Two cell lines have been used for virus cultivation and growth kinetics. Ap61 cells (Aedes pseudocutellaris) were grown in L15 (Leibovitz’s 15) medium (10% heat-inactivated fetal bovine serum [FBS], 1% penicillin-streptomycin, 0.05% amphotericin B [Fungizone] (GIBCO by life technologies; USA) and 10% tryptose phosphate (Becton, Dickinson and Company Sparks, USA) and incubated at 28°C without CO2. Vero cells (African green monkey kidney epithelial cells; Cercopithecus aethiops) (obtained from Sigma Aldrich, France) were grown using the same medium without tryptose phosphate and CO2. Furthermore, PS (Porcine Stable kidney cell line, American type Culture Collection, Manassas, USA) cells were grown in same conditions than Vero cells and have been used for plaque assay.
Tryptose phosphate
Manufactured by Merck Group
Sourced in United States
Tryptose phosphate is a nutrient medium component used in microbiology and cell culture applications. It provides a source of tryptose, which is a peptone derived from the enzymatic digestion of casein. Tryptose phosphate serves as a general growth-promoting supplement for a variety of microorganisms and cell lines.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
M199 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Bio Basic (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Vero cells, by Merck Group (1 mentions)
Vero cell, by American Type Culture Collection (1 mentions)
4 hne, by Merck Group (1 mentions)
L 15 medium, by Thermo Fisher Scientific (1 mentions)
Minimal essential medium mem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
α mem, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
S modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Leibovitz l 15 media, by Merck Group (1 mentions)
10 protocols using tryptose phosphate
1
Virus Cultivation and Growth Kinetics
2
Serum Starvation of QM7 Cells
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QM7 cells were grown in M199 medium (Life Technologies, Grand Island, NY) with 10% fetal bovine serum (Life Technologies), 10% tryptose phosphate (Sigma-Aldrich, St. Louis, MO), and 1% penicillin-streptomycin (Biobasic, Amherst, NY) at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. At 80–90% confluence, cells were synchronized overnight in serum-free medium.
3
Zika Virus Strain Propagation and Characterization
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ZIKV strains were provided by CRORA at the Institute Pasteur of Dakar. The strains were obtained from mosquitoes, humans and other mammals isolated in Burkina Faso, Central African Republic, Côte d'Ivoire and Senegal in West Africa (Table S1 ). Viral stocks were prepared by inoculating viral strains into Aedes pseudoscutellaris clone 61 monolayer in Leibovitz 15 (L-15) growth medium (GibcoBRL, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) (GibcoBRL, Grand Island, NY, USA), 10% Tryptose Phosphate and antibiotics (Sigma, Gmbh, Germany). Viral infection was confirmed after seven days of propagation by an indirect immunofluorescence assay (IFA) using specific hyper-immune mouse ascitic fluid, as described previously [27] (link). Cultures supernatants were collected for virus RNA isolation.
4
Quail Muscle Cell Stress Responses
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Quail muscle (QM7, Antin and Ordahl, 1991 (link)) cell lines were purchased from American Type Culture Collection (ATCC CRL-1962, Manassas, VA) and were grown in M199 medium (Life Technologies, Grand Island, NY) complemented with 10% FBS (Life Technologies), 10% tryptose phosphate (Sigma-Aldrich, St. Louis, MO), and 1% penicillin-streptomycin (Biobasic, Amherst, NY) at 37°C under a humidified atmosphere of 5% CO2 and 95% air. The medium was changed every 48 h and cells were subjected, during their exponential phase of growth, to the following treatments:
Acute heat stress exposure (HS): QM7 cells were exposed to HS (45°C) for 0.5, 1, 2, or 4 h. The control cells were maintained at 37°C.
Hydrogen peroxide (H2O2) treatment: Cells were treated with 10, 50, 100, or 200 μM of H2O2 (Sigma-Aldrich, St. Louis, MO) for 3 h. Untreated cells were used as control.
4-Hydroxynonenal (4-HNE) treatment: Cells were treated with 10, 20, or 30 μM of 4-HNE (Sigma-Aldrich, St. Louis, MO) for 24 h. Untreated cells were used as control.
The dose and time of the above mentioned treatments were chosen based on pilot and previous experiments (Piekarski et al., 2014 (link)).
5
Cell Line Maintenance for Arboviruses
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Ae. aegypti (AA-A20 and AE) and Ae. albopictus (C6/36 and U4.4) cells were maintained in L-15 medium (Life Technologies) with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin (PS; 5000 U/ml and 5000 µg/ml; Life technologies) and 1% tryptose phosphate (29.5 g/L; Sigma-Aldrich) at 30 °C. African green monkey cells (Vero) cells were maintained in Minimal Essential medium (MEM; Life Technologies) with 10% FBS, 1% P/S at 37 °C with 5% CO2.
6
Cell Culture Protocols for Various Cell Lines
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Baby hamster kidney cells (BHK-21; ATCC CCL-10) were maintained in alpha minimal essential medium (αMEM) (Gibco) supplemented to contain 10% fetal bovine serum (FBS) (VWR) and 10% tryptose phosphate (Sigma). Vero 81 cells (ATCC CCL-81) were maintained in αMEM supplemented to contain 5% FBS. Human osteosarcoma cells (U-2 OS; ATCC HTB-96) were maintained in McCoy’s 5A medium (Gibco) supplemented to contain 10% FBS. Culture media for BHK-21, Vero-81, and U-2 OS cells also were supplemented with 0.29 mg/ml l -glutamine (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), and 25 ng/ml amphotericin B (Sigma). WT and B3GAT3−/− human HapI cells (41 (link)) were provided by Yusuke Maeda (Osaka University) and Atsushi Tanaka (Thailand-Japan RCC-ERI). HapI cells were maintained in Iscove’s modified Dulbecco’s medium (Gibco) supplemented to contain 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. All cells were cultivated at 37°C in an atmosphere of 5% CO2.
7
BHK-21 and Vero Cell Culture
8
Aedes albopictus Cell Culture Protocol
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The
Aedes albopictus cell C636, kindly provided by Dr. Amílcar Tanuri from Universidade Federal do Rio de Janeiro, was cultivated in Leibovitz L-15 media (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), 0.26% tryptose phosphate (Sigma-Aldrich), 100 units/mL of Penicillin and 100 μg/mL of Streptomycin (Sigma-Aldrich), at 28°C
13 (link).
Aedes albopictus cell C636, kindly provided by Dr. Amílcar Tanuri from Universidade Federal do Rio de Janeiro, was cultivated in Leibovitz L-15 media (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), 0.26% tryptose phosphate (Sigma-Aldrich), 100 units/mL of Penicillin and 100 μg/mL of Streptomycin (Sigma-Aldrich), at 28°C
13 (link).
9
Optimal Cell Culture Conditions
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Vero, Huh7, 293T and BHK cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal calf serum (FCS), 1% penicillin/streptomycin (P/S; Thermo Fisher) and 1% non essential amino-acid (Thermo Fisher) in a humidified atmosphere at 37°C with 5% CO2. U4.4 and Aag2 cells were maintained in Leibovitz's L-15 medium with 10% FCS, 1% P/S, 1% non-essential amino acids (Sigma) and 1% tryptose phosphate (Sigma) at 28° C.
10
Zika Virus Strains Propagation in Cells
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ZIKV strains were provided by WHO collaborating Center at the Institute Pasteur of Dakar in Senegal. The monkey strain MR766 and the human strain HD78788 were isolated in 1947 (in Uganda) and 1991 (in Senegal) in Africa, respectively, during surveillance. Viral stocks were prepared by inoculating viral strains into Aedes pseudoscutellaris clone 61 (AP61) monolayer. Cells were grown in cell culture flasks (25 cm2) until they reached a confluence of approximately 80%. The medium was discarded, and 150 µl virus solution was added to the cells. The flasks were gently agitated every 15 min during incubation to enhance viral infection. After 1 h, 5 ml of Leibovitz 15 (L-15) growth medium (GibcoBRL, Grand Island, NY, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (GibcoBRL, Grand Island, NY, USA), 10% Tryptose Phosphate 1% glutamine, 1% penicillinstreptomycin, 0.05% amphotericin B [Fungizone] (Sigma, Gmbh, Germany) was added and the infected cells were incubated at 28°C without CO2 until a cytopathic effect was observable.. Viral infection was confirmed by an indirect immunofluorescence assay (IFA) using specific hyper-immune mouse ascitic fluid, as described previously (Digoutte et al., 1992) .
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