Introduction

Chikungunya virus (CHIKV) is an arbovirus primarily transmitted to humans through the bites of the Aedes aegypti and A. albopictus mosquitoes1,2. Upon infection with CHIKV, individual succumbs to Chikungunya disease which manifests as sudden-onset fever, severe and persisting arthralgia and/or fatal encelphalitis1,3,4. In recent years, CHIKV has resurged as a significant human pathogen in various tropical and temperate regions worldwide. Since 2005, CHIKV has caused severe unprecedented outbreaks in Indian Ocean islands5,6,7, India8,9,10 and countries in the South-East Asia11,12,13,14,15. Imported cases of CHIKV infection are increasingly reported in other previously non-endemic areas such as Australia16, Europe17,18,19,20,21 and America22,23,24. Currently, local CHIKV transmission is active in various Carribean countries23,25. Given its expanding geographical range, CHIKV disease is an emerging key concern to the world public health.

To date, no specific treatment and commercial vaccines for CHIKV infection is available. Symptomatic relief of CHIKV disease through the use of corticosteriods and non-steroidal anti-inflammatory drugs are accompanied with side effects4. Broad-spectrum antiviral drugs such as chloroquine and ribavirin are shown effective against CHIKV replication in cultured cells and animal models. However, these drugs showed limited success in human clinical trials26,27,28,29. Pharmaco-safety of promising host specific compounds against CHIKV replication have yet to be investigated in vivo26,30, while live attenuated CHIKV vaccines are still in need of further clinical evaluation31,32,33. In this regard, it is of pressing concern for the development of a highly safe and potent therapeutic alternative for CHIKV disease.

CHIKV is an enveloped RNA virus containing a single-strand, positive-sense, 11.8 Kb RNA genome34. As with other alphavirues, the viral genomic RNA consists of two open reading frames (ORF). Following receptor-mediated endocytosis of CHIKV into its host cell, the virus mediate mediates synthesis of positive and negative-strand viral RNA and proteins. At the first ORF, CHIKV genomic RNA is translated into a non-structural polyprotein (nsP1–4) which consequently is post-translationally cleaved into non-structural proteins (nsP1, 2, 3 and 4). These non-structural proteins formed a replication complex required to drive the synthesis of a 26S subgenomic RNA. Translation initiation at the second ORF of CHIKV subgenomic RNA leads to formation of structural proteins, namely, Capsid, E1 and E234. Together with newly-replicated viral RNA and structural proteins, mature virions are formed and released. Given that virus replication is dependent on viral RNA synthesis, antisense oligomers targeting specific viral RNA sequence can be effective strategies to inhibit virus replication.

As a recent advance in antisense technology, the Phosphorodiamidate Morpholino oligomer (PMO), is highly efficient in blocking cellular gene expression. PMO is a single-stranded oligonucleotide made up of novel six-membered morpholine ring with purine and pyrimidine bases linked by unique phosphorodiamidate bond35,36. By high affinity-binding to complementary sequence in the translational region, PMO forms a steric block to ribosome assembly on the target RNA molecule and prevents RNA translation. PMO-mediated inhibition has been widely demonstrated in cell cultures infected with Dengue virus (DENV)37, West Nile virus (WNV)38, multiple strains of Influenza A virus39, Severe Acute Respiratory Syndrome Coronavirus40, Enterovirus 7141, as well as Alphavirus members such as Sindbis virus (SINV) and Venezuelan Equine Encephalitis virus (VEEV)42. In murine models, PMO was also strongly protective against WNV,43, Influenza A virus44, VEEV42, Japanese Encephalitis virus45,46, Respiratory Syncytial virus47 and Ebola virus infection48. Notably, PMO therapeutic has also successfully reached human clinical trials49,50.

In prospect of its promising antiviral efficacy, anti-CHIKV PMO, namely, CPMO1 and CPMO2, were designed to target a 25-mer sequence in the AUG region of the first and second ORF of CHIKV RNA genome. This will prevent translation initiation of the non-structural and structural proteins, respectively and thus inhibit CHIKV replication. Antiviral efficacy was evaluated by viral plaque assays, Western blot detection of viral protein expression, ultrastructural analysis, virus titration and immunohistological analysis of mice organs as well as mice survival study. Our findings collectively showed that CPMO1 was highly potent against CHIKV replication on both cell-based and the murine model for CHIKV.

Results

Effective Construct and Efficient Uptake of CPMOs with Absence of Cellular Cytotoxicity

In this study, CPMO1 and CPMO2 were designed to bind to the AUG region of ORF1 and ORF2 of CHIKV genomic RNA, respectively (Fig. 1). Both CPMO target sequences were structurally accessible with no complex secondary folding (Fig. 1a) and the sequences were also predicted to be highly conserved among different geographical strains of CHIKV (Fig. 1b; Table 1). Quantification of cell viability was done following incubation with a combination of PMOs and Endo-Porter (EP) delivery reagent, EP or individual CPMO on confluent HeLa cells for at least 24 h. More than 96% of the cells remained viable across PMO + EP concentration range at 24 h post-treatment (supplementary Figure S1a). Consistently, cell viablity was also close to 100% for EP control treatment (supplementary Figure S1b) and at least 94% was observed for CPMO treatment at 24 h, 48 h and 72 h post-treatment (Fig. 2a). Taken together, this suggests that EP delivery, the designed CPMO and their combination had no apparent toxcity on HeLa cells. In order to visualize CPMO intracellular distribution following cellular uptake, cells were incubated with fluoresceinated-CPMOs (10 μM) and EP (6 μM) for 24 h, stained with DAPI dye and observed under fluorescence microscopy. None of the mock-treated and EP-treated cells have any visible fluorescence signal under FITC channel (Fig. 2b,c). In contrast, 99% of CPMO1 and 97% of CPMO2-treated cells showed diffused fluorescence occuring individually or freely in clusters around the cytosol, thereby suggesting an efficient EP-mediated cellular uptake of CPMO. Next, CPMO-treated cells were subjected to CHIKV infection (M.O.I. 0.1) in an attempt to investigate whether intracellular stability of CPMO is affected during post-CHIKV infection. Fluorescence signals of CPMO1 and CPMO2 were similarly detected in at least 84% of CHIKV-infected cells at day 1, 2 and 3 p.i (Fig. 2d). Similar to post-treatment, CPMO signals were only located in the cell cytosol and most of them colocalized with CHIKV E2 protein stain. This suggests that CPMO could be distributed to the Endoplasmic reticulum where it inhibits CHIKV protein expression. Though CPMO signals were comparatively reduced at day 3 p.i., they have remained intracellularly stable among CHIKV-infected cells as observed by the evident fluorescence signal.

Table 1 Antisense CPMOs and scrambled CPMO sequences (25-mer).
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Figure 1
figure 1

Design of CPMO and scrambled CPMO constructs against CHIKV genomic RNA.

(a) CPMO1 binds to a sequence (25-mer) upstream of the first Open Reading Frame (ORF), which encodes the non-structural proteins (nsP1–4) while CPMO2 targets a sequence (25-mer) in the second ORF which is important for the expression of Capsid (C), E1 and E2 structural proteins. These structural proteins are required for the formation of the mature CHIKV virion. Black arrows indicate translation initiation. Secondary structures of CPMO target region in the CHIKV genome are predicted by mfold program (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form). Both CPMOs are conjugated to a carboxyflurocesin tag (oval) at the 3’ end. AUG translational start site is outlined in box. CPMO1 target site appears to be more accessible compared to CPMO2. (b) NCBI blast nucleotide alignment with representative geographical strains of CHIKV showed high similarity in CPMO target sequences except for a few mismatch base. CPMO; Anti-CHIKV Phosophorodiamidate Morpholino Oligomers. *denotes matched nucleotide base.

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Figure 2
figure 2

Assessing Cytotoxicity and Efficiency of PMO uptake into HeLa cells.

(a) CPMO1 and CPMO2 of 10 μM were tested for their cytotoxic effect after day 1, 2 and 3 treatment on confluent HeLa CCL2 cells. At each incubation time point, cell viability was quantitated by AlamarBlue assay and calculated using mock-treated cells as baseline control of 100% viability. (b) Cellular uptake of CPMO1 or CPMO2 (10 μM) was quantitated under fluorescence microscopy. HeLa cells were treated with 0 μM (mock-treated) or 6 μM Endo-Porter delivery reagent as vehicle control. Number of cells with intracellular PMO fluorescence signal, n, was indicated on each bar. (c) CPMO1 or CPMO2-treated cells were nuclei-stained with dapi dye and observed under DAPI and FITC channels at day 1 post-treatment. (d,e) Stability of PMO in CHIKV-infected cells was assessed following PMO treatment. Cells pre-treated with CPMO1 or CPMO2 (10 μM) were infected with CHIKV (M.O.I. 0.1), fixed at day 1, 2 and 3 p.i and stained with rabbit anti-CHIKV envelope E2 serum followed by anti-rabbit 594 IgG to assess the stability of PMO in presence of CHIKV infection. (d) Cells with positive PMO-FITC signal were expressed as a percentage over the total number of CHIKV-infected cells with E2-594 signal. n number of cells with 594 signal was quantitated and indicated on the top of each bar. Error bars denote the average mean ± s.e.m. expressed from at least two independent set of experiments. (e) Representative images under DAPI + FITC, DAPI + TRITC and tri-colours overlay channels were shown under ×100 magnification with scale bar of 10 μm. p.i., post infection; Inf, CHIKV-infected cells; EP, Endo-Porter.

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Significant Reduction of CHIKV titre and viral Protein level in CPMO1-treated Cells

After pretreatment of HeLa cells with CPMO1 targeting the synthesis of CHIKV non-structural polyprotein or CPMO2 that is targeting the synthesis of structural polyprotein, CHIKV infection was carried out at M.O.I. 0.1 and virus titre in the culture supernatant was quantitated by viral plaque assays. Post-infection time-points, day 1, 2 and 3 were chosen based on CHIKV growth kinetic in HeLa cells, where the highest CHIKV titre was produced at day 3 p.i (supplementary Figure S2). Mock, EP and sCPMOs treatments produced similarly high levels of CHIKV titre from Day 1–3 p.i (Fig. 3a). Relative to mock-treated control, CPMO1 (10 μM) significantly reduced CHIKV titre by 2 log10 PFU/ml at day 1 p.i and 3 log10 PFU/ml at day 2 and 3 p.i. In contrast, pre-treatment with equal concentration of CPMO2 achieved only a 1 log10 PFU/ml reduction in CHIKV titre at day 2 and 3 p.i. In parallel with quantification of virus titre, the total cell lysates of CPMO or sCPMO treated-cells were harvested at all three days p.i for Western blot detection of CHIKV E2 and nsP3 protein expression level. In mock, EP or sCPMO-treated cells, increasing level of expression of CHIKV E2 (Fig. 3b,c) and nsP3 protein (Fig. 3d,e) were observed from day 1 to day 3 p.i, correlating to the increasing virus titres (Fig. 3a). On the other hand, viral E2 and nsP3 protein level was almost completely suppressed at day 1–3 p.i in cells pre-treated with 10 μM of CPMO1. Similarly, CPMO2-treated cells showed strong reduction in both E2 and nsP3 protein expression relative to CHIKV-infected control (Fig. 3b–e). However, CPMO2 treatment seemed less effective in silencing the production of E2 protein and infectious CHIKV titre relative to CPMO1. Taken together, these findings highlight the strong antiviral efficacy of CPMO1 against CHIKV replication.

Figure 3
figure 3

Antiviral efficacy of CPMO1 and CPMO2 in HeLa cells.

(a) Quantification of CHIKV production from CPMO-treated cells by viral plaque assays. Treatment with Endo-Porter reagent (EP) and scrambled CPMOs (sCPMO1 and sCPMO2) did not interfere with CHIKV replication. CPMO1 treatment (10 μM) produced significant and sustained inhibition against CHIKV replication (M.O.I 0.1) from day 1 (p < 0.01), 2 and 3 p.i (p < 0.001), relative to mock-treated and infected cells. CPMO2 (10 μM) exhibited a lower inhibition relative to the non-treated control. (b) Western blot detection of CHIKV E2 protein (50 kDa) expression in total cell lysate of mock-treated, Endo-Porter (EP-inf), scrambled CPMO, or CPMO treated and infected cells across day 1, 2 and 3 p.i. (D1, D2, D3). (c) Quantification of relative band density of CHIKV E2 protein. (d) Western blot analysis and (e) quantification of CHIKV nsP3 protein (78 kDa) level in total cell lysate. β-actin (42 kDa) serves as loading control. Gel image presented was cropped from several original images and all gels were electrophoresed under the same conditions. Treatment of HeLa cells with CPMO1 10 μM did not produce specific inhibition against (f) Sindbis and (g) Dengue virus replication (M.O.I. 0.1 infection) when compared to the non-treated and scrambled CPMO1 controls. Error bars are indicative of mean ± s.e.m. expressed from three independent set of experiments. Statistical analysis is performed using one-way ANOVA across all groups followed by unpaired one-tailed t-test (*p < 0.05, **p < 0.001).

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In the interest of whether CPMO1 is specific in its inhibitory effect to CHIKV per se, we carried out similar treatment assay of CPMO1 on HeLa cells and subjected them to Sindbis virus (SINV) or Dengue virus (DENV) infection at M.O.I 0.1 for day 1–3 p.i. SINV was used in this study as it belongs to the Togaviridae family similar to CHIKV while DENV is a representative member of the flaviviruses. Quantification of virus titre by viral plaque assays demonstrated no significant difference in SINV or DENV titre for CPMO1-treatment relative to the mock-treated cells (Fig. 3f,g). In support of this data, homology alignment of CPMO1 target sequence in CHIKV RNA (GenBank; FJ445502) to SINV (GenBank; NC_001547) and DENV genome (GenBank; M29095.1) revealed no significant similarity (data not shown). Therefore, we deduce that CPMO1 is highly specific against CHIKV with no cross-reactivity to the replication of other alphaviruses or flaviviruses.

Ultrastructural Analysis of CPMO1-treated Cells Reveals Absence of CHIKV Replication

To further validate the efficacy of CPMO1, transmission electron microscopy (TEM) was carried out on day 3 post-infected cells for ultrastructural observation of CHIKV replication. Mock-infected HeLa cells maintained healthy cellular morphology with intact organelles such as a well-defined nucleus, mitochondria and endoplasmic reticulum (Fig. 4a). The cell surface membrane was smooth and there was no sign of CHIKV virions. In CHIKV-infected (Fig. 4b–d), EP- (Fig. 4e) and sCPMO1-treated cells (Fig. 4f), extensive virus replication has occurred as indicated by the formation of numerous CHIKV replication complexes, namely, the cytopathic vacuoles type II (CPV-II, arrowheads) in the cytosol (Fig. 4d). During the late phase of CHIKV replication, infected cells showed assembly and budding of mature CHIKV virions from cell surface and intercellular junctions (Fig. 4c,e,f). In contrast, CPMO1-treated cells showed no sign of CHIKV replication with the absence of CPVs at say 3 p.i. (Fig. 4g,h). The mitochondria and endoplasmic reticulum remained intact and there was no virus budding from cell surface and in between cell junctions, similar to the mock-treated cells (Fig. 4a). These ultrastructural analysis further strengthen our finding that treatment of HeLa cells with CPMO1 has effectively prevented CHIKV infection within the cells.

Figure 4
figure 4

Transmission electron microscopy is performed at day 3 p.i. (M.O.I. 0.1) of (a) mock-treated and mock-infected, (b–d) mock-treated and CHIKV-infected, (e) treated with 6 μM Endo-Porter, (f) treated with 10 μM sCPMO1 and (g,h) 10 μM CPMO1. (d) CHIKV-induced cytopathic vacuoles, CPV II, are indicated by arrowheads and virus budding from cell surface membrane is indicated by *. Representative images are shown here with corresponding scale bar in μm. CPV II, cytopathic vacuole II; ER, endoplasmic reticulum; G, Golgi apparatus; L, lysosome; M, mitochondria; N, nucleus; NM, nuclear membrane; PM, plasma membrane.

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Strong Protection of CPMO1v against CHIKV Infection in Mice

Having shown that CPMO1 could strongly suppress cellular CHIKV replication, we further assessed the antiviral efficacy of CPMO1v in suitable murine model (6-day old BALB/c mice) that was previously established for CHIKV infection in our research laboratory. In this 6-day old BALB/c model, mice succumbed to CHIKV infection with a range of 10–65% lethality in day 5–9 p.i. relative to mock-infected wildtype control group. There could be variability in survival due to the development of innate immunity that help mice to overcome lethality. Therefore, it is more notable for the use of this mouse model to analyze the acute phase of CHIKV infection, where the latter was well-characterized by high viremia, virus burden and pathogenesis in CHIKV-targeted tissues including the hind limb muscles, liver, spleen and brain (data not shown). In this study, Vivo-PMOs, namely, CPMO1v and scrambled CPMO1v (sCPMO1v), were synthesized where their fluorescence dye was replaced with an octaguanidium dendrimer to mediate effective in vivo delivery to mice tissues. Following CPMO1v or sCPMO1v administration via intraperitoneal (i.p) injection at 5, 10 and 15 μg/g to our murine model, serum Lactate dehydragenase (LDH) level in the mice remained low and insignificant compared to the LDH positive control. Activity of LDH is commonly used as a stable biomarker in cytotoxicity assay51,52. Based on this finding, we inferred an absence of dose-dependent toxicity from vivo-PMO treatment (Fig. 5a). Next, the antiviral efficacy of CPMO1v at 5 μg/g or 15 μg/g was investigated where the compound was administered to the mice for two consecutive days, followed by CHIKV infection and subsequent treatment with CPMO1v for two more consecutive dose at 18 h and 42 h p.i. A dose-inhibition effect was observed where 5 μg/g of CPMO1v protected 75% of the mice from CHIKV-induced morbidity and 15 μg/g has further enhanced the survival of all mice for two weeks p.i. (Fig. 5b). In contrast, PBS- and sCPMO1v control groups showed 65% survival at Day 3 p.i. and 50% survival at Day 4 p.i, respectively. On average daily basis, 15 μg/g CPMO1v-treatment also conferred the mice healthy weight gain similar to mock-infected mice while 5 μg/g CPMO1v-treated mice showed retarded growth similar to PBS- and sCPMO1v-treatment groups (Fig. 5c). As this mouse model did not provide evaluation of absolute lethality, quantification of CHIKV titre in mice serum and several organs was further evaluated at day 2 p.i. to analyze the protective efficacy of CPMO1v during the acute phase of CHIKV infection. Indeed, strong suppression of CHIKV production in various tissues of CPMO1v-treated mice relative to PBS- and sCPMO1v-treated groups (Fig. 5d–h). In line with high potency of protection seen in survival study, CPMO1v given at 15 μg/g had the most notable reduction of viremia (2.83 log10; Fig. 5d), CHIKV load in the spleen (3.85 log10; Fig. 5e), liver (2.09 log10; Fig. 5f), brain (2.45 log10; Fig. 5g) and limbs (2.91 log10; Fig. 5h). Taken together, these data showed that the administration of 15 μg/g CPMO1v strongly protects against CHIKV disease in our murine model during the acute phase.

Figure 5
figure 5

Evaluation of in vivo toxicity and anti-CHIKV efficacy of CPMO1v in neonate murine model.

(a) BALB/c mice (6-day old, n = 6 per group) were intraperitoneally (i.p) injected with sterile PBS, anti-CHIKV vivo-PMO (CPMO1v) or scrambled vivo-PMO (sCPMO1v) at 5 μg/g, 10 μg/g or 15 μg/g consecutively for seven days at every 24 h interval. Mice were sacrificed at 6 h after the last treatment and whole blood was harvested for quantification of LDH activity level. (b) In survival study, two dose of sterile PBS, CPMO1v or sCPMO1v at 5 μg/g or 15 μg/g were given via i.p to neonate mice (n = 6 per group) at 24 h interval, followed by CHIKV infection at 4 × 105 PFU. Treatment was given at two more equivalent doses at 18 h p.i. and 42 h p.i. and mice were monitored daily for sign or symptom of CHIKV morbidity as well as (c) their daily weight gain over two weeks. At the end of two weeks p.i., the number of surviving mice was recorded and presented on Kaplan-Meier chart. A dose-dependent protection is observed in CPMO1v-treated mice relative to the PBS- and sCPMO1v- control groups. (d–h) Mice (n = 5 per group) were subjected to the same treatment regimen above. At 48 h p.i., (d) whole blood, (e) spleen, (f) liver, (g) brain and (h) hind limbs were harvested and homogenized for viral plaque assays. Significant reduction in viremia and CHIKV load were observed. Statistical analysis is done using one-way ANOVA across all groups followed by unpaired one-tailed t-test. (*p < 0.05, **p < 0.005, ***p < 0.0005; Graphpad prism 6).

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Absence of CHIKV Pathology in Tissues of Mice pre-treated with CPMO1v

To further validate the antiviral activity of CPMO1v against CHIKV disease development in mice, histological analysis was performed on PBS-, sCPMO1v- and CPMO1v-treated mice tissues at day 7 p.i. PBS-treated and CHIKV infected mice tissues showed evident morphological abnormities. In line with studies on CHIKV muscle tropism50,53,Viral Plaque Assay

BHK cells were seeded at 90% confluency on 24-well plates for quantification of the infectious virus titre in the harvested culture supernatants, mice serum or homogenized organs. At the end of infection, cells were maintained in an overlay media (1% Carboxymethyl cellulose and RPMI-1640 media supplemented with 2% FCS) and plates were incubated at 37 °C in 5% CO2. At three days p.i. of CHIKV infection, cells were stained with crystal violet dye. For SINV or DENV titration, cells were stained with crystal violet dye at two days p.i. or seven days p.i., respectively. Virus titre was then quantitated by the number of plaques formed in the stained monolayer.

Western blot Analysis

Total cell lysates harvested at day 1, 2 and 3 p.i. were quantitated by BCA assay (Thermoscientific) and 25 μg of each lysate was subjected to SDS-PAGE. CHIKV E2 protein was probed using rabbit anti-CHIKV E2 13893 B3 polyclonal antibody (in-house produced) at 1:3000 dilution and goat anti-rabbit secondary antibody conjugated to HRP (Thermoscientific) at 1:6000 dilution. On the other hand, CHIKV nsP3 protein was probed using rabbit anti-nsP3 antibody (in-house produced) at 1:100 dilution followed by secondary anti-rabbit HRP antibody at 1:10,000 dilution. Beta-actin detection was carried out using mouse monoclonal anti-actin at 1:6000 or 1:10,000 (Millipore, USA). CHIKV E2 blot was probed with antibodies in TBST with 5% skim milk while CHIKV nsP3 blot was probed with PBST with 5% BSA. Blots were washed using TBS or PBS buffer with 0.1% Tween-20. Following incubation with antibodies, protein bands were developed by Enhanced Chemiluminescence (ECL) method using Supersignal® West Pico Chemiluminescent Substrate (Thermoscientific). Quantification of relative band density was performed using Image J67, followed by one-way ANOVA analysis with reference to CHIKV-infected control bands.

Transmission Electron Microscopy

HeLa cells were seeded at 90% confluency on 6-well plates and treated with CPMO1 or sCPMO1 at 10 μM, Endo-Porter or mock-treated, respectively. At 24 h post-treatment, cells were washed with PBS and infected with CHIKV at M.O.I. 0.1. At day 3 p.i., cells were fixed and processed for TEM as described in our laboratory-established protocol56.

Ethics statement

All mice experiments were approved and performed in accordance to the guidelines provided by Institutional Animal Care and Use Committee of National University of Singapore (Protocol No. 023/12).

Mice and CPMO1v Treatment

Neonate BALB/c mice were used in this study. The animals were housed in a pathogen-free BSL2-facility in Vivarium, CeLS building in National University of Singapore. Six-day-old mice (n = 5 or 6 per group) were treated with 5 μg/g or 15 μg/g of CPMO1v, sCPMO1v diluted in sterile PBS or mock-treated with sterile PBS only by intraperitoneal (i.p) injection for two consecutive dose at every 24 h interval. At 6 h following the second dose, mice were i.p injected with 105 PFU of CHIKV (strain LK (EH) CH6708, GenBank; FJ513654). At 18 h and 42 h p.i, equivalent dose of vivo-PMO or PBS was given. At the end-point of 48 h p.i., mice were sacrificed under anesthesia and whole blood, brain, spleen, liver and limbs were harvested for quantification of CHIKV titre. Tissues were homogenized using Precellys CK beads and centrifuged at 14,000 g × 10 min to obtain clarified supernatant for viral plaque assay. For survival analysis, mice (n = 6) were pretreated as described above. Following CHIKV infection, mice were monitored daily and scored for sign and symptoms of CHIKV disease. At the end of two weeks, the number of surviving mice was noted and all mice were humanely euthanized and sacrificed.

Toxicity Evaluation of Vivo-PMO in Mice

BALB/c mice (6-day old, n = 6) were i.p injected with sterile PBS, CPMO1v or sCPMO1v at 5, 10 or 15 μg/g at every 24 h for 7 days. At 6 h after the last treatment, mice were sacrificed and whole blood was collected for evaluation of LDH level following the manufacturer’s protocol in the LDH Cytotoxicity Assay kit (Biovision, USA).

Histological and Immunohistochemical Examination of Infected Mice tissues

BALB/c mice (6-day old, n = 3) were pretreated with sterile PBS, CPMO1v or sCPMO1v and subjected to CHIKV infection as described above. At day 7 p.i., mice were sacrificed and their limbs and liver were harvested and formalin fixed, dehydrated and paraffin embedded. Sectioning of tissue samples was done at 4 μm and samples were routinely stained with haematoxylin and eosin (H&E). For IHC, tissue samples were labeled with primary rabbit E2 antibody diluted to 1:100, followed by a secondary goat anti-rabbit HRP conjugate (Thermoscientific). The assay was subsequently performed using automated Bond-Max System (Leica Biosystems, Germany) based on a customized protocol with several steps of dehydration and differentiation. Final slides were then mounted with PermountTM mounting medium (Fisher Chemical, UK) and viewed under Olympus microscope. Images were captured at 400× magnification to visualize CHIKV antigen.

Additional Information

How to cite this article: Lam, S. et al. Antiviral Phosphorodiamidate Morpholino Oligomers are Protective against Chikungunya Virus Infection on Cell-based and Murine Models. Sci. Rep. 5, 12727; doi: 10.1038/srep12727 (2015).