Vero cells

ZIKV strains ATCC® VR-84 (GenBank: NC012532.1) and PLCal_ZV (GenBank: KF993678) provided by Public Health Agency of Canada [16 (link)] were used to prepare viral stocks. Viral stocks were prepared by inoculating each isolate into Vero cells (African Green Monkey Kidney) in M199 growth medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum, 1% L-glutamine solution and 100 mM Penicillin/Streptomycin (Sigma-Aldrich, St. Louis, MO, USA). After five days of propagation, viral titres were quantified by using the plaque assay performed on BHK-21 cells as described elsewhere [17 ].

HFFF, HeLa (both obtained from European Collection of Authenticated Cell Cultures -ECACC) and HaCaT (obtained from Prof J. Breuer) cells were cultured in DMEM supplemented with 10% foetal bovine serum (Invitrogen). Vero cells (obtained from ECACC) were grown in DMEM supplemented with 10% newborn calf serum (Invitrogen). Viruses were routinely propagated in Vero cells, with titrations carried out in DMEM supplemented with 2% human serum. HSV1 strain 17 (s17) was used routinely. The s17 derived VP22 deletion mutant (Δ22) and the vhs knockout virus (Δvhs) have been described before [28 (link), 38 (link)]. HSV1 strain Kos with a deletion of the C-terminal 36 residues of VP16 (RP3v) and its revertant (RP3vR) have been described elsewhere [64 (link)] and were kindly provided by Steve Triezenberg (Van Andel Institute). The s17 derived ΔUL13 and ΔICP34.5 knockout viruses have been described previously [84 (link), 85 (link)]. The construction of viruses expressing GFP-tagged VP22 (GFP1-301), and GFP-tagged VP22 subdomains (GFP192-301, GFP108-301, GFP1-212, GFP1-165 and GFPΔ213–226) has also been described before [61 (link), 62 (link), 86 (link)].

The human embryonic kidney epithelial cell line 293T (Invitrogen, Grand Island, NY, USA) was kindly provided by Dr. Sansanee Noisakran, National Center for Genetic Engineering and Biotechnology, Thailand. 293T cells were cultured at 37 °C, 5 % CO₂ in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Invitrogen, USA) supplemented with 10 % heat-inactivated fetal bovine serum (FBS; Gibco, Invitrogen, USA), 37 μg ml⁻¹ of penicillin, 60 μg ml⁻¹ of streptomycin, 25 mM HEPES, 2 mM L-glutamine, and 0.37 % NaH₂CO₃. African green monkey kidney Vero cell (American Type Culture Collection; ATCC, Manassas, VA) and Aedes albopictus C6/36 cell [22 (link)], were kindly provided by Ms. Tanapan Pruksamas, National Center for Genetic Engineering and Biotechnology, Thailand. Vero cells were cultured at 37 °C, 5 % CO₂ in Minimum Essential Medium (MEM; Gibco, Invitrogen, USA) supplemented with 10 % heat-inactivated FBS, and 37 μg ml⁻¹ of penicillin, 60 μg ml⁻¹ of streptomycin. C6/36 cells were cultured at 28 °C in Leibovitz’s L-15 medium (Gibco, Invitrogen, USA) supplemented with 10 % heat-inactivated FBS and 10 % tryptose-phosphate.

The HSV1 F strain was used in this study. The strain was grown in Vero cells (ATCC, Manassas, VA, USA). The virus was harvested and then frozen at −80 °C when typical cytopathic effects (CPE) developed and was produced at a concentration of 10^6.0–10^7.050% cell culture infectious doses (CCID₅₀)/mL. The Vero cells were maintained in minimum essential medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and were used for viral proliferation and titration.

Vero cells (ATCC number CCL-81) were maintained at 37°C/5 % CO₂ in minimal essential medium (Life Technologies) with 7 % heat-inactivated foetal bovine serum (FBS; Life Technologies), 1 % penicillin/streptomycin (PS; 5000 U.ml⁻¹ and 5000 μg.ml⁻¹; Life Technologies) and 1% Glutamine (Gln; 200 mmol.l⁻¹; Life Technologies). HEK-293 cells (ATCC number CCL-1573) were maintained at 37°C/ 5 % CO₂ in the same medium supplemented with 1 % non-essential amino acids (Life Technologies).

CHIKV strain CP10 (ECSA genotype) was propagated in Vero cells maintained in Minimum Essential Medium (Life Technologies, Inc., USA) supplemented with 10% (v/v) heat-inactivated Foetal Bovine Serum (Life Technologies). The virus was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) [9 ], using a laboratory-generated strain (IND/DEL/2010–01) cloned in pGEM-T vector and serially diluted from 100 ng to 1 pg as reference to determine viral copy number of CHIKV viral RNA isolated from patients sera using the formula.
Number of VNA copies = (amount of VNA in nanograms × 6.022 × 10²³) / (length of VNA amplicon (in basepairs) × 1 × 10⁹ × 330).
The CHIKV strain CP10 was also titrated using standard plaque assay [10 (link)].