Figure 1.
Schematic overview of the synthetic infectious clone pCHIKV-LS3-GFP.
The ‘wild type’ full-length clone pCHIKV-LS3, which lacks the eGFP reporter gene, was generated by deleting the SacI fragment; the variant pCHIKV-LS3-MCS, containing a multiple cloning site (MCS) preceded by subgenomic promoter 1, was generated by removing the AsiSI-PacI fragment.
Table 1.
CHIKV E1-226V strains that were aligned to produce the CHIKV LS3 consensus sequence.
Table 2.
Comparison of the amino acid sequences of CHIKV LS3 and various natural isolates.
Figure 2.
Growth kinetics of CHIKV LS3, LS3-GFP and ITA07-RA1 on various cell lines.
Growth kinetics of CHIKV on Vero E6 (A), 293/ACE2 (B) and mosquito C6/36 cells (C). Cells were infected at an MOI of 5 and the viral progeny titers in the supernatant were determined at various time points post infection. (D) Plaque morphology of ITA07, LS3 and LS3-GFP on Vero E6 cells. (E) Induction of CPE by ITA07 (upper panel) and LS3 (lower panel) on Vero E6 cells at different time points post infection.
Figure 3.
Transcriptional and translational shut-off induced by CHIKV.
293/ACE2 cells were infected with CHIKV LS3 or ITA07-RA1 at an MOI of 5, and at the indicated time points post infection metabolic labeling with 3H-uridine (A, B) or 35S-Cys and 35S-Met (C) was performed to analyze total RNA and protein synthesis, respectively. (A) Incorporation of 3H-uridine into viral and cellular RNA as measured by liquid scintillation counting of total RNA samples taken at various time points post infection. (B) 3H-uridine incorporation into viral and cellular RNA during CHIKV LS3 infection as detected by denaturing gel electrophoresis and fluorography. In control samples (ActD) 5 ug/ml Actinomycin D was added 30 min. prior to metabolic labeling to inhibit cellular transcription. (C) Synthesis of 35S-labeled viral and cellular proteins during CHIKV LS3 infection. The control lane labeled CHX contains proteins from cells treated with the translation inhibitor cycloheximide prior to metabolic labeling. CHIKV-specific proteins are indicated with a *.
Figure 4.
Accumulation of negative- and positive-strand CHIKV RNA in infected cells.
(A) 293/ACE2 cells were infected with CHIKV LS3, LS3-GFP or ITA07-RA1 at an MOI of 5, total RNA was isolated at different time points post infection and strand-specific detection was performed with radioactively labeled oligonucleotides complementary to the 3′ end of either negative- (top panel) or positive-strand (middle panel) CHIKV RNA. Cellular 18S ribosomal RNA was probed as a loading control (lower panel). The positions of genomic RNA, the 26S sgRNA and the second eGFP-encoding sgRNA are indicated to the right of the middle panel. (B) Plot representing the kinetics of CHIKV negative-strand RNA accumulation, based on quantification of data from panel A. After correction for variations in sample loading based on the 18S rRNA signal, the relative abundance of the RNAs was determined by normalizing to the highest value observed (CHIKV LS3-GFP, 8 h p.i.). (C) Kinetics of CHIKV positive-strand RNA accumulation. The relative abundance of RNA was calculated as before, except that data were normalized to the value measured for LS3 sgRNA at 12 h p.i (100%). Genomic RNA levels are indicated with black lines, sgRNA levels with gray lines. The total level of both sgRNAs expressed by LS3-GFP is indicated with the gray dotted line.
Figure 5.
Western blot analysis of CHIKV nsP1 and E2 expression at different time points post infection.
293/ACE2 cells were infected with CHIKV ITA07, LS3 or LS3-GFP at an MOI of 5. At the indicated time points cells were lysed, proteins were separated by SDS-PAGE and viral proteins were detected by Western blotting. The anti-E2 antiserum also recognized the E3E2 (p62) precursor of E2. Actin and the transferrin receptor were used as loading controls.
Figure 6.
Immunofluorescence analysis of dsRNA and E2 expression in time.
Vero E6 cells grown on coverslips were infected with CHIKV ITA07, LS3 or LS3-GFP at an MOI of 5. At the indicated time points the coverslips were fixed and stained with antibodies specific for dsRNA (A) or E2 (B). (C) eGFP fluorescence in CHIKV LS3-GFP infected cells (green). Nuclei (blue) were visualized by Hoechst staining.
Figure 7.
Neutralization of CHIKV ITA07 and LS3 by mouse monoclonal antibodies raised against ITA07-RA1.
100 PFU of the CHIKV strains were incubated with serially diluted antibodies in 96-well clusters containing confluent monolayers of VeroE6 cells. After 2 days the wells were fixed with formaldehyde and stained with crystal violet. Either the neutralizing antibody 1H7 or non-neutralizing control antibody 3H9 were used.
Figure 8.
Replication of synthetic CHIKV strains in vivo.
(A) Survival of mice after intraperitoneal injection with 100 TCID50 of CHIKV LS3, LS3-GFP or S27 (5 mice per group). (B) CHIKV titers (in TCID50 equivalents per mg of tissue) in the brains of the mice infected with the 3 CHIKV strains.
Figure 9.
Effect of antiviral compounds on the replication of various CHIKV strains.
Dose dependent reduction of CHIKV-induced CPE by (A) 3-deaza-adenosine, (B) 6-aza-uridine, (C) chloroquine and (D) mycophenolic acid in Vero E6 cells infected with CHIKV strains ITA07-RA1, LS3 and LS3-GFP (MOI 0.005). (E) Antiviral effect of ribavirin on CHIKV replication in BHK-21 (black lines) and Vero E6 cells (gray lines). Cell viability was normalized to untreated uninfected cells (100%). The 50% cytotoxic concentration (CC50) of the compounds is indicated in the top left of each panel. (F) Dose-response curves showing the effect of five antiviral compounds on the eGFP expression in Vero E6 cells infected with CHIKV LS3-GFP (MOI 0.05) at 42 h p.i. Values were normalized to eGFP fluorescence in untreated infected cells (100%).