CHIKV infection in mice pre-treated with shRNA E1.

<p>In contrast to the wildtype and scrambled E1 (sE1)-treated groups, pre-treatment of mice with single doses of plasmid-shRNA E1 via intraperitoneal (i.p.) route was shown to confer strong protective effect against CHIKV disease (<i>n = 5</i> per treatment group). Survival of these shRNA E1-treated mice suggested a dose-dependent inhibition against CHIKV pathology throughout 15 days p.i. CHIKV infection was carried out using 10⁶ PFU. WT refers to the non-treated group; For the mock-inf, sterile PBS was inoculated in replacement of infectious CHIKV; shRNA sE1 refers to the shRNA scrambled E1 plasmid.</p

Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication

<div><h3>Background</h3><p>Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection.</p> <h3>Methods</h3><p>Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The <em>in vivo</em> efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection.</p> <h3>Results</h3><p>Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1.</p> <h3>Conclusion</h3><p>Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.</p> </div

Growth kinetics of CHIKV in HeLa S3 cell line.

<p>CHIKV infection was performed at M.O.I. 1 and viral supernatants of the infected cells were harvested at the respective time points. Viral plaque assays were performed to quantitate the infectious CHIKV titre produced. The average ±S.E. (standard error) is expressed from three independent set of experiments.</p

Quantification of CHIKV titre produced from shRNA E1-expressing cell clones.

<p>(<b>A</b>) At higher M.O.I. 10 of CHIKV infection, shRNA E1 expression showed a strong suppression against CHIKV replication in shRNA HeLa cell clones as compared to the non-transfected and sE1-expressing cell clones. (<b>B</b>) <b>&</b> (<b>C</b>) Similar inhibitory trend of low CHIKV production was noted at Day 1–3 p.i. in RD and BHK cell clones with shRNA E1 activity. (<b>D</b>) Broad-spectrum silencing effect of shRNA E1 was notably significant at Day 2 and 3 p.i. where there was complete inhibition on CHIKV Ross strain of the ECSA genotype. The average ±S.E. (standard error) is expressed from three independent set of experiments. Using Student’s T-test analysis, *indicates significant difference (p<0.05) and **indicates a greater significant difference (p<0.005) from control set.</p

Ultrastructural analysis of shRNA cell clones and non-transfected HeLa cells upon CHIKV infection.

<p>At Day 3 p.i., extensive CHIKV replication in non shRNA-expressing HeLa cells was detected with (<b>A</b>) formation of viral replication complexes (Cytopathic vacuoles type I, CPV-I →) as well as (<b>B</b>) numerous CPV-II (▸) containing CHIKV particles in the cytosol. Similar trend of CHIKV infection was observed in (<b>C</b>) stable shRNA scrambled E1 and (<b>D</b>) shRNA scrambled nsP1 cell clones where CHIKV virions were detected to be budding off (*) at the plasma membrane. (<b>E</b>) Stable shRNA E1 and (<b>F</b>) nsP1 cell clones maintained healthy morphology in their membranous organelles structure. There was an absence of CHIKV-induced replication complexes and virus particles in these cell clones. ER, endoplasmic reticulum; Mito, mitochondrion; Nu, nuclei.</p

Quantification of SINV and DENV infectious titres produced from shRNA cell clones and non-transfected HeLa cells.

<p>(<b>A</b>) SINV infection produced consistently high virus yield throughout Day 1–3 p.i., relative to their shRNA scrambled E1 and non-transfected HeLa cell controls. (<b>B</b>) DENV infection showed an increasing trend of virus replication during Day 1–3 p.i, relative to their controls. Taken together, both data indicate the non-target specificity of shRNA E1 against SINV and DENV replication. The average ±S.E. is expressed from three independent set of experiments. Using Student’s T-test analysis, *indicates significant difference (p<0.05) and **indicates a greater significant difference (p<0.005) from control set.</p

Predicted structures of shRNA target sequences in CHIKV genomic RNA and anti-CHIKV shRNA sequences designed.

<p>(<b>A</b>) Region of shRNA E1 target site (location: 10614–10632), (<b>B</b>) Region of shRNA nsP1 target site (location: 694–712) and (<b>C</b>) Region of Capsid shRNA target site (location: 7743–7761). The exact target site in each region is indicated by black bold lines. (<b>D–F</b>) shRNA was expressed from the pSilencer vector as an oligonucleotide duplex construct (55-mer) that contains the antisense sequence which is complementary to its target in the CHIKV genome. Antisense sequences of (<b>D</b>) shRNA E1, (<b>E</b>) shRNA nsP1 and (<b>F</b>) shRNA Capsid shown here are not predicted to form considerable secondary structures. All structures are predicted using mfold web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046396#pone.0046396-Zuker1" target="_blank">[29]</a>.</p

Sequencing of shRNA E1 target region upon extensive passaging of CHIKV in shRNA-expressing cell clones.

<p>Continuous maintenance of CHIKV in shRNA E1 cell clones at the 15^th (P15) and 50^th (P50) passage did not indicate presence of CHIKV resistant mutations in the shRNA target sequence of the viral RNA. Sequences are comparable to the sequence of the initial passage (P0) CHIKV RNA genome.</p

Mosquito Cellular Factors and Functions in Mediating the Infectious entry of Chikungunya Virus

<div><p>Chikungunya virus (CHIKV) is an arthropod-borne virus responsible for recent epidemics in the Asia Pacific regions. A customized gene expression microarray of 18,760 transcripts known to target <em>Aedes</em> mosquito genome was used to identify host genes that are differentially regulated during the infectious entry process of CHIKV infection on C6/36 mosquito cells. Several genes such as epsin I (EPN1), epidermal growth factor receptor pathway substrate 15 (EPS15) and Huntingtin interacting protein I (HIP1) were identified to be differentially expressed during CHIKV infection and known to be involved in clathrin-mediated endocytosis (CME). Transmission electron microscopy analyses further revealed the presence of CHIKV particles within invaginations of the plasma membrane, resembling clathrin-coated pits. Characterization of vesicles involved in the endocytic trafficking processes of CHIKV revealed the translocation of the virus particles to the early endosomes and subsequently to the late endosomes and lysosomes. Treatment with receptor-mediated endocytosis inhibitor, monodansylcadaverine and clathrin-associated drug inhibitors, chlorpromazine and dynasore inhibited CHIKV entry, whereas no inhibition was observed with caveolin-related drug inhibitors. Inhibition of CHIKV entry upon treatment with low-endosomal pH inhibitors indicated that low pH is essential for viral entry processes. CHIKV entry by clathrin-mediated endocytosis was validated via overexpression of a dominant-negative mutant of Eps15, in which infectious entry was reduced, while siRNA-based knockdown of genes associated with CME, low endosomal pH and RAB trafficking proteins exhibited significant levels of CHIKV inhibition. This study revealed, for the first time, that the infectious entry of CHIKV into mosquito cells is mediated by the clathrin-dependent endocytic pathway.</p> </div

Effects of low endosomal pH inhibitors on the entry of CHIKV into C6/36 cells.

<p>C6/36 cells were pre-treated with different drug inhibitors for 3 hours before CHIKV infection. Supernatants were harvested 24 hours p.i for viral plaque assays. Low endosomal pH inhibitors show dose-dependent inhibition of CHIKV entry into (a) bafilomycin A-, (b) concanamycin A-treated cells, infected with CHIKV Singapore/07/2008 strain, (c) concanamycin A-treated infected with CHIKV SGEHICHD122508 strain and (d) concanamycin A-treated cells infected with CHIKV SGEHIDSD67Y2008 strain. The log virus titre is plotted against the concentrations of drug used. Cell viability upon drug treatments is represented by the line graphs. The asterisk indicates *<i>p</i> values<0.05, **<i>p</i> values of <0.01 and ***<i>p</i> values<0.0001 by Student's <i>t</i> test. Asterisks indicate statistically significant results relative to control group (▪).</p