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

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

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

Prevalence of intestinal helminths of the Silver grunt (Pomadasys Kaakan) and black finned sea bream (Spondyliosoma cantharus) fish collected from Kuwait

Climate change has caused extensive changes to marine ecosystems in recent decades; this might lead to an increase in marine disease. Parasite infestation in silver grunt and black finned sea bream is still few. This study aimed to determine the prevalence and intensity of intestinal helminthes of 60 sample of Silver grunt and 60 samples of black finned sea bream species collected from Kuwait fish markets. The result revealed that the total infection rate in each species was 58.3% and 33.3% respectively.  The endoparasite worms found in the intestine of Silver grunt were Procamallanus sinespinis which recorded the highest infestation rate 26.6% with intensity 2, followed by Aephnidiogenes senegalensis (25%) followed by Prosorhynchus indicus (18.3%) with intensity 4 for each, Anisakis physeteris and Aniskais simplex with infestation rate 11.6, 8.3%, respectively and intensity of 2 and 4, respectively. In addition, the endoparasite worms found in the intestine of black finned sea bream was Hysterothylacium fabri (8.3%). Histopathological examination of infested intestine showed necrosis, degenerative changes, and congestion of intestinal blood vessels

Inhibition of dengue virus entry into target cells using synthetic antiviral peptides

Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6 and 40.6 respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection

Isolation and Screening of Extracellular Protease Enzyme from Fungal Isolates of Soil

Protease is an enzyme that conducts proteolysis and receives high attention as the most important enzyme in many industries. The enzymes find applications in waste treatment, bioremediation process, detergents and leather industry. However, the search for microbial sources of novel proteases in natural diversity is a promising area of interest for researchers. In the present research work, protease producing microorganisms were isolated from soil samples collected from three different locations in Petaling Jaya region from soil surface and at depths of 15 cm. The isolated organisms screened for their protease generating abilities by using the skim milk agar experiment. The isolates that gave high positive results in screening identified as Aspergillus sp., Rhizopus sp., Fusarium sp., and Mucor sp. by analyzing the colonies morphology and using lacto- phenol cotton blue (LPCB) staining technique. Among these isolates, Aspergillus sp. 14L3S which isolated from soil surface demonstrated the highest protease activity indicated by the clear zone around the colony which is incubated at room temperature and maintained a pH of about 6.0 for 96 hours. The results showed that among the isolates four fungi had great potential to be used for the production of protease enzymes and upscale for industrial production

Primer sequences.

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

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