Ae. aegypti (PAEA strain) mosquitoes were maintained at 28±1°C under 80% relative humidity with a light/dark cycle of 16 h/8 h. Larvae were reared in pans containing a tablet of yeast in 1 L of tap water. Adults were provided with 10% sucrose solution ad libitum.
The CHIKV 06.21 strain isolated in November 2005 from a new-born male in La Reunion who presented meningo-encephalitis symptoms was used for all experiments . This strain contained an A→V change at position 226 in the E1 glycoprotein (E1-226V). Stock virus was produced following passages on Aedes albopictus C6/36 cells then harvested and stored at −80°C in aliquots as described . The titre of the frozen stock virus was estimated to be 109 plaque-forming units (PFU)/mL.
Oral infections of mosquitoes and dissections
Seven day-old female mosquitoes were deprived of sucrose for 24 h prior to the infectious blood meal. They were then allowed to feed for 15 min through chicken skin membranes covering glass feeders maintained at 37°C. The infectious blood meal was comprised of two thirds washed rabbit erythrocytes, one third viral suspension, with ATP as a phagostimulant at a final concentration of 5x10-3 M. The infectious blood was at a titre of 107.5 PFU/ml CHIKV 06.21. Three independent infections were performed.
Salivary gland collection
Salivary glands were collected from female mosquitoes, infected or not infected with CHIKV, 3 and 5 days after the blood meal. They were placed into a tube containing 100 μl of 150 mM sodium chloride (NaCl) and protease inhibitors (Complete, Roche). Samples were stored at −80°C until use.
Reverse transcription and quantitative PCR (RT-q PCR)
Total RNA from mosquitoes or salivary glands was extracted using the Nucleospin® RNA II kit (Macherey-Nagel) according to the manufacturer's instructions. RNA was eluted in 40 μl of RNAse-free H20 by centrifugation at 11,000 g for 1 min.
Synthetic RNA transcript for CHIKV was generated to construct a standard curve. The targeted region in the CHIKV sequence was amplified by PCR and ligated into the pCR II TOPO vector (Invitrogen). The plasmid was then linearized using the EcoRI restriction enzyme and purified using the QIAquick PCR purification kit. RNA transcripts were prepared in vitro using the RiboMAX™ Large Scale RNA Production Systems (Promega) appropriate for either SP6 or T7 RNA polymerase. The transcript size was 1,356 bp. Residual DNA was eliminated by several DNAse treatments (Turbo DNA-free (Ambion)). After quantification by spectrophotometery, RNA transcript solutions were stored at −80°C.
One-step reverse transcription quantitative PCR (RT-qPCR) was performed using the Power Sybr Green RNA-to-Ct one step kit (Applied Biosystems). CHIKV primers were selected in the E2 structural protein coding region: sense Chik/E2/9018/+ (CACCGCCGCAACTACCG); anti-sense Chik/E2/9235/- (GATTGGTGACCGCGGCA).
RT-qPCR was performed using Applied Biosystem’s Fast Real-Time PCR 7500 System with the v.2.0.1 7500 software. The thermal cycling conditions comprised: a reverse transcription step at 48°C for 30 min, an inactivation step of RT/RNAse enzyme at 95°C for 10 min followed by 40 cycles of 95°C 15 s and 60°C 1 min, a final denaturation step where the temperature was increased from 60°C to 95°C during 20 min and a step of 15 sec at 95°C. Signals were normalized to the standard curve using serial dilutions of RNA synthetic transcripts. Using ΔCt analysis, normalized data were used to estimate the transcript copy number in infected mosquitoes.
After dissection, salivary glands were placed on a slide. PBS was removed and salivary glands were fixed in 4% paraformaldehyde for 1 h, dried and kept at 4°C until use. For indirect immunofluorescent assay (IFA) experiments, salivary glands were rehydrated in PBS for 3 x 5 min, and then incubated for 15 min with Triton X100 (0.2%). They were washed again with PBS (3 x 5 min) and incubated for 30 min with PBS that contained 1% BSA. The slides were drained and incubated overnight at 4°C with Cy3 conjugated anti-chikungunya E 3E4 protein diluted 1:500 in PBS, then washed with PBS (3 x 5 min) under shaking. The actin network was stained with Phalloidin Alexafluor 488 (Invitrogen) (diluted 1/40 in PBS). After washing, a drop of Prolong gold antifade (Invitrogen) was dropped onto each slide and a coverslide was placed on top. All preparations were examined by confocal microscopy (Zeiss LSM 510 Meta and TCS SP5 Leica Microsystems).
Preparation of salivary gland protein extracts
3 and 5 days after feeding, pools of 200 salivary glands were dissected in 100 μl PBS that contained protease inhibitors (Complete, Roche Diagnostics). These were kept at −80°C until use. Salivary glands were disrupted by ultrasound (Cup Horn, Sonics & Material) for 20 min with 2 sec pulse on and 2 sec pulse off, at the maximum amplitude. Salivary gland homogenates were then centrifuged for 30 min at 130,000 g and proteins were quantified using the BCA protein assay (Pierce). Aliquots of salivary gland proteins were then lyophilized either for immediate use or for storage at −80°C. The protein concentration of salivary gland extracts (SGE) was determined by spectrophotometry using a Nanodrop ND-1000 Spectrophotometer (Nyxor Biotech).
SGE samples were lyophilized (Christ, ALPHA 1–4 LD model; vacuum pump: Vaccubrand, MZ2-SE 220V 60 Hz model) and either resuspended in 20 μl of sterile distilled water for immediate use or stored dried at −80°C.
Two-dimensional gel electrophoresis
Three 130 μg samples were analyzed for each of infected and non-infected salivary glands. The first dimension of electrophoresis was by isoelectric focusing and the second dimension by SDS/polyacrylamide gel electrophoresis. Samples containing 130 μg (30 μl) of SGE were placed on ice for 20 min, then vortexed and centrifuged for 5 sec. 1.2% benzonase (Sigma) was added and samples incubated for 1 min at 4°C. They were then mixed with 320 μl of rehydration solution containing 1% of pH 3–10 carrier ampholytes (Invitrogen), 7M urea (Bio-Rad), 2M thiourea (Sigma), 4% CHAPS (Sigma), 100 mM DTT (Bio-Rad) and 0.002% bromophenol blue (Sigma), vortexed and centrifuged for 5 sec. In-gel rehydration was performed using strip holders, the strip (18 cm, pH 3–10 NL, GE Healthcare) being covered with 3–4 ml of oil (Cover Fluid, GE Healthcare). The first dimension separation protocol was conducted stepwise, according to the following protocol: an active rehydration for 5 h at 30 V, 500 V for 2 h, 1000 V for 30 min, 1500 V for 30 min, 2500 V for 30 min, 5000 V for 10 h and 8000 V for 2 h. 50 μA maximum were applied to the strips at 20°C (Ettan IPGphor III, GE Healthcare). The strips were then equilibrated for 15 min in buffer containing 6 M urea (Bio-Rad), 50 mM Tris–HCl pH 8.8 (Sigma), 30% glycerol (Prolabo), 2% SDS (Prolabo), 0.002% bromophenol blue with 1% DTT (Bio-Rad) for the first step and 2.5% iodoacetamide (Sigma), for the second step. For the second dimension, the strip was loaded on a 1 mm-thick 12% SDS PAGE gel with no stacking gel, and embedded into the SDS PAGE gel using 1% agarose. Gel electrophoresis was performed for 7 h 45 min at 45 mA and 200 V/gel. The slab gels were then stained with SYPRO Ruby (Invitrogen).
Analysis of gel patterns
SYPRO Ruby stained gels were scanned with a Typhoon 9400 variable-mode imager (GE Healthcare) and compared using ImageMaster 2D Platinum software (GE Healthcare). All spots identified by the software were verified by eye. All spots reported had at least a 1.8 fold intensity (normalized volume) difference between infected and non-infected samples, and were all statistically significant according to the statistical tool built into the software (Anova test, with p<0.05).
Protein preparation for mass spectrometry
After SYPRO Ruby staining, all visible gel bands were excised using the ProPic Investigator robotic work station (Genomic Solutions, Ann Arbor, MI), then plugs were collected in a 96-well plate. Proteins were reduced, alkylated, and digested overnight with porcine-modified trypsin (Promega Sequencing grade, ratio 1:100) at 37°C using the ProGest Investigator (Genomic Solutions, Ann Arbor, MI,USA). The trypsin digests were desalted with C18 tips (μZipTip, Millipore). Peptides were directly eluted, using the ProMS Investigator (Genomic Solutions, Ann Arbor, MI,USA), onto a 96-well stainless steel MALDI target plate (Applied Biosystems/MDS SCIEX) with 0.5 μl of ∝-Cyano-4-Hydroxy Cinnaminic Acid (2.5 mg/ml in 70% acetonitrile, 30% H2O, 0.1% trifluoroacetic acid).
Mass spectrometry analysis
Raw data for protein identification was obtained using the 4800 Proteomics Analyzer (Applied Biosystems/MDS SCIEX, Framingham, MA, USA) and was analyzed using the GPS Explorer 2.0 version 3.6 software (Applied Biosystems/MDS SCIEX, Framingham, MA, USA). For positive ion reflector mode spectra, 2000 laser shots were averaged. For monoisotopic (MS) calibration, autolysis trypsin peaks ([M + H]+ = 842.5100 and 2211.1046) were used as internal calibrators. MS peak masses were automatically determined within the 800–4000 Da range with a signal to noise ratio minimum set to 30. Up to 25 of the most intense ion signals were selected as precursors for MS/MS acquisition, excluding common trypsin autolysis peaks and matrix ion signals. In MS/MS positive ion mode, 4000 spectra were averaged; the collision energy was 2 kV, the collision gas was air, and the default calibration was set using Glu1-fibrinopeptide B ([M + H]+ = 1570.6696) spotted onto 13 positions on the MALDI target. Combined Peptide Mass Fingerprint (PMF) and MS/MS queries were performed using the MASCOT 2.1 search engine (Matrix Science Ltd., London, UK) embedded into GPS Explorer software on the NCBInr database (downloaded on 2010 01 19; 10348164 sequences; 3529470745 residues) with the following parameter settings: 50-ppm mass accuracy for MS, trypsin cleavage with one missed cleavage allowed, carbamidomethylation set as fixed modification, oxidation of methionines and formation of Pyro-Glu (N-term E and N-term Q) allowed as variable modifications, and MS/MS fragment tolerance set to 0.3 Da. Protein hits with MASCOT protein score ≥ 83 and peptide hits with Ionscore ≥ 53 and a GPS Explorer protein confidence index ≥ 95% were used for further manual validation.