-
10 μl, 20 μl, 200 μl, 1,000 μl pipette tips (Thermo Fisher Scientific, catalog numbers: 2140-05, 72830-440, 72830-044, 72830-042)
-
1 ml, 5 ml, 10 ml, 25 ml, 50 ml sterile serological pipettes (Thermo Fisher Scientific, catalog numbers: 14672-918, 14672-920, 4101, 14672-900, 53384-451)
-
6-well tissue culture dishes (Corning, Falcon®, catalog number: 353046)
-
96-well tissue culture flat bottom plate with sterile lid (CostarTM, catalog number: 29442-056)
-
Deep well 96-well plates, sterile (Life Technologies, catalog number: 11388-566)
-
15 and 50 ml Conical tubes (Corning, Falcon®, catalog numbers: 353110 and 352098)
-
Polypropylene microcentrifuge tubes (Life Technologies|AB/Invitrogen, catalog number: AM12400)
-
2 ml screw cap tubes (Sarstedt, catalog number: 72.694.006)
-
T-75 flask (Corning, catalog number: 3276CS)
-
T-150 flask (Corning, catalog number: 430825)
-
WYPALL Absorbent toweling (Kimberly-Clark® Professional, catalog number: KIM41200)
-
Reservoirs (Costar Reagent Reservoir, Corning®, catalog number: 29442-474)
-
Freezing vial (Corning, catalog number: 431386)
-
MA-104 Clone 1 cells were obtained by American Tissue Culture Collection (ATCC, catalog number: CRL-2378.1)
-
Dulbecco’s Phosphate Buffered Saline (DPBS) 1× no calcium, no magnesium, pH 7.0-7.3 (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-250)
-
Gamma Irradiated Fetal bovine serum (GE Healthcare, HycloneTM, catalog number: SH3007103IREA-HYL)
-
1× antibiotic-Antimycotic (Life Technologies|AB/Invitrogen, catalog number: 15240062)
-
0.25% Trypsin 0.53 mM EDTA (Life Technologies|AB/Invitrogen, catalog number: 12604-013)
-
DMEM (Fisher Scientific, catalog number: 11965-118)
-
Recovery Freezing Medium (Thermo Fisher, catalog number: 12648-010)
-
Acetone (Fisher Scientific, catalog number: A18-4)
-
Methanol (Fisher Scientific, catalog number: A412P4)
-
Isopropanol (Fisher Scientific, catalog number: A415-4)
-
Ethanol (Sigma-Aldrich, catalog number: E7023)
-
Horse serum (Vector Laboratories, catalog number: S-2000)
-
ASFV p30 monoclonal antibody produced by APHIS, USDA (Wu et al., 2020)
-
Vectastain® ABC kit, Peroxidase Mouse IgG (Vector Laboratories, catalog number: PK4002)
-
Vector® VIP, Peroxidase substrate kit SK-4600 (Vector Laboratories, catalog number: ZF0517)
-
Bovine Albumin Fraction V Low endotoxin, BSA (MP Biomedicals, catalog number: 810681)
-
ASFV field strain: For example an ASFV-Georgia field strain (ASFV-G)
-
MA-104 media (see Recipes)
-
Blocking buffer (see Recipes)
-
Fixing Solution (see Recipes)
-
25% v/v suspension of swine red blood cells (see Recipes)
-
0.1% BSA (see Recipes)
-
70% Ethanol (see Recipes)
-
2 L beaker
-
Mr. Frosty Freezing Container (Nalgene, catalog number: 5100-0001)
-
Class II biological safety cabinet (LABCONCO®, NSF®, model: D045939)
-
Humidified 37±2 °C, 5±1% CO2 incubator (Panasonic, catalog number: KM-CC17RU1A)
-
-70 °C freezer (Panasonic MDF-U76VA-PA, catalog number: 17117N0425)
-
-20 °C freezer (Kenmore, model: 2539261110)
-
Refrigerator, 4 °C, with an acceptable range of 2 °C to 8 °C (Sanyo Medicool, model: MPR-513)
-
Liquid nitrogen Freezer < -120 °C storage temp.
-
Water bath, 37 °C (Lab-line AquabathTM, model: 18052AQ)
-
Pipet aid (Drummond Scientific, catalog number: 4-000-101)
-
Micropipettors: Single channel, 1-10 µl, 2-20 µl, 50-200 µl, 100-1,000 µl (Eppendorf catalog numbers: Q11390H, K12770H, Q24196H, L18745H)
-
Micropipettors: Multi-channel, 0.5-10 µl, 5-50 µl, 50-300 µl (Lab System, catalog numbers: N87539, D23745, E21512)
-
Multi-channel for larger volumes (RaininTM Pipet-Life XLS, L-1200, 100-1,200 µl, catalog number: 17014497)
-
Inverted Microscope (Carl-Zeiss, model: Axio Observer 3)
-
Bright line Hematocytometer (Sigma, catalog number: Z359629)
-
Tabletop Centrifuge (Eppendorf, model: 5417R)
-
Sorvall® LYNX 6000 (Thermo Fisher Scientific, catalog number: 75006590)
-
Vortex mixer (Daigger vortex Genie2TM, catalog number: G22220)
-
Plate shaker (Gene Mate, BioExpress Rocker Variable, catalog number: R-3200-1)
To achieve 80% confluency of MA-104 cells 2 days after splitting a ratio of 1:3 is used.
To achieve 80% confluency of MA-104 cells 4-5 days after splitting a ratio of 1:10 is used.
Requirement of virus culture in primary swine macrophages is a technically challenging process and was historically done for detecting and quantifying infectious ASFV. We previously identified MA-104 cells as a commercially available cell line which supports robust ASFV growth, allowing the detection of infectious ASFV from clinical and field samples (Rai et al., 2020). MA-104 cells have a doubling time of 72 h and, importantly, there are no requirements of any special media for growing these cells nor conducting virus culture in these cells. MA-104 cells can be easily frozen in large quantities, allowing timely expansion of the cells in quantities required in a disease outbreak when a large number of samples needs to be processed to confirm whether samples contain just ASFV DNA or infectious ASFV virus. Therefore, MA-104 cells can aid in the rapid diagnosis of ASFV by bypassing the need for producing primary cell cultures of swine macrophages (which require fresh swine blood, something not readily available at common veterinary diagnostic laboratories). Infectious ASFV in MA-104 cells can be easily detected by either HA or IIPA staining using anti-ASFV specific antibodies. IPA staining can further allow the detection of ASFV field isolates that has mutations in CD2, and would be unable to form HA. IPA staining would be used in instances where PCR was positive but no HA was observed to determine the presence of live infectious virus in a sample (Borca et al., 2018). The clear staining of ASFV-infected cells by IPA is advantageous over using swine macrophages as a substrate, because swine macrophages have intrinsic peroxidase activity leading to a high staining background in uninfected cells, leading to false positive reactions (Rai et al., 2020).
-
When thawing MA-104 cells never leave cell vial unattended, because at 37 °C, cell thawing completes within 1-2 min, and long exposure at 37 °C causes cryoprotectant toxicity. Remove the vial from the water bath when still a few ice crystals remain.
-
Cells are particularly sensitive immediately post thaw. They should be seeded into pre-warmed medium.
-
Low seeding densities should be avoided for starting the cells taken out of liquid nitrogen storage or vendor supplied vial.
-
While waiting for the cells to detach, do not agitate the cells by hitting or shaking the flask to avoid cell clumping. Place cells that are difficult to detach for extended time at 37 °C to facilitate dispersal.
-
Don’t store the cells at -70 °C for more than 2 days. It will decrease cell viability.
-
When freezing the cells, do not leave them in Recovery freezing medium at room temp for long time because cryoprotectants in the media are toxic to cells.
-
This protocol shall be conducted in an animal Biosafety level 3 laboratory, because ASFV is a select agent, which can only be handled in a designated animal Biosafety level 3 laboratory.
-
Exterior of the container need to be surface decontaminated by wiping with 10% bleach soaked towel.
-
To transfer virus containing vials, plates or storage boxes between different work areas and the storage, always use an air-sealed secondary container.
-
Open the secondary container only in a Biosafety cabinet. Before taking out of the Biosafety cabinet surface contaminate both the vial/plate/box and the secondary container.
-
Contain all the solid and liquid waste in approved Biohazard container. Dispose of all material used in the experiment after autoclaving at appropriate cycle.
The identification of Ma-104 cells funded through an interagency agreement with the Science and Technology Directorate of the U.S. Department of Homeland Security under Award Number: 70RSAT18KPM0000138.
This research was supported in part by an appointment to the Plum Island Animal Disease Center (PIADC) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664.
The authors Ayushi Rai, Manuel Borca and Douglas Gladue have filed a patent for using MA-104 cells as a substrate for detection of ASFV.