Inhibition of Dengue Virus Entry and Multiplication into Monocytes Using RNA Interference

Prevention and treatment of dengue infection remain a serious global public health priority. Extensive efforts are required toward the development of vaccines and discovery of potential therapeutic compounds against the dengue viruses. Dengue virus entry is a critical step for virus reproduction and establishes the infection. Hence, the blockade of dengue virus entry into the host cell is an interesting antiviral strategy as it represents a barrier to suppress the onset of infection. This study was achieved by using RNA interference to silence the cellular receptor, and the clathrin mediated endocytosis that enhances the entry of dengue virus in monocytes. Results showed a marked reduction of infected monocytes by flow cytometry. In addition, both intracellular and extracellular viral RNA load was shown to be reduced in treated monocytes when compared to untreated monocytes. Based on these findings, this study concludes that this therapeutic strategy of blocking the virus replication at the first stage of multiplication might serve as a hopeful drug to mitigate the dengue symptoms, and reduction the disease severity

RNA Interference Mediated Inhibition of Dengue Virus Multiplication and Entry in HepG2 Cells

Background: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells. Methodology/Principal Findings: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8 % for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells

Nigella sativa (Black Seed) as a Natural Remedy against Viruses

The currently available antiviral agents are associated with serious adverse effects, coupled with the increasing rate of viral resistance to the existing antiviral drugs. Hence, the search for alternative natural remedies is gaining momentum across the globe. Nigella sativa Linnen, also called Black seed, is a medicinal plant that is gaining worldwide recognition and has been extensively investigated. The present work is aimed to review the existing literature on the antiviral efficacy of Nigella sativa extracts (oil & bioactive compounds). The findings reveal that numerous articles have been published on Nigella sativa and its beneficial effects against different kinds of diseases. However, the antiviral efficacy of Nigella sativa is yet to be given the proper research attention it deserves

Primer sequences.

<p>Primer sequences of the target genes, positive control gene, optimal reference genes and DENV NS5.</p

Genetic Diversity of Fungi Producing Mycotoxins in Stored Crops

Mycotoxins are a variety of critical secondary metabolites for the defense, that produced by multiple types of fungi. These metabolites are toxins where metabolic pathways that produce these toxins are found in adjacent gene groups in the fungal genome when they have adequate environmental and dietary conditions. Mainly, they found in commodities stored by the wrong ways. Mycotoxins are the most potent known toxins that cause serious diseases with minimal concentrations. Genetic diversity was detected using polymorphic randomized amplification technique for DNA fragments between fungal isolates from different crops. This review article aims to review the current status of genetically diverse of mycotoxigenic fungi in various contaminated food. Several studies that have focused on the determination of prevalence and frequency of varies types of toxic fungi were reviewed. Also, the articles that study the toxicity of stored crops such as cereals and oilseeds were considered. The high contrast between findings of these works was presented in terms of the genetic diversity of fungal isolates produced toxins. Aspergillus, Fusarium, and Penicillium were observed among the most common fungus producing toxins. This study which derived from previous researches observed that Aflatoxin was the most toxin produced by most fungi. Aspergillus was the most genetically modified fungus, carrying the most genes responsible for producing the fungal toxins

Quantification of infected cells by flow cytometry.

<p>At 72 h post-transfection, monocytes were infected by DENV-2 at MOI of 2. Marked reduction in percentage of infected cells was observed. This figure shows the percentage of DENV infected cells at different conditions. (a) Transfected non-infected monocytes (0.0%) represents the negative control. (b) Transfected mock-infected monocytes as a staining control (0.0%). (c) Non-transfected infected monocytes (34.9%) as a positive control. (d) CD-14 associated molecule silenced infected monocytes (14.3%). (e) CLTC silenced infected monocytes (11.1%). (f) DNM2 silenced infected monocytes (18.5%). (g) CD-14 associated molecule, CLTC, and DNM2 combined silenced infected monocytes (5.1%). (h) Summarized the results of the flow cytometry experiments. Data is expressed as a percentage of infected cells compared with non-transfected infected monocytes (NTI) which was defined as 100%. The percentages of the infected cells are 41.0%, 36.2%, 52.9% and 14.8% for CD-14 associated molecule, CLTC, DNM2, and combined silenced monocytes, respectively (One-way ANOVA with Dunnett's post-test, P<0.0001, Error bars are ± SD).</p

Silencing efficiency of target genes.

<p>siRNA pools were proven to be specific and potent in silencing target genes. Each gene of the target genes was targeted with a pool of siRNAs consisted of three different siRNAs. Results showed an efficient gene knockdown when compared with non-transfected control and normalized to reference genes (One-way ANOVA with Dunnett's post-test, P<0.0001, Error bars are ± SD). The effect of a combined transfection of the siRNA pools in monocytes is consistent, and there are no significant differences between separated transfection, (82.4%±1.9), (86.0%±0.6), and (78.5%±3.0), and combined transfection (82.7%±2.4), (84.9%±1.2), and (76.3%±1.7), on expression of CD-14 associated molecule, CLTC, and DNM2, respectively (Two-way ANOVA with Bonferroni post-test, P>0.05, Error bars are ± SD). Scrambled siRNA had no inhibitory effect on any gene expression and showed a similar expression to the non-transfected control.</p

Quantification of extracellular dengue virus RNA load.

<p>RT-qPCR was used to quantify the DENV RNA in the culture supernatant of silenced and non-silenced HepG2 cells. Marked reduction in viral RNA was achieved (65.1%±1.7), (65.4%±9.8), (60.9%±10.6), and (71.4%±5.7) for GRP78, CLTC, DNM2, and combined silenced HepG2 cells respectively. This result is statistically significant (One-way ANOVA with Dunnett's post-test, P<0.0001, Results are expressed as mean ± SD from a representative experiment performed in triplicate). There is no significant difference between scramble siRNA transfected and non-transfected infected HepG2 cells (One-way ANOVA with Dunnett's post-test, P>0.05). (TNI, Transfected Non-Infected; NTI, Non-Transfected Infected; NTNI, Non-Transfected Non-Infected).</p

Efficiency of silencing target genes.

<p>Each of target genes was targeted with a specific pool of siRNA. An efficient gene silencing was achieved in both separated and combined transfection when compared with non-transfected control and normalized to reference genes (One-way ANOVA with Dunnett's post-test, P<0.0001, Results are expressed as mean ± SD from a representative experiment performed in triplicate). There are no significant differences between separated transfection (87.1%±0.9, 90.5%±0.5, and 87.4%±1.0) and combined transfection (87.2%±1.0, 90.3%±0.9, and 87.8%±1.6) on gene expression of GRP78, CLTC, and DNM2 respectively (Two-way ANOVA with Bonferroni post-test, P>0.05, Results are expressed as mean ± SD from a representative experiment performed in triplicate). Scrambled siRNA control had no inhibitory effect on any gene expression, and a similar expression to the non-transfected control was observed (One-way ANOVA with Dunnett's post-test, P>0.05).</p

Sequences of siRNA oligonucleotide template.

a<p>The position refers to the siRNA oligonucleotide position on the target gene.</p>b<p>Scrambled siRNA is a control used to discount any changes to the gene expression profile that may result from the siRNA delivery method.</p><p>Three siRNAs for each target gene were designed. The experiment includes also one Positive control siRNA for human β-actin gene and one scramble siRNA oligonucleotide.</p