Jefree Johari

Antiviral Activity of Baicalein and Quercetin against the Japanese Encephalitis Virus

Japanese encephalitis (JE), a mosquito-borne viral disease, is endemic to the entire east and southeast Asia, and some other parts of the world. Currently, there is no effective therapeutic available for JE; therefore, finding the effective antiviral agent against JEV replication is crucial. In the present study, the in vitro antiviral activity of baicalein and quercetin, two purportedly antiviral bioflavonoids, was evaluated against Japanese encephalitis virus (JEV) replication in Vero cells. Anti-JEV activities of these compounds were examined on different stages of JEV replication cycle. The effects of the compounds on virus replication were determined by foci forming unit reduction assay (FFURA) and quantitative RT-PCR. Baicalein showed potent antiviral activity with IC50 = 14.28 μg/mL when it was introduced to the Vero cells after adsorption of JEV. Quercetin exhibited weak anti-JEV effects with IC50 = 212.1 μg/mL when the JEV infected cells were treated with the compound after virus adsorption. However, baicalein exhibited significant effect against JEV adsorption with IC50 = 7.27 μg/mL while quercetin did not show any anti-adsorption activity. Baicalein also exhibited direct extracellular virucidal activity on JEV with IC50 = 3.44 μg/mL. However, results of quantitative RT-PCR experiments confirmed the findings from FFURA. This study demonstrated that baicalein should be considered as an appropriate candidate for further investigations, such as the study of molecular and cellular mechanism(s) of action and in vivo evaluation for the development of an effective antiviral compound against Japanese encephalitis virus.

Early Detection of Dengue Virus by Use of Reverse Transcription-Recombinase Polymerase Amplification

ABSTRACT 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.

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

BackgroundEarly 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.MethodsA 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).ResultsAcute 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.ConclusionThe 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.

Dengue virus type 1 clade replacement in recurring homotypic outbreaks

BackgroundRecurring 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.ResultsWe 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.ConclusionDENV-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.

Colorimetric Detection of Dengue by Single Tube Reverse-Transcription-Loop-Mediated Isothermal Amplification.

Dengue is usually diagnosed by isolation of the virus, serology or molecular diagnostic methods. Several commercial kits for the diagnosis of dengue are existing, but concerns have arisen regarding to the affordability and performance characteristics of these kits. Hence, the loop-mediated isothermal amplification (LAMP) is potentially ideal to be used especially in resource limited environments. Serum was collected from healthy donors and patients diagnosed with dengue infection. RNA extracted from the serum samples were tested by reverse-transcription-LAMP assay developed based on 3′-NCR gene sequences for DENV 1–4. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. Sensitivity and specificity of RT-LAMP results were calculated and compared to qRT-PCR and ELISA. RT-LAMP is highly sensitive with the detection limit of 10 RNA copies for all serotypes. Dengue virus RNA was detected in all positive samples using RT-LAMP and none of the negative samples within 30–45 minutes. With continuing efforts in the optimization of this assay, RT-LAMP may provide a simple and reliable test for detecting DENV in areas where dengue is prevalent.

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.

Senescence affects endothelial cells susceptibility to dengue virus infection.

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.

The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen

Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2–94.8%) than in those from primary dengue (21.7–64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.

Synovial Tissue Infection with Burkholderia fungorum

To the Editor: The genus Burkholderia, first proposed in 1992, has now expanded to consist of many species (1). Bacteria of this genus exhibit extensive ubiquity; they have been isolated from human, animal, and environmental sources (1,2). Although rare, Burkholderia fungorum has been implicated in several human infections (2–4). We isolated B. fungorum from the synovial tissue of a patient’s knee.