Development and validation of cost-effective SYBR Green-based RT-qPCR and its evaluation in a sample pooling strategy for detecting SARS-CoV-2 infection in the Indonesian setting.

A low-cost SYBR Green-based RT-qPCR method to detect SARS-CoV-2 were developed and validated. Primers targeting a conserved and vital region of the N genes of SARS-CoV-2 were designed. In-silico study was performed to analyse the compatibility of the selected primer pair with Indonesian SARS-CoV-2 genome sequences available from the GISAID database. We determined the linearity of our new assay using serial dilution of SARS-CoV-2 RNA from clinical samples with known virus concentration. The assay was then evaluated using clinically relevant samples in comparison to a commercial TaqMan-based test kit. Finally, we applied the assay in sample pooling strategies for SARS-CoV-2 detection. The SYBR Green-based RT-qPCR method was successfully developed with sufficient sensitivity. There is a very low prevalence of genome variation in the selected N primer binding regions, indicating their high conservation. The validation of the assay using clinical samples demonstrated similar performance to the TaqMan method suggesting the SYBR methods is reliable. The pooling strategy by combining 5 RNA samples for SARS-CoV-2 detection using the SYBR RT-qPCR methods is feasible and provides a high diagnostic yield. However, when dealing with samples having a very low viral load, it may increase the risk of missing positive cases.

Low-cost SYBR Green-based RT-qPCR assay for detecting SARS-CoV-2 in an Indonesian setting using WHO-recommended primers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for the ongoing coronavirus disease 2019 (COVID-19) pandemic. For laboratory diagnosis, low-cost detection of SARS-CoV-2 is urgently needed, particularly in developing countries with limited resources. Probe- or TaqMan-based real-time reverse transcription polymerase chain reaction (RT-qPCR) is currently the gold standard for diagnosing infected individuals, as recommended by the World Health Organization (WHO). However, this assay is expensive, making it difficult to use for diagnosis on a large scale. Therefore, in this study, we develop and validate an alternative approach for RT-qPCR diagnosis by employing the DNA intercalating dye SYBR Green. We evaluate and use two WHO-recommended primers, namely CCDC-N and HKU-ORF1b-nsp14. The compatibility of the two primers was tested in silico with Indonesian SARS-CoV-2 genome sequences retrieved from the GISAID database and using bioinformatic tools. Using in vitro-transcribed RNA, optimization, sensitivity, and linearity of the two assays targeting the N and Nsp-14 genes were carried out. For further evaluation, we used clinical samples from patients and performed the SYBR Green-based RT-qPCR assay protocol in parallel with TaqMan-based commercial assay. Our results show that our methodology performs similarly to the broadly used TaqMan-based detection method in terms of specificity and sensitivity and thus offers an alternative assay for the detection of SARS-CoV-2 RNA for diagnostic purposes.

The old pharmaceutical oleoresin labdanum of Cistus creticus L. exerts pronounced in vitro anti-dengue virus activity.

ETHNOPHARMACOLOGICAL RELEVANCE: The haemorrhagic dengue fever affects up to 500 million patients, annually causing 20.000 deaths, with no chemotherapeutic agent available. The oleoresin labdanum of Cistus creticus L. has been established as an anti-infective agent since antiquity in Mediterranean ethnopharmacology. MATERIALS AND METHODS: We tested several extracts and fractions of labdanum - standardised on labdane-type diterpenes via GC-MS - on their activity against the dengue virus (DENV-2 strain 00st-22A) in in vitro Vero cell cultures (96-well-plates, 5 days). Preliminary experiments with a labdanum diethyl ether raw-extract did not yield measureable results due to cytotoxic effects against Vero cells. In all following experiments, cell viability was constantly checked using the MTT-test. Fractionation of this raw-extract by liquid-liquid-extraction and column-chromatography on silica-gel (gradient elution with hexane, EtOAc, CHCl3, MeOH) succeeded in separating the anti-viral activity of labdanum from its cytotoxic effect. RESULTS: In the most active fraction GS5 at 30 µg/ml, dengue virus proliferation was 100% suppressed and cell viability was over 90%. Structural elucidation of major constituents of GS5 is currently ongoing, but thin-layer chromatography showed that this fraction is mainly dominated by manoyloxides, a class of labdane-type diterpenes with known antimicrobial activity. Claims concerning the antiviral activity of above ground parts of C. creticus have been made previously, but these generally ascribe this activity to hot water soluble polyphenols and propose an unspecific tanning effect of the viral surface proteins as the mechanism of action. However, the water soluble fraction enhanced viral proliferation. CONCLUSION: We therefore describe a direct, pharmacological, antiviral activity of a diethyl ether extract of labdanum against a virulent haemorrhagic fever like dengue for the first time.

iPS cell serves as a source of dendritic cells for in vitro dengue virus infection model.

The lack of an appropriate model has been a serious concern in dengue research pertinent to immune response and vaccine development. It remains a matter of impediment in dengue virus (DENV) studies when it comes to an in vitro model, which requires adequate quantity of dendritic cells (DC) with uniform characters. Other sources of DC, mostly monocyte derived DC (moDC), have been used despite their limitations such as quantity, proliferation, and donor dependent characters. Recent development of human iPS cells with consistent proliferation for long, stable, functional characteristics and desired HLA background has certainly offered added advantages. Therefore, we hypothesised that iPS derived cells would be a reliable alternative to the traditional DCs to be used with an in vitro DENV system. To develop a DENV infection and T cell activation model, we utilised iPS cells (HLA-A*24) as the source of DC. iPS-ML-DC was prepared and DENV infectivity was assessed apart from the major surface markers expression and cytokine production potential. Our iPS-ML-DC had major DC markers expression, DENV infection efficiency and cytokine production properties similar to that of moDC. Moreover, DENV infected iPS-ML-DC demonstrated the ability to activate HLA-matched T cell (but not mismatched) in vitro as evidenced by significantly higher proportion of IFN-γ+ CD69+ T cells compared to non-infected iPS-ML-DC. This affirmed the antigen-specific T cell activation by iPS-ML-DC as a function of antigen presenting cells. To conclude, maturation potential, DENV infection efficiency and T cell activation ability collectively suggest that iPS-ML-DC serves as an attractive option of DC for use in DENV studies in vitro.

A Simple Mechanism Based on Amino Acid Substitutions is not a Critical Determinant of High Mortality of Japanese Encephalitis Virus Infection in Mice.

For the development of effective treatment strategies for Japanese encephalitis (JE), it is important to identify the viral factors causing severe disease during JE virus (JEV) infection. In this study, we assessed whether amino acid substitutions are critical factors for higher mortality of JaTH160 compared with JaOArS982 in mice using the technique of infectious cDNA clones. We raised the possibility that two amino acids of C124 and NS3482 of JaTH160 may contribute to increased mortality in mice. However, simultaneous substitutions of these amino acids did not significantly increase the virulence of JaOArS982, suggesting that high mortality due to JaTH160 viral infection cannot be simply attributed to the specific amino acids. Multiple and complex, but not simple, mechanisms may induce the high mortality of JaTH160 infection in mice.

Inhibitory effect of the green tea molecule EGCG against dengue virus infection.

Dengue virus (DENV) infection is a major public health problem worldwide; however, specific antiviral drugs against it are not available. Hence, identifying effective antiviral agents for the prevention of DENV infection is important. In this study, we showed that the reportedly highly biologically active green-tea component epigallocatechin gallate (EGCG) inhibited dengue virus infection regardless of infecting serotype, but no or minimal inhibition was observed with other flaviviruses, including Japanese encephalitis virus, yellow fever virus, and Zika virus. EGCG exerted its antiviral effect mainly at the early stage of infection, probably by interacting directly with virions to prevent virus infection. Our results suggest that EGCG specifically targets DENV and might be used as a lead structure to develop an antiviral drug for use against the virus.

Identification of novel antiviral of fungus-derived brefeldin A against dengue viruses.

Microbial natural products possess a wide range of biological and biochemical potential. Among them, fungal secondary metabolites are one of the most important sources for discovering new drugs or lead compounds. In the present study, we explored substances produced by the strain Penicillium sp. FKI-7127 for its antiviral activity. We identified brefeldin A as a novel antiviral agent against dengue viruses. The inhibitory effect of brefeldin A was confirmed by virus titer and immunofluorescence assay. Brefeldin A inhibited dengue viruses regardless of serotypes and other related viruses including Zika virus and Japanese encephalitis virus. Time-of-addition study showed that brefeldin A exerts its antiviral effect at an early stage of the dengue virus (DENV) life cycle. These studies demonstrate that (i) brefeldin A could be used as a lead compound for drug development of anti-DENV and other related viruses and (ii) fungal metabolites are a potential and valuable source for dengue virus drug discovery.

NS1' Protein Expression in the JaOArS982 Strain of Japanese Encephalitis Virus Does Not Enhance Virulence in Mice.

Using a mouse model, we previously demonstrated that subcutaneous infection with the JaTH160 strain of Japanese encephalitis virus (JEV) causes significantly higher virulence and stronger virus propagation in the brain compared with that of the JaOArS982 strain. We also showed that the JaTH160 strain, but not JaOArS982, expresses the NS1' protein and that NS1' enhances JEV production in avian cells and embryonated chicken eggs. In this study, we examined whether NS1' expression affects virulence in mice infected with the JaOArS982 and JaTH160 strains using the corresponding recombinant viruses S982-IC and JaTH-IC. Expression of the NS1' protein in S982-IC diminished the mortality in mice, whereas S982-IC viruses without NS1' caused 40-60% mortality. However, the viral loads in the brains of these mice were not significantly different despite the dvariation in NS1' expression. JaTH-IC viruses depleted of the NS1' protein exhibited high mortality levels, similar to those of the virus expressing NS1'. Previous studies showed that the NS1' protein plays a role in the enhanced virulence of the JEV SA14 strain in mice. However, our current data suggest that NS1' protein expression in S982-IC reduces, rather than enhances, the mortality in mice. Thus, the effect of NS1' on pathogenicity in vivo may vary among virus strains. Our data also suggest that the reduced mortality resulting from NS1' expression in S982-IC is not simply due to viral replication in the brains. Further investigation is needed to uncover the mechanism by which NS1' affects pathogenicity in JEV-infected animals.

Dengue virus neither directly mediates hyperpermeability nor enhances tumor necrosis factor-α-induced permeability in vitro.

The mechanisms of endothelial barrier dysfunction in dengue disease remain poorly understood. Endothelial cell (EC) death due to virus infection or in combination with an infection-induced cytokine storm is deemed as one of the major causes of plasma leakage. Using an in vitro model of human endothelia and several dengue virus (DENV) strains (including a clinical isolate), the direct consequence of infection on endothelial permeability was investigated throughout the course of the infection. All employed DENV-2 strains were able to infect and replicate in ECs. Rather than increase endothelial permeability, DENV infection alone enhanced cell barrier integrity up to 7 days postinfection. Improved cell barrier function was mediated by type I interferon activation at the early phase of infection and by the survival advantage of the infected cells at the late phase of infection. Consistent with this phenomenon, DENV infection did not augment tumor necrosis factor-α-induced permeability. Our results prove that DENV infection does not directly account for vascular permeability; DENV neither induces hyperpermeability nor exacerbates the permeabilizing effect of cytokines. The contributory role of other factors on plasma leakage during dengue disease warrants further investigation.

Dengue virus strain DEN2 16681 utilizes a specific glycochain of syndecan-2 proteoglycan as a receptor.

Dengue virus (DENV) causes fever and severe haemorrhagic symptoms in humans. The DEN2 16681 strain, derived from a dengue haemorrhagic fever patient, has been widely used in studies related to DENV pathogenesis, such as mouse and non-human primate haemorrhagic models and human vascular endothelial-cell permeability. To clarify the entry mechanism of the 16681 strain, we characterized a novel cell receptor for this strain. Our two major findings were as follows: firstly, the SDC2 membrane protein was an effective DEN2 16681 receptor in a cloned K562 cell line. Secondly, a heparan sulfate (HS) glycochain (of four glycochains in SDC2) is the specific binding site of DENV and seems to be involved in tissue-culture adaptation. Our findings present an entry mechanism that could be implicated for DENV adaptation and HS-mediated DENV infection.

Genetic variations and relationship among dengue virus type 3 strains isolated from patients with mild or severe form of dengue disease in Indonesia and Thailand.

Sequence analysis was conducted on structural and non-structural genes of 7 strains of dengue virus type-3 (DENV-3 virus) isolated in Indonesia and Thailand in the year 1973, 1994, and 1998 from patients with different clinical manifestations. In general, sequence similarity among isolates was greater than 93%, indicating that the mutation rate of DENV-3 circulating in this region was not more than 7% in the last 3 decades and suggesting that sequences that may responsible for viral architectures and/or biological function were strictly conserved. Mutations unique to viral strains associated with specific clinical manifestations were not found. Alignment of PrM/M and E nucleic acid sequences followed by parsimony analysis of sequences obtained in this study and published elsewhere allowed generation of phylogenetic trees, demonstrating that DENV-3 strains isolated in Indonesia in 1998 belonged to a separate cluster (subtype 2) from those isolated between 1973-1985 (subtype 1).