Tryptose

CHIKVs were isolated from either human serum or CSF (
Table 1).
A. albopictus C6/36 cells were inoculated with 1 ml of serum or CSF diluted 1:10 in Leibovitz-L15 medium (Invitrogen/Gibco, Carlsbad, California, United States). The cells were grown at 28 °C in Leibovitz-L15 medium supplemented with 5% heat-inactivated foetal bovine serum (FBS) and 10% tryptose-phosphate. Cells and supernatants were harvested after the first passage (5 d) and the second passage (7 d). The virus isolates were identified as CHIKV by indirect immunofluorescence using anti-CHIKV HMAF. In the case of clinical isolates 05.115, 06.21, 06.27, and 06.49, whose genomes were sequenced, absence of yellow fever virus, dengue type-1 virus, and West Nile virus was confirmed by immunofluorescence assay using specific HMAF.
Extraction of viral RNA from the CHIKV isolates was performed using the NucleoSpin RNA II kit (Machery-Nagel, Düren, Germany) or the QIAAmp Viral Minikit (Qiagen, Courtaboeuf Cedex, France) according to manufacturer's recommended procedures. The sequence of the non-structural region of isolates 05.115, 06.21, 06.27, and 06.49 was determined from RNA extracted from supernatants harvested after the second passage. All other CHIKV isolates sequences were obtained using template RNA extracted from the first passage. Extraction of viral RNA from biological specimens was performed using the QIAAmp Viral Minikit.

Viral strains used were provided by WHO Collaborating Center for arboviruses and viral hemorrhagic fever (CRORA) at the Institut Pasteur de Dakar. ZIKV and other flaviviruses strains isolated from mosquitoes and non-human vertebrates used in this study are described in Tables 1 and 2. Viral stocks were prepared by inoculating viral strains into AP 61 monolayer continuous cell lines in Leibovitz 15 (L-15) growth medium (GibcoBRL, Grand Island, NY, USA) supplemented with 5% foetal bovine serum (FBS) (GibcoBRL, Grand Island, NY, USA), 10% tryptose phosphate, penicillin-streptomycin and fungizone (Sigma, Gmbh, Germany). After 7 days of propagation, viral infection was tested by an indirect immunofluorescence assay (IFA) using specific hyperimmune mouse ascitic fluids as previously described [23 (link)] and supernatants from infected cells were collected as stocks for virus RNA isolation. ZIKV stocks were used for sequencing and evaluation of the sensitivity of the rRT-PCR assay. Other flaviviruses were used to evaluate the specificity of the assay.

The different CHIKV isolates provided by the French National Reference Center for Arbovirus in Lyon have been entirely sequenced [19] (link). All the four strains were isolated on Ae. albopictus cells C6/36 [20] (link) from human serum: (i) strain 05.115 in June 2005 from a 24-year old female from La Réunion presenting classical CHIK symptoms, (ii) strain 06.21 in November 2005 from a new-born male from La Réunion presenting meningo-encephalitis symptoms, (iii) strain 06.111 collected in February 2006 from a patient from Mayotte presenting classical CHIK symptoms; and (iv) strain 06.117 collected during the 1999–2000 outbreak in the Democratic Republic of Congo identified as a member of Eastern/Central/Southern African group [6] (link). CHIKV 05.115 isolated at the beginning of the outbreak had E1-226A and CHIKV 06.21 isolated later in the outbreak had E1-226V [19] (link). CHIKV 06.111 contained the change A->V in E1-226 [19] (link) and CHIKV 06.117 has an Alanine at the position 226. Compared to the three other strains, CHIKV 06.117 had a change at the position 284 in the E1 glycoprotein from an Asp to a Glu. Ae. albopictus cells C6/36 were infected at a MOI of 5 and maintained at 28°C on L-15 medium supplemented with 10% fetal bovine serum (FBS), 1000 units/mL penicillin, 1 mg/mL streptomycin, and Tryptose phosphate broth 1×. Cell infection was checked by indirect immunofluorescence assays (IFA) using mouse ascitic fluid directed against CHIKV. Cells were fixed with methanol/acetone (7∶3) on glass spots at −20°C for 20 min. The fixed cells were incubated with specific ascitic fluids at a dilution of 1∶200 in PBS 1× at 37°C for 20 min. After washing with PBS 1×, cells were incubated at 37°C for 20 min with FITC-conjugated goat anti-mouse IgG antibody (Sigma) at a 1∶100 dilution in PBS 1×. Slides were examined using a fluorescence microscope. When 80% of cells were infected, the supernatant fluid was collected and viral titer estimated by serial 10-fold dilutions on Vero cells. Briefly, cells were incubated for 3 days under an overlay consisting of DMEM (Dulbecco's modified Eagle's medium), 2% FBS, antibiotics and 1% Indubiose (IBF Biotechnics) at 37°C. The lytic plaques were counted after staining with a solution of crystal violet (0.2% in 10% formaldehyde and 20% ethanol). Viral stocks which have been constituted after two passages on C6/36 cells were divided into aliquots and stored at −80°C until used. The genotypic characteristics of CHIKV inoculums have been verified by sequencing.

In accordance with the guidelines on routine culture media from the World Health Organization, all urine samples were cultured using a semiquantitative method (Vandepitte 2003 ). To isolate E. cloacae bacteria, one mL of each urine sample was plated on nutritional agar, tryptose soya agar, and two differential selective media, including Xylose Lysine Deoxycholate agar (XLD) and Salmonella Shigella agar (SS). The culture plates were then incubated at 37 °C for 18–24 h. The presence of E. cloacae was determined by observing the appearance of rough and smooth colonies on tryptose-soya agar. In this study, biochemical assays and cultural features were utilized to identify and classify the bacteria, utilizing the BD Phoenix™ automated identification system (BD—Mississauga, Canada) and Analytical profile index (API) system (bioMerieux UK Ltd, Basingstoke, UK).