accuracy of the results was also not affected by user laboratory experience. The concordance of the assay results against that of the expected was at 89.3%. The user satisfaction towards the RT-RPA protocol and interpretation of results was 90% and 100%, respectively. CONCLUSIONS: The dengue RT-RPA assay can be successfully performed by simply following the provided written instructions. Deviations from the written protocols did not adversely affect the outcome of the assay. These suggest that the RT-RPA assay is indeed a simple, robust and efficient laboratory method for detection of dengue virus. Furthermore, high new user acceptance of the RT-RPA assay suggests that this assay could be successfully deployed into new laboratories where RT-RPA was not previously performed.

Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification.

A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue.

High producing tumor necrosis factor alpha gene alleles in protection against severe manifestations of dengue.

UNLABELLED: Dengue virus (DENV) infection usually presents with mild self-limiting dengue fever (DF). Few however, would present with the more severe form of the disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the present study, the association between IL-12B, IL-10 and TNF-α gene polymorphisms and dengue severity was investigated. METHODS: A case-control study was performed on a total of 120 unrelated controls, 86 DF patients and 196 DHF/DSS patients. The polymorphisms in IL-12B, IL-10 and TNF-α genes were genotyped using PCR-RFLP and PCR-sequencing methods. RESULTS: A protective association of TNF-α -308A allele and -308GA genotype against DHF/DSS was observed, while TNF-α -238A allele and -238GA genotype were associated with DHF/DSS. A combination of TNF-α -308GA+AA genotype and IL-10 non-GCC haplotypes, IL-12B pro homozygotes (pro1/pro1, pro2/pro2) and IL-12B 3'UTR AC were significantly correlated with protective effects against DHF/DSS. An association between the cytokine gene polymorphisms and protection against the clinical features of severe dengue including thrombocytopenia and increased liver enzymes was observed in this study. CONCLUSION: The overall findings of the study support the correlation of high-producer TNF-α genotypes combined with low-producer IL-10 haplotypes and IL-12B genotypes in reduced risk of DHF/DSS.

Dengue virus type 1 clade replacement in recurring homotypic outbreaks.

BACKGROUND: Recurring dengue outbreaks occur in cyclical pattern in most endemic countries. The recurrences of dengue virus (DENV) infection predispose the population to increased risk of contracting the severe forms of dengue. Understanding the DENV evolutionary mechanism underlying the recurring dengue outbreaks has important implications for epidemic prediction and disease control. RESULTS: We used a set of viral envelope (E) gene to reconstruct the phylogeny of DENV-1 isolated between the periods of 1987-2011 in Malaysia. Phylogenetic analysis of DENV-1 E gene revealed that genotype I virus clade replacements were associated with the cyclical pattern of major DENV-1 outbreaks in Malaysia. A total of 9 non-conservative amino acid substitutions in the DENV-1 E gene consensus were identified; 4 in domain I, 3 in domain II and 2 in domain III. Selection pressure analyses did not reveal any positively selected codon site within the full length E gene sequences (1485 nt, 495 codons). A total of 183 (mean dN/dS = 0.0413) negatively selected sites were found within the Malaysian isolates; neither positive nor negative selection was noted for the remaining 312 codons. All the viruses were cross-neutralized by the respective patient sera suggesting no strong support for immunological advantage of any of the amino acid substitutions. CONCLUSION: DENV-1 clade replacement is associated with recurrences of major DENV-1 outbreaks in Malaysia. Our findings are consistent with those of other studies that the DENV-1 clade replacement is a stochastic event independent of positive selection.

Outbreak of human infection with Sarcocystis nesbitti, Malaysia, 2012.

An outbreak of fever associated with myalgia and myositis occurred in 2012 among 89 of 92 college students and teachers who visited Pangkor Island, Malaysia. The Sarcocystis nesbitti 18S rRNA gene and sarcocysts were obtained from muscle tissues of 2 students. Our findings indicate emergence of S. nesbitti infections in humans in Malaysia.

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification.

BACKGROUND: Early and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic. METHODS: A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). RESULTS: Acute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses. CONCLUSION: The RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.

Extract of Scutellaria baicalensis inhibits dengue virus replication.

BACKGROUND: Scutellaria baicalensis (S. baicalensis) is one of the traditional Chinese medicinal herbs that have been shown to possess many health benefits. In the present study, we evaluated the in vitro antiviral activity of aqueous extract of the roots of S. baicalensis against all the four dengue virus (DENV) serotypes. METHODS: Aqueous extract of S. baicalensis was prepared by microwave energy steam evaporation method (MEGHE™), and the anti-dengue virus replication activity was evaluated using the foci forming unit reduction assay (FFURA) in Vero cells. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to determine the actual dengue virus RNA copy number. The presence of baicalein, a flavonoid known to inhibit dengue virus replication was determined by mass spectrometry. RESULTS: The IC(50) values for the S. baicalensis extract on Vero cells following DENV adsorption ranged from 86.59 to 95.19 µg/mL for the different DENV serotypes. The IC(50) values decreased to 56.02 to 77.41 µg/mL when cells were treated with the extract at the time of virus adsorption for the different DENV serotypes. The extract showed potent direct virucidal activity against extracellular infectious virus particles with IC(50) that ranged from 74.33 to 95.83 µg/mL for all DENV serotypes. Weak prophylactic effects with IC(50) values that ranged from 269.9 to 369.8 µg/mL were noticed when the cells were pre-treated 2 hours prior to virus inoculation. The concentration of baicalein in the S. baicalensis extract was ~1% (1.03 µg/gm dried extract). CONCLUSIONS: Our study demonstrates the in vitro anti-dengue virus replication property of S. baicalensis against all the four DENV serotypes investigated. The extract reduced DENV infectivity and replication in Vero cells. The extract was rich in baicalein, and could be considered for potential development of anti-DENV therapeutics.

The clinical utility of CD163 in viral diseases.

Macrophage activation and hypercytokinemia are notable presentations in certain viral infections leading to severe disease and poor prognosis. Viral infections can cause macrophage polarization into the pro-inflammatory M1 or anti-inflammatory M2 phenotype. Activated M1 macrophages usually restrict viral replication whereas activated M2 macrophages suppress inflammation and promote tissue repair. In response to inflammatory stimuli, macrophages polarize to the M2 phenotype expressing hemoglobin scavenger CD163 surface receptor. The CD163 receptor is shed as the soluble form, sCD163, into plasma or tissue fluids. sCD163 causes detoxification of pro-oxidative hemoglobin which produces anti-inflammatory metabolites that promote the resolution of inflammation. Hence, increased CD163 expression in tissues and elevated circulatory levels of sCD163 have been associated with acute and chronic inflammatory diseases. CD163 and other macrophage activation markers have been commonly included in the investigation of disease pathogenesis and progression. This review provides an overview of the involvement of CD163 in viral diseases. The clinical utility of CD163 in viral disease diagnosis, progression, prognosis and treatment evaluation is discussed.

Epidemiological serosurvey of chikungunya fever post outbreak at Tanjung Sepat, Malaysia.

INTRODUCTION: Chikungunya fever is a mosquito-borne viral disease that usually presents with prominent arthralgia. An outbreak of chikungunya fever was reported in Tanjung Sepat, Malaysia in 2019. The outbreak was limited in size with a low number of cases being reported. The present study sought to determine the possible variables that could have affected the transmission of the infection. METHODOLOGY: A cross-sectional study involving 149 healthy adult volunteers from Tanjung Sepat was performed soon after the outbreak had subsided. All the participants donated blood samples and completed the questionnaires. Laboratory detection of anti-CHIKV IgM and IgG antibodies was performed using enzyme-linked immunoassays (ELISA). Risk factors associated with chikungunya seropositivity were determined using logistic regression. RESULTS: The majority (72.5%, n = 108) of the study participants tested positive for CHIKV antibodies. Only 8.3% (n = 9) of the participants out of all the seropositive volunteers had an asymptomatic infection. Participants who resided with a febrile (p < 0.05, Exp(B) = 2.2, confidence interval [CI] 1.3-3.6) or a CHIKV-diagnosed person (p < 0.05, Exp(B) = 2.1, CI 1.2-3.6) in the same household were found likely to be tested positive for CHIKV antibodies. CONCLUSIONS: Findings from the study support that asymptomatic CHIKV infections and indoor transmission occurred during the outbreak. Hence, widespread community testing and indoor use of mosquito repellent are among the possible measures that can be implemented to reduce CHIKV transmission during an outbreak.

MERS-CoV seroconversion amongst Malaysian Hajj pilgrims returning from the Middle East, 2016-2018: results from the MERCURIAL multiyear prospective cohort study.

Prospective cohort study to investigate the potential exposure to the Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) following Hajj pilgrims is still very limited. Here, we report the antibody seroconversion study results obtained from successive three years cohort studies (2016-2018) involving the Malaysian Hajj pilgrims returning from the Middle East. A cohort study of Hajj pilgrims from Malaysia enrolled 2,863 participants from 2016-2018, all of whom consented to provide paired blood samples for both pre- and post-Hajj travel to the Middle East. ELISAs and micro-neutralization assays were performed to detect the presence of MERS-CoV IgG antibodies. Sociodemographic data, symptoms experienced during Hajj, and history of exposure to camels or camel products were recorded using structured pre- and post-Hajj questionnaires. A 4-fold increase in anti-MERS-CoV IgG between paired pre-Hajj and post-Hajj serum samples in twelve participants was observed. None of the twelve ELISA-positive sera had detectable levels of virus-neutralizing antibodies. All reportedly had mild symptoms of respiratory symptoms at a certain point during the pilgrimage, implying mild or asymptomatic infections. No association between post-Hajj serum positivity and a history of exposure to camels or camel products was obtained. Findings from the study suggest that serologic conversion to MERS-CoV occurred in at least 0.6% of the Hajj pilgrims returning from the Middle East. Since all the seroconvertants had mild to no symptoms during the sampling period, it highlights the likelihood of occurrence of only low infectivity spillover infections among the Hajj pilgrims.

Novel antiviral activity of baicalein against dengue virus.

BACKGROUND: Dengue is a serious arboviral disease currently with no effective antiviral therapy or approved vaccine available. Therefore, finding the effective compound against dengue virus (DENV) replication is very important. Among the natural compounds, bioflavonoids derived mainly from plants are of interest because of their biological and medicinal benefits. METHODS: In the present study, antiviral activity of a bioflavonoid, baicalein, was evaluated against different stages of dengue virus type 2 (DENV-2) replication in Vero cells using focus forming unit reduction assay and quantitative RT-PCR. RESULTS: Baicalein inhibited DENV-2 replication in Vero cells with IC50= 6.46 µg/mL and SI= 17.8 when added after adsorption to the cells. The IC50 against DENV-2 was 5.39 µg/mL and SI= 21.3 when cells were treated 5 hours before virus infection and continuously up to 4 days post infection. Baicalein exhibited direct virucidal effect against DENV-2 with IC 50= 1.55 µg/mL and showed anti-adsorption effect with IC50 = 7.14 µg/mL. CONCLUSIONS: Findings presented here suggest that baicalein exerts potent antiviral activity against DENV. Baicalein possesses direct virucidal activity against DENV besides its effects against dengue virus adsorption and intracellular replication of DENV-2. Baicalein, hence, should be considered for in vivo evaluation in the development of an effective antiviral compound against DENV.

Flavone enhances dengue virus type-2 (NGC strain) infectivity and replication in vero cells.

This study investigates the effects of 2-phenyl-1-benzopyran-4-one (flavone) on DENV-2 infectivity in Vero cells. Virus adsorption and attachment and intracellular virus replication were investigated using a foci forming unit assay (FFUA) and quantitative RT-PCR, respectively. Addition of flavone (100 µg/mL) significantly increased the number of DENV-2 foci by 35.66% ± 1.52 and 49.66% ± 2.51 when added during and after virus adsorption to the Vero cells, respectively. The average foci size after 4 days of infection increased by 33% ± 2.11 and 89% ± 2.13. The DENV-2 specific RNA copy number in the flavone-treated infected cells increased by 6.41- and 23.1-fold when compared to the mock-treated infected cells. Flavone (100 µg/mL) did not promote or inhibit Vero cell proliferation. The CC50 value of flavone against Vero cells was 446 µg/mL. These results suggest that flavone might enhance dengue virus replication by acting antagonistically towards flavonoids known to inhibit dengue virus replication.

Genomic and In Vitro Phenotypic Comparisons of Epidemic and Non-Epidemic Getah Virus Strains.

Getah virus is an emerging mosquito-borne animal pathogen. Four phylogenetic groups of GETV, Group I (GI), GII, GIII and GIV, were identified. However, only the GETV GIII was associated with disease epidemics suggesting possible virulence difference in this virus group. Here, we compared the genetic and in vitro phenotypic characteristics between the epidemic and non-epidemic GETV. Our complete coding genome sequence analyses revealed several amino acid substitutions unique to the GETV GIII and GIV groups, which were found mainly in the hypervariable domain of nsP3 and E2 proteins. Replication kinetics of the epidemic (GIII MI-110 and GIII 14-I-605) and non-epidemic GETV strains (prototype GI MM2021 and GIV B254) were compared in mammalian Vero cells and mosquito C6/36 and U4.4 cells. In all cells used, both epidemic GETV GIII MI-110 and GIII 14-I-605 strains showed replication rates and mean maximum titers at least 2.7-fold and 2.3-fold higher than those of GIV B254, respectively (Bonferroni posttest, p < 0.01). In Vero cells, the epidemic GETV strains caused more pronounced cytopathic effects in comparison to the GIV B254. Our findings suggest that higher virus replication competency that produces higher virus titers during infection may be the main determinant of virulence and epidemic potential of GETV.

Intracellular translocation of HMGB1 is important for Zika virus replication in Huh7 cells.

Neonatal microcephaly and adult Guillain-Barré syndrome are severe complications of Zika virus (ZIKV) infection. The robustly induced inflammatory cytokine expressions in ZIKV-infected patients may constitute a hallmark for severe disease. In the present study, the potential role of high mobility group box 1 protein (HMGB1) in ZIKV infection was investigated. HMGB1 protein expression was determined by the enzyme-linked immunosorbent assay (ELISA) and immunoblot assay. HMGB1's role in ZIKV infection was also explored using treatment with dexamethasone, an immunomodulatory drug, and HMGB1-knockdown (shHMGB1) Huh7 cells. Results showed that the Huh7 cells were highly susceptible to ZIKV infection. The infection was found to induce HMGB1 nuclear-to-cytoplasmic translocation, resulting in a > 99% increase in the cytosolic HMGB1 expression at 72-h post-infection (h.p.i). The extracellular HMGB1 level was elevated in a time- and multiplicity of infection (MOI)-dependent manner. Treatment of the ZIKV-infected cells with dexamethasone (150 µM) reduced HMGB1 extracellular release in a dose-dependent manner, with a maximum reduction of 71 ± 5.84% (P < 0.01). The treatment also reduced virus titers by over 83 ± 0.50% (P < 0.01). The antiviral effects, however, were not observed in the dexamethasone-treated shHMGB1 cells. These results suggest that translocation of HMGB1 occurred during ZIKV infection and inhibition of the translocation by dexamethasone coincided with a reduction in ZIKV replication. These findings highlight the potential of targeting the localization of HMGB1 in affecting ZIKV infection.

Antiviral activity of four types of bioflavonoid against dengue virus type-2.

BACKGROUND: Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic concentration 50 (CC50) to inhibitory concentration 50 (IC50) for each compound. RESULTS: The half maximal inhibitory concentration (IC50) of quercetin against dengue virus was 35.7 µg mL-1 when it was used after virus adsorption to the cells. The IC50 decreased to 28.9 µg mL-1 when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. The SI values for quercetin were 7.07 and 8.74 µg mL-1, respectively, the highest compared to all bioflavonoids studied. Naringin only exhibited anti-adsorption effects against DENV-2 with IC50 = 168.2 µg mL-1 and its related SI was 1.3. Daidzein showed a weak anti-dengue activity with IC50 = 142.6 µg mL-1 when the DENV-2 infected cells were treated after virus adsorption. The SI value for this compound was 1.03. Hesperetin did not exhibit any antiviral activity against DENV-2. The findings obtained from Foci Forming Unit Reduction Assay (FFURA) were corroborated by findings of the qRT-PCR assays. Quercetin and daidzein (50 µg mL-1) reduced DENV-2 RNA levels by 67% and 25%, respectively. There was no significant inhibition of DENV-2 RNA levels with naringin and hesperetin. CONCLUSION: Results from the study suggest that only quercetin demonstrated significant anti-DENV-2 inhibitory activities. Other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of DENV-2 virus replication. These findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. This group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication.