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Jul 2020
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Detection and Quantification of African Swine Fever Virus in MA-104 Cells
非洲猪瘟病毒在MA-104细胞中的检测与定量   

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Abstract

Detection of live African swine fever virus (ASFV) has historically relied on the use of primary swine macrophages (PSM). PSM do not replicate and have to be isolated fresh from donor swine. We previously identified that a MA-104 cells (ATCC #CRL-2378.1), a commercially available cell line isolated from African green monkey (Cercopithecus aethiops) kidney epithelial cells, supports the detection of ASFV from field samples with a sensitivity comparable to that of primary swine macrophages. Collection of swine blood or lungs is time costing, which is often not readily available in most veterinary diagnostic laboratories. MA-104 cells could thus be used as substitute for primary swine macrophages to save significant lead time by avoiding the production of primary swine macrophages.

Keywords: ASFV (非洲猪瘟病毒), ASF (非洲猪), African swine fever cell line (非洲猪瘟细胞系), Diagnostics (诊断学), MA-104 (MA-104)

Background

African swine fever virus (ASFV), a member of Asfarviridae family, causes a highly contagious and lethal hemorrhagic fever, namely African swine fever (ASF) in wild boars and domesticated pigs. The mature virus particle (virion) has 175-215 nm diameter, with a lipid bilayer enclosing an icosahedral capsid and a 180-190 kilobase pairs long double-stranded DNA genome. The virus causes a wide-spectrum of symptoms including highly lethal to sub-clinical, depending on host characteristics and the virus strain (Tulman et al., 2009). Preliminary diagnosis of ASFV in clinical samples (typically whole blood) is performed using real-time polymerase chain reaction (rt-PCR), which detects pieces of viral genome but active infection, the latter is confirmed via virus isolation and further downstream analyses, such as whole genome sequencing. Before the publication of our recent work identifying MA-104 cells as a substrate for ASFV detection (Rai et al., 2020), infectious virus detection could only be performed in primary macrophage cultures prepared from fresh pig blood. This protocol uses commercially available African green monkey cell line MA-104 (Whitaker and Hayward, 1985) as a substitute for detection and quantification of infectious ASFV either by hemadsorption (HA) or by staining with specific antibody for ASFV.


Materials and Reagents

  1. 10 μl, 20 μl, 200 μl, 1,000 μl pipette tips (Thermo Fisher Scientific, catalog numbers: 2140-05, 72830-440, 72830-044, 72830-042)

  2. 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)

  3. 6-well tissue culture dishes (Corning, Falcon®, catalog number: 353046)

  4. 96-well tissue culture flat bottom plate with sterile lid (CostarTM, catalog number: 29442-056)

  5. Deep well 96-well plates, sterile (Life Technologies, catalog number: 11388-566)

  6. 15 and 50 ml Conical tubes (Corning, Falcon®, catalog numbers: 353110 and 352098)

  7. Polypropylene microcentrifuge tubes (Life Technologies|AB/Invitrogen, catalog number: AM12400)

  8. 2 ml screw cap tubes (Sarstedt, catalog number: 72.694.006)

  9. T-75 flask (Corning, catalog number: 3276CS)

  10. T-150 flask (Corning, catalog number: 430825)

  11. WYPALL Absorbent toweling (Kimberly-Clark® Professional, catalog number: KIM41200)

  12. Reservoirs (Costar Reagent Reservoir, Corning®, catalog number: 29442-474)

  13. Freezing vial (Corning, catalog number: 431386)

  14. MA-104 Clone 1 cells were obtained by American Tissue Culture Collection (ATCC, catalog number: CRL-2378.1)

  15. Dulbecco’s Phosphate Buffered Saline (DPBS) 1× no calcium, no magnesium, pH 7.0-7.3 (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-250)

  16. Gamma Irradiated Fetal bovine serum (GE Healthcare, HycloneTM, catalog number: SH3007103IREA-HYL)

  17. 1× antibiotic-Antimycotic (Life Technologies|AB/Invitrogen, catalog number: 15240062)

  18. 0.25% Trypsin 0.53 mM EDTA (Life Technologies|AB/Invitrogen, catalog number: 12604-013)

  19. DMEM (Fisher Scientific, catalog number: 11965-118)

  20. Recovery Freezing Medium (Thermo Fisher, catalog number: 12648-010)

  21. Acetone (Fisher Scientific, catalog number: A18-4)

  22. Methanol (Fisher Scientific, catalog number: A412P4)

  23. Isopropanol (Fisher Scientific, catalog number: A415-4)

  24. Ethanol (Sigma-Aldrich, catalog number: E7023)

  25. Horse serum (Vector Laboratories, catalog number: S-2000)

  26. ASFV p30 monoclonal antibody produced by APHIS, USDA (Wu et al., 2020)

  27. Vectastain® ABC kit, Peroxidase Mouse IgG (Vector Laboratories, catalog number: PK4002)

  28. Vector® VIP, Peroxidase substrate kit SK-4600 (Vector Laboratories, catalog number: ZF0517)

  29. Bovine Albumin Fraction V Low endotoxin, BSA (MP Biomedicals, catalog number: 810681)

  30. ASFV field strain: For example an ASFV-Georgia field strain (ASFV-G)

  31. MA-104 media (see Recipes)

  32. Blocking buffer (see Recipes)

  33. Fixing Solution (see Recipes)

  34. 25% v/v suspension of swine red blood cells (see Recipes)

  35. 0.1% BSA (see Recipes)

  36. 70% Ethanol (see Recipes)

Equipment

  1. 2 L beaker

  2. Mr. Frosty Freezing Container (Nalgene, catalog number: 5100-0001)

  3. Class II biological safety cabinet (LABCONCO®, NSF®, model: D045939)

  4. Humidified 37±2 °C, 5±1% CO2 incubator (Panasonic, catalog number: KM-CC17RU1A)

  5. -70 °C freezer (Panasonic MDF-U76VA-PA, catalog number: 17117N0425)

  6. -20 °C freezer (Kenmore, model: 2539261110)

  7. Refrigerator, 4 °C, with an acceptable range of 2 °C to 8 °C (Sanyo Medicool, model: MPR-513)

  8. Liquid nitrogen Freezer < -120 °C storage temp.

  9. Water bath, 37 °C (Lab-line AquabathTM, model: 18052AQ)

  10. Pipet aid (Drummond Scientific, catalog number: 4-000-101)

  11. Micropipettors: Single channel, 1-10 µl, 2-20 µl, 50-200 µl, 100-1,000 µl (Eppendorf catalog numbers: Q11390H, K12770H, Q24196H, L18745H)

  12. Micropipettors: Multi-channel, 0.5-10 µl, 5-50 µl, 50-300 µl (Lab System, catalog numbers: N87539, D23745, E21512)

  13. Multi-channel for larger volumes (RaininTM Pipet-Life XLS, L-1200, 100-1,200 µl, catalog number: 17014497)

  14. Inverted Microscope (Carl-Zeiss, model: Axio Observer 3)

  15. Bright line Hematocytometer (Sigma, catalog number: Z359629)

  16. Tabletop Centrifuge (Eppendorf, model: 5417R)

  17. Sorvall® LYNX 6000 (Thermo Fisher Scientific, catalog number: 75006590)

  18. Vortex mixer (Daigger vortex Genie2TM, catalog number: G22220)

  19. Plate shaker (Gene Mate, BioExpress Rocker Variable, catalog number: R-3200-1)

Procedure

  1. Thaw frozen vial of MA-104 cells received from ATCC on dry ice or frozen in liquid nitrogen

    1. Remove the frozen vial of MA-104 cells either from dry ice or liquid nitrogen storage.

    2. Thaw MA-104 cells in a 37 °C water bath.

    3. Keep the lid of the MA-104 cell frozen vial above the surface of the water to lessen the chances of contamination.

    4. When the MA-104 cells are almost thawed (only a small piece of ice) move the vial to the tissue culture hood.

    5. Wipe the surface of the vial with 70% ethanol and remove the top.

    6. Carefully remove the MA-104 cell suspension using a sterile Pasteur pipette.

    7. Gently add MA-104 cells in T75 flask containing 15 ml of pre-warmed MA-104 media at 37 °C

    8. Place the MA-104 cells in T75 flasks in a 5% CO2 incubator.

    9. After 24 h discard the old media.

    10. Add 15 ml of MA-104 media in T-75 flask and place the flask in a 5% CO2 incubator.


  2. MA-104 cell sub culturing when confluent (Figure 1) at ratios 1:3 to 1:10

    1. Remove and discard culture media.

    2. Rinse the MA-104 cell monolayer with 10 ml 1× DPBS.

    3. To remove traces of serum that contains trypsin inhibitor, briefly rinse the cell monolayer with a 0.25% (w/v) Trypsin-0.53 mM EDTA solution.

    4. Add 3.0 ml Trypsin-EDTA to the flask and keep the flask in 5% CO2 incubator and observe every 2 min under an inverted microscope until cell monolayeris completely dispersed (typically takes 5-15 min).

    5. Add 8.0 ml MA-104 media and break cells by gently pipetting up and down

    6. Add approximately 1 ml aliquot of the cell suspension to new T-150 flask

    7. Incubate culture in an 5% CO2 incubator at 37 °C



      Figure 1. MA-104 cells at 100% confluency and ready to be subcultured into new flasks


Subculture and plating ratios used to prepare MA-104 cells:

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.

To culture MA-104 cells in 6-well culture dishes, seed at 1 × 10cells/well.

To culture MA-104 cells in 96-well culture dishes, seed at 1 × 10cells/well.



  1. Procedure to freeze MA-104 cells

    1. Before freezing MA-104 cells check for bacterial, yeast, or fungal contamination under inverted microscope.

    2. Freeze only healthy cells in the log phase of growth (between 70-90% confluency; Figure 2).



      Figure 2. MA-104 cells shown growing in active log phase ready to be prepared for frozen stocks


    3. Split MA-104 cells: Discard old growth media. Wash cell monolayer with 10 ml 1× DPBS then discard 1× DPBS. Rinse cells with trypsin/EDTA and discard it and then add 3 ml of trypsin/EDTA. Once cells start detaching, give the flask a gentle pat and re-suspend the cells with 8 ml of growth media. Disperse cells by gently pipetting up and down to break clumps and get a homogeneous suspension.

    4. Count the cells using hemacytometer under inverted microscope.

    5. Number of cells used to freeze should be in the range of 5 × 106-1 × 107 cells/vial. Calculate the volume of freezing media needed for a final cell concentration to be in this range.

    6. Transfer cell suspension to sterile centrifuge tubes, balance the centrifuge, and centrifuge at 201 × g (1,000 rpm) for 5 min.

    7. Carefully remove as much supernatant as possible without disturbing the pellet

    8. Quickly re-suspend pellet by adding the calculated amount of recovery freezing medium (Thermo Fisher, catalog number: 12648-010) in a 2 ml screw cap tube, and label the tube.

    9. Place the vials in Mr. Frosty Freezing Container, which allows the cells to cool down slowly.

    10. Close the freezing container and place at -70 °C for at least overnight.

    11. Next day, transfer the vials of frozen MA-104 to liquid nitrogen.


  2. ASFV titration in MA-104 cells detection by Hemadsorption (HA)

    1. For titration in 96-well culture plates use 1 × 104 cells/well prepared in MA-104 media; recommended volume of cell suspension/well is 100 µl.

    2. Prepare ten-fold serial dilutions of each ASFV sample (swine blood) using MA-104 media.

    3. Add 50 µl of each dilution of ASFV samples in order from 10-7 to 10-1 to the plate wells containing 100 µl of MA-104 cells.

    4. Incubate cells at 37 °C with 5% CO2.

    5. 24 h post infection (hpi), add 1 µl of 25% v/v suspension of swine red blood cells to each well of MA-104 cells in the plate.

    6. Incubate at 37 °C with 5% CO2 for seven days of incubation.

    7. Observe the plates under the inverted microscope for Hemadsorption to confirm the infection.

    8. Calculate virus titer using the Reed and Muench method (Reed and Muench, 1938) by Hemadsorping dose (HAD) and express it as log10HAD50/ml.


  3. Detection of ASFV infection in MA-104 by HA

    1. For HA, plate 2 ml of MA-104 cell suspension prepared in MA-104 media at a density of 1 × 106 cells/well in 6-well tissue culture dishes.

    2. Add ASFV field sample to three wells of the 6-well plate with 1 µl, 10 µl and 100 µl.

    3. 1 hpi, add 100 µl of 25% v/v suspension of swine red blood cells were to each well.

    4. 24 hpi, observe ASFV-infected cells for rosette formation in MA-104 cells (Figure 3). Depending on quantity and quality of the ASF sample, rosettes may take longer to form, if no rosettes are observed at 24 hpi, check daily for 7 days.



      Figure 3. MA-104 cells infected with ASFV-G and incubated with red blood cells showing clear HA


  4. Immunoperoxidase (IPA) staining for ASFV in MA-104 cells

    *Perform on Biosafety cabinet*

    1. Plate 2 ml of MA-104 cell suspension prepared in MA-104 media at a density of 1 × 106 cells/well in 6-well tissue culture dishes.

    2. Add ASFV field sample to three wells of the 6-well plate with 1 µl, 10 µl and 100 µl.

    3. Incubate the plates at 37 °C in an incubator for 72 h.

    4. Take the plates out of the incubator.

    5. Discard media by inverting plate and dumping media into 2 L beaker and then the rest of the media soaked by inverting plates onto thick paper towels kept in tray container

    6. Fix the cells using 2 ml of fixing solution (1:1 solution of acetone and methanol) in each well.

    7. Incubate the plate(s) at room temperature for 15-20 min.

    8. Prepare solutions:

      1. Blocking solution: DMEM + 2% normal horse serum kept at 4 °C

      2. Antibody solution: DMEM + 2% normal horse serum + ASFV p30 monoclonal antibody (1:200 dilution) kept 4 °C

      3. Anti-mouse IgG solution: DMEM + 2% normal horse serum + 0.25% anti­mouse IgG (Vectastain ABC kit, Peroxidase Mouse IgG) kept at 4 °C

      4. 1× DPBS + BSA + A + B solution: 1× DPBS + 0.1% BSA + 0.6% A + 0.6% B ('A' and 'B' are components of Vectastain ABC kit) kept at 4 °C

      5. Peroxidase substrate solution: 1× DPBS (from lab container) + Vector® VIP reagents kept at 4 °C (Use 3 drops from each of the four bottles per 5 ml solution)


    *Perform on benchtop*

    1. Discard fixing solution in a 2 L beaker, invert the plate over thick paper towel to remove the leftover fixing solution and air-dry plates for 10 min.

    2. Add 2 ml/well Blocking solution. Gently tap or shake the plate to ensure the entire surface of each well is covered by the solution.

    3. Incubate the plate(s) at 37 °C for 30 min.

    4. Discard of Blocking solution in 2 L beaker and invert the plates over thick paper towel to remove the leftover Blocking solution.

    5. Add 2 ml/well Antibody solution.

    6. Incubate the plate(s) at 37 °C for 30 min.

    7. Discard of Antibody solution in a 2 L beaker and rinse 2 times with 1XDPBS by using squeeze bottle.

    8. Add 2 mI/ well of Anti-mouse lgG solution.

    9. Incubate the plate(s) at 37 °C for 30 min.

    10. During the 30 min incubation, prepare 1× DPBS + BSA + A + B solution. Add 0.1% BSA w/v in 1× DPBS and mix thoroughly, next add 0.6% v/v solution A and solution B from Vectastain ABC kit stock. Keep the freshly prepared 1× DPBS + BSA + A + B solution at room temperature.

    11. Discard Anti-mouse IgG solution in a 2 L beaker and rinse 2 times with 1× DPBS using squeeze bottle.

    12. Add 2 ml/well 1× DPBS + BSA + A + B solution.

    13. Incubate the plate(s) at 37 °C for 30 min.

    14. Discard 1× DPBS + BSA + A + B solution in a 2 L beaker and wash 2 times with 1× DPBS by using squeeze bottle.

    15. Add 2 mI/well Peroxidase substrate solution.

    16. Incubate the plates at room temperature for 10 min.

    17. Discard Peroxidase substrate solution in a 2 L beaker and rinse the plates with tap water.

    18. Air-dry the plate for 10 min.

    19. Calculate the titer using Reed and Muench method (Reed and Muench, 1938). Figure 4 shows positive ASFV staining.

      Note: Take extra caution in performing the washes, because each wash step requires a different solution and cross contamination between solutions could potentially lead to failed experiment and erroneous readings.



    Figure 4. Fixed MA-104 cells infected with ASFV-G that were IPA stained for ASFV using monoclonal antibody against a structural protein p30, the MA-104 cells show clear staining for ASFV

Data analysis

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).

Notes

  1. 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.

  2. Cells are particularly sensitive immediately post thaw. They should be seeded into pre-warmed medium.

  3. Low seeding densities should be avoided for starting the cells taken out of liquid nitrogen storage or vendor supplied vial.

  4. 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.

  5. Don’t store the cells at -70 °C for more than 2 days. It will decrease cell viability.

  6. 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.

  7. 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.

  8. Exterior of the container need to be surface decontaminated by wiping with 10% bleach soaked towel.

  9. To transfer virus containing vials, plates or storage boxes between different work areas and the storage, always use an air-sealed secondary container.

  10. 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.

  11. Contain all the solid and liquid waste in approved Biohazard container. Dispose of all material used in the experiment after autoclaving at appropriate cycle.

Recipes

  1. MA-104 Media

    500 ml DMEM

    50 ml FBS

    5 ml Antibiotic-Antimycotic

  2. Blocking Buffer

    500 ml DMEM

    10 ml (2%) Horse serum

  3. Fixing Solution (1:1 solution of acetone and methanol)

    50 ml Acetone

    50 ml Methanol

  4. 0.1% BSA

    Add 0.5 g BSA (4 °C) to 500 ml 1× DPBS bottle

  5. 25% v/v suspension of swine red blood cells

    Add 9 ml of red cells pellet and 27 ml of 1× DPBS to make 25% blood in 1× DPBS

    Store at 4 °C label % of red blood cells in pbs and date

  6. 70% Ethanol

    70 ml 100% Ethanol

    30 ml dH2O

Acknowledgments

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.

Competing interests

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.

References

  1. Borca, M. V., Holinka, L. G., Berggren, K. A. and Gladue, D. P. (2018). CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses.Sci Rep 8: 3154.
  2. Rai, A., Pruitt, S., Ramirez-Medina, E., Vuono, E. A., Silva, E., Velazquez-Salinas, L., Carrillo, C., Borca, M. V. and Gladue, D. P. (2020). Identification of a Continuously Stable and Commercially Available Cell Line for the Identification of Infectious African Swine Fever Virus in Clinical Samples. Viruses 12(8). doi:10.3390/v12080820.
  3. Reed, L. J. and Muench, H. (1938). A simple method of estimating fifty percent endpoints.The American J Hygiene 27(3): 493-497.
  4. Tulman, E. R., Delhon, G. A., Ku, B. K. and Rock, D. L. (2009). African Swine Fever Virus. In: Lesser Known Large dsDNA Viruses. Springer-Verlag Berlin Heidelberg Vol. 328 pp. 43-87.
  5. Whitaker, A. M. and Hayward, C. J. (1985). The characterization of three monkey kidney cell lines. Dev Biol Stand 60: 125-131.
  6. Wu, P., Lowe, A. D., Rodriguez, Y. Y., Murgia, M. V., Dodd, K. A., Rowland, R. R. and Jia, W. (2020). Antigenic regions of African swine fever virus phosphoprotein P30. Transbound Emerg Dis doi:10.1111/tbed.13533.

简介

[摘要]活的非洲猪瘟病毒(ASFV)的检测在历史上一直依赖于原代猪巨噬细胞(PSM)的使用。PSM不能复制,必须从供体猪中新鲜分离。我们先前发现,MA-104细胞(ATCC#CRL-2378.1)是一种从非洲绿猴(Cercopithecus aethiops )肾上皮细胞分离的商业细胞系,支持从野外样品中检测ASFV,其灵敏度可与ASFV媲美。原发性猪巨噬细胞。Ç的猪的血液或肺ollection是时间成本计算,这往往是在大多数兽医诊断实验室容易获得的。MA-104细胞因此可以用作原代猪巨噬细胞的替代品,通过避免原代猪巨噬细胞的产生来节省大量的准备时间。

[背景]非洲猪瘟病毒(ASFV)的成员,非洲猪瘟病毒科的家庭,导致野猪和家猪具有高度传染性和致命性出血热,即非洲猪瘟(ASF)。成熟的病毒颗粒(病毒粒子)直径为175-215 nm,脂质双层包裹了二十面体衣壳和180-190千碱基对的双链DNA基因组。根据宿主特征和病毒株,该病毒会引起多种症状,包括高度致死性至亚临床性(Tulman等,2009 )。使用实时聚合酶链反应(rt-PCR)对临床样品(通常是全血)中的ASFV进行初步诊断,该方法可以检测病毒基因组片段,但可以主动感染,可以通过病毒分离和进一步的下游分析来确认后者作为全基因组测序。在我们最近发表的鉴定MA-104细胞为ASFV检测底物的研究发表之前(Rai等,2020),传染性病毒检测只能在用新鲜猪血制备的原代巨噬细胞培养物中进行。该方案使用可商购的非洲绿猴细胞系MA-104(Whitaker和Hayward,1985)作为替代品,用于通过吸血(HA)或用针对ASFV的特异性抗体染色来检测和定量感染性ASFV。

关键字:非洲猪瘟病毒, 非洲猪, 非洲猪瘟细胞系, 诊断学, MA-104

材料和试剂


10μl,20μl,200μl,1,000μl移液器吸头(Thermo Fisher Scientific,目录号:2140-05、72830-440、72830-044、72830-042)
1 ml,5 ml,10 ml,25 ml,50 ml无菌血清移液管(Thermo Fisher Scientific,目录号:14672-918、14672-920、4101、14672-900、53384-451 )
6孔组织培养皿中(Corning,隼® ,目录号:353046 )
                             带无菌盖的96孔组织培养平底板(Costar TM ,目录号:29442-056)
深孔96孔无菌平板(Life Technologies,目录号:11388-566)
15和50毫升锥形管中(Corning,隼® ,目录号:353110和352098)
聚丙烯微量离心管(Life Technologies | AB / Invitrogen,目录号:AM12400)
2 ml螺帽管(Sarstedt,目录号:72.694.006)
T-75烧瓶(Corning,目录号:3276CS)
T-150烧瓶(Corning,目录号:430825)
WYPALL吸水纸巾(金佰利®专业版,目录号:KIM41200)
水库(Costar公司试剂水库,康宁® ,目录号:29442-474)
冷冻小瓶(Corning,目录号:431386)
通过美国组织培养物保藏中心(ATCC,目录号:CRL-2378.1)获得MA-104克隆1细胞。
Dulbecco磷酸盐缓冲盐水(DPBS)1 ×无钙,无镁,pH 7.0-7.3(Thermo Fisher Scientific,Gibco TM ,目录号:14190-250 )
伽马射线照射的胎牛血清(GE Healthcare,Hyclone TM ,目录号:SH3007103IREA-HYL )
1 ×抗生素-抗真菌药(Life Technologies | AB / Invitrogen,目录号:15240062 )
0.25%胰蛋白酶0.53 mM EDTA(Life Technologies | AB / Invitrogen,目录号:12604-013)
DMEM (Fisher Scientific,目录号:11965-118)
恢复冷冻介质(Thermo Fisher,目录号:12648-010)
丙酮(Fisher Scientific,目录号:A18-4)
甲醇(Fisher Scientific,目录号:A412P4)
异丙醇(Fisher Scientific,目录号:A415-4)
乙醇(Sigma - Aldrich,目录号:E7023 )
马血清(Vector Laboratories,目录号:S-2000)
由APHIS,USDA产生ASFV P30单克隆抗体(吴等人。,2020)
的Vectastain ® ABC试剂盒,过氧化物酶小鼠IgG(Vector Laboratories公司,目录号:PK4002)
矢量® VIP,过氧化物酶底物试剂盒SK-4600(Vector Laboratories公司,目录号:ZF0517)
牛白蛋白组分V低内毒素,BSA(MP生物医学,目录号:810681)
ASFV野毒株:例如ASFV-乔治亚州野毒株(ASFV-G)
MA-104媒体(请参阅食谱)
阻塞缓冲区(请参见配方)
固定解决方案(请参阅食谱)
猪血红细胞的25%v / v悬浮液(请参阅食谱)
0.1%BSA(请参阅食谱)
70%乙醇(请参阅食谱)


设备


2 L烧杯
弗罗斯蒂冷冻箱先生(Nalgene,目录号:5100-0001)
II级生物安全柜(LABCONCO ® ,NSF ® ,型号:D045939)
加湿的37±2°C,5±1%CO 2培养箱(Panasonic,目录号:KM-CC17RU1A)
-70°C冰箱(Panasonic MDF-U76VA-PA ,目录号:17117N0425)
-20℃的冰箱(Kenmore的,米Odel等:2539261110 )
4°C的冰箱,可接受范围为2°C至8°C(Sanyo Medicool ,型号:MPR-513)
                            液氮冷冻室<-120°C储存温度。
                            37°C水浴(Lab-line Aquabath TM ,型号:18052AQ)
移液器(Drummond Scientific,目录号:4-000-101)
微量移液器:单通道,1-10 µl,2-20 µl,50-200 µl,100-1,000 µl (Eppendorf目录编号:Q11390H,K12770H,Q24196H,L18745H)
                        移液器:多通道,0.5-10微升,5-50微升,50-300微升(实验室小号ystem ,Ç atalog数字:N87539,D23745,E21512)
                        多通道用于更大体积(Rainin TM Pipet-Life XLS,L-1200,100-1,200 µl,目录号:17014497)
倒置显微镜(Carl-Zeiss ,型号:Axio Observer 3)
亮线血细胞计数器(Sigma,目录号:Z359629)
台式离心机(Eppendorf,型号:5417R)
SORVALL ® LYNX 6000(赛默飞世尔科技,目录号:75006590)
涡旋混合器(Daigger vortex Genie2 TM ,目录号:G22220)
平板振荡器(Gene Mate,BioExpress Rocker Variable,目录号:R-3200-1)


程序


在干冰或液氮中冷冻从ATCC融化的MA-104细胞的冷冻小瓶
从干冰或液氮存储中取出MA-104细胞的冷冻小瓶。
在37解冻MA-104细胞 °C水浴。
将MA-104细胞冷冻小瓶的盖子放在水面上方,以减少污染的机会。
当MA-104细胞几乎融化时(只有一小块冰),将小瓶移至组织培养罩。
用70%的乙醇擦拭小瓶的表面,然后除去顶部。
用无菌巴斯德吸管小心取出MA-104细胞悬液。
在37 °C的T75烧瓶中轻轻加入MA-104细胞,其中装有15 ml预热的MA-104培养基
将MA-104细胞放在5%CO 2培养箱中的T75烧瓶中。
24小时后,丢弃旧媒体。
在T-75烧瓶中加入15 ml MA-104培养基,并将烧瓶放入5%CO 2培养箱中。


MA-104细胞培养子当共nfluent (图1)在比1:3至1:10
除去并丢弃培养基。
用10 ml 1 × DPBS冲洗MA-104细胞单层。
要去除痕量的含胰蛋白酶抑制剂的血清,只需用0.25%(w / v)胰蛋白酶-0.53 mM EDTA溶液冲洗细胞单层即可。
向烧瓶中加入3.0 ml胰蛋白酶-EDTA,并将烧瓶保持在5%CO 2培养箱中,在倒置显微镜下每2分钟观察一次,直到细胞单层完全分散(通常需要5-15分钟)。
加入8.0 ml MA-104培养基并通过上下轻轻移液来破坏细胞
向新的T-150烧瓶中加入约1 ml细胞悬液的等分试样
在5%CO 2培养箱中于37 °C孵育培养物






图1. 100%融合的MA-104细胞,准备将其传代到新的烧瓶中


用于制备MA-104细胞的传代培养和铺板比率:


为了在分裂后2天达到80%的MA-104细胞融合度,使用1:3的比例。


为了在分裂后4-5天达到MA-104细胞80%的融合,使用1:10的比例。


培养MA-104细胞在6 -孔培养皿,在种子1 × 10 6个细胞/孔。


培养MA-104细胞在96 -孔培养皿,在种子1 × 10 4个细胞/孔。


冷冻MA-104细胞的程序
乙EFORE冷冻MA-104细胞检查细菌,酵母,或倒置显微镜下真菌污染。
                 在生长的对数期(70-90%融合之间;图2 ),仅冷冻健康的细胞。






图2. MA-104细胞显示处于活跃对数生长期,可随时准备用于冷冻原种


分裂MA-104细胞:丢弃旧的生长培养基。用10 ml 1 × DPBS洗涤细胞单层,然后弃去1 × DPBS。用胰蛋白酶/ EDTA冲洗细胞并丢弃,然后加入3 ml胰蛋白酶/ EDTA。一旦细胞开始分离,轻轻摇动烧瓶,并用8 ml生长培养基重悬细胞。轻轻地上下吹打以分散团块,分散细胞,得到均匀的悬浮液。
使用血细胞计数器在倒置显微镜下计数细胞。
用于冷冻的细胞数应在5 × 10 6 -1 × 10 7细胞/小瓶的范围内。计算最终细胞浓度在此范围内所需的冷冻培养基体积。
转移细胞悬液至无菌离心管,平衡离心机,和在离心机201 ×克(1 ,000 rpm)离心5分钟。
小心地去除尽可能多的上清液,而不会干扰沉淀
通过将计算量的回收冷冻培养基(Thermo Fisher,目录号:12648-010)添加到2 ml螺帽管中,快速重悬沉淀,并在管上贴上标签。
将小瓶放入Mrs. Frosty Freezing容器中,该容器可使细胞缓慢冷却。
关闭冷冻容器,并在-70 °C放置至少过夜。
第二天,将冷冻的MA-104小瓶转移到液氮中


血液吸附(HA)检测MA-104细胞中的ASFV滴定
对于在96滴定-孔培养板用1 × 10 4个细胞/在MA-104介质以及制备 推荐的细胞悬液/孔体积为100 µl。
制备吨烯倍使用MA-104介质的每一非洲猪瘟病毒样品(猪血)的连续稀释液。
从10 -7至10 -1的顺序将50 µl ASFV样品的每种稀释液添加到含有100 µl MA-104细胞的平板孔中。
在37°C和5%CO 2下孵育细胞。
感染(hpi)后24小时,将1微升猪红细胞的25%v / v悬浮液添加到平板中的MA-104细胞的每个孔中。
在37°C和5%CO 2下孵育7天。
在倒置显微镜下观察培养皿的吸血情况,以确认感染。
使用Reed and Muench方法(Reed and Muench,1938年)通过Hemadsorping剂量(HAD)计算病毒滴度,并将其表示为log 10 HAD50 / ml。


HA检测MA-104中的ASFV感染
对于HA,将2 ml在MA-104培养基中制备的MA-104细胞悬液平板接种在6孔组织培养皿中,密度为1 × 10 6个细胞/孔。
非洲猪瘟病毒字段样品添加到6孔板的三个孔用1 μ升,10 μ升和100 μ升。
1 hpi,将100μl猪红细胞25%v / v悬浮液分别添加到每个孔中。
感染后24 h,观察ASFV感染的细胞在MA-104细胞中的玫瑰花结形成(图3)。根据ASF样品的数量和质量,玫瑰花结的形成时间可能会更长,如果在24 hpi时未观察到玫瑰花结,则每天检查7天。






图3 。被ASFV-G感染并与显示清晰HA的红细胞一起孵育的MA-104细胞


我mmunoperoxidase(IPA)在MA-104细胞染色ASFV
*在生物安全柜上执行*


将2 ml在MA-104培养基中制备的MA-104细胞悬液以1 × 10 6个细胞/孔的密度平板接种在6孔组织培养皿中。
非洲猪瘟病毒字段样品添加到6孔板的三个孔用1 μ升,10 μ升和100 μ升。
将板在培养箱中于37°C孵育72小时。
从培养箱中取出培养皿。
丢弃媒体通过反相板和倾倒到媒体2升烧杯中,然后将介质的其余部分浸泡通过反转镀到厚纸张保持毛巾在盘容器
固定用2-细胞毫升定影液:在每个孔中(1丙酮和甲醇的1:1溶液)。
将板在室温下孵育15-20分钟。
准备解决方案:
封闭溶液:DMEM + 2%正常马血清保持在4°C
抗体溶液:DMEM + 2%正常马血清+ ASFV p30单克隆抗体(1:200稀释)保持在4 °C
抗小鼠IgG溶液:DMEM + 2%正常马血清+ 0.25%抗小鼠IgG (Vectastain ABC试剂盒,过氧化物酶小鼠IgG)保持在4 °C
1 × DPBS + BSA + A + B溶液:1 × DPBS + 0.1%BSA + 0.6%A + 0.6%B(“ A”和“ B”是Vectastain ABC试剂盒的成分),保持在4 °C
过氧化物酶底物溶液:1 × DPBS(从实验室容器)+矢量® VIP试剂保持在4 ℃(使用来自每个每5ml溶液中的四瓶的3滴)


*在台式机上执行*


丢弃定影溶液在2升烧杯中,转化平板上厚纸巾以除去剩余的定影液和风干板为10分钟。  
加入2 ml /孔的封闭液。轻轻敲击或摇动所述板,以确保在整个表面的每个井被覆盖由该溶液。
将板在37°C下孵育30分钟。
将封闭溶液弃于2 L烧杯中,将板倒置在厚纸巾上,以除去剩余的封闭溶液。
添加2毫升/孔抗体溶液。
将板在37°C下孵育30分钟。
将抗体溶液丢弃在2 L的烧杯中,并使用挤压瓶用1XD PBS冲洗2次。
添加2米I /孔的抗小鼠的lgG溶液。
将板在37°C下孵育30分钟。
在30分钟的孵育过程中,准备1 × DPBS + BSA + A + B溶液。在1 × DPBS中添加0.1%BSA w / v ,并充分混合,然后从Vectastain ABC试剂盒库存中添加0.6%v / v溶液A和溶液B。将新鲜配制的1 × DPBS + BSA + A + B溶液保持在室温下。
将抗小鼠IgG溶液倒入2 L烧杯中,并使用挤瓶用1 × D PBS冲洗2次。
加入2 ml /孔的1 × D PBS + BSA + A + B溶液。
孵育所述板(一个或多个)在37℃下进行30分钟。
将1 × DPBS + BSA + A + B溶液丢弃在2 L的烧杯中,并使用挤瓶用1 × DPBS洗涤2次。
加入2 m I /孔过氧化物酶底物溶液。
在室温下孵育平板10分钟。
将过氧化物酶底物溶液倒入2 L的烧杯中,并用自来水冲洗板。
风干该板用于10分钟。
使用里德和穆恩奇方法(Reed and Muench,1938)计算滴度。图4显示阳性ASFV染色。
注意:执行洗涤时要格外小心,因为每个洗涤步骤需要使用不同的溶液,并且溶液之间的交叉污染可能会导致实验失败和读数错误。







图4 。使用抗结构蛋白p30的单克隆抗体对固定感染了ASFV-G的MA-104细胞进行IPA染色以进行ASFV染色,MA-104细胞显示出清晰的ASFV染色


数据分析


Requir的EMENT在初级猪巨噬细胞病毒培养是一种技术上的挑战过程和用于检测和定量感染非洲猪瘟病毒是历史上完成的。我们先前将MA-104细胞鉴定为可支持ASFV强劲生长的市售细胞系,从而可从临床和现场样品中检测出感染性ASFV(Rai等人,2020)。MA-104细胞的倍增时间为72小时,重要的是,不需要任何特殊培养基来培养这些细胞或在这些细胞中进行病毒培养。MA-104细胞可大批量容易冻结,从而允许在疾病爆发所需数量的细胞的及时扩展时需要大量的样本的小号要被处理,以确认样本是否只包含非洲猪瘟病毒DNA或感染性病毒ASFV。因此,MA-104细胞可以绕过产生猪巨噬细胞的原代细胞培养物(需要新鲜的猪血,这是普通兽医诊断实验室不容易获得的),从而可以帮助快速诊断ASFV。可以使用抗ASFV特异性抗体通过HA或IPA染色轻松检测MA-104细胞中的感染性ASFV 。IPA染色可以进一步检测在CD2中具有突变且无法形成HA的ASFV野外分离株。IPA染色将用于PCR呈阳性但未观察到HA来确定样品中是否存在活感染性病毒的情况下(Borca等,2018 )。通过IPA对ASFV感染的细胞进行清晰染色优于使用猪巨噬细胞作为底物,因为猪巨噬细胞具有固有的过氧化物酶活性,导致未感染细胞的染色背景高,从而导致假阳性反应(Rai等人,2020) 。


笔记


解冻MA-104细胞时,切勿使细胞瓶无人看管,因为在37°C下,细胞解冻会在1-2分钟内完成,并且在37°C下长时间暴露会导致冷冻保护剂毒性。当仍有少量冰晶残留时,从水浴中取出小瓶。
解冻后立即对细胞特别敏感。应将它们播种到预热的培养基中。
应避免低接种密度,以启动从液氮存储或供应商提供的小瓶中取出的细胞。
等待细胞分离时,请勿通过敲打或摇动烧瓶来搅动细胞,以免细胞结块。地方细胞是难以在37到分离延长的时间℃,以促进分散。
不要将细胞在-70°C下保存2天以上。它将降低细胞活力。
冷冻细胞时,请勿在室温下长时间放置于Recovery冷冻培养基中,因为培养基中的冷冻保护剂会对细胞有毒。
该协议应在动物生物安全3级实验室中进行,因为ASFV是一种选择剂,只能将其指定为动物生物安全3级实验室进行处理。 
容器的外部需要通过用10%漂白剂浸泡的毛巾擦拭来进行表面消毒。
要在不同的工作区域和存储区之间转移包含病毒的小瓶,培养皿或存储箱,请始终使用空气密封的辅助容器。
仅在生物安全柜中打开辅助容器。在从生物安全柜中取出表面之前,请同时污染样品瓶/板/盒和辅助容器。
将所有固体和液体废物容纳在经过批准的Biohazard容器中。以适当的周期进行高压灭菌后,处置实验中使用的所有材料。


菜谱


MA-104媒体
500毫升DMEM


50毫升FBS


5毫升抗生素-抗真菌药


阻塞缓冲
500毫升DMEM


10毫升(2%)马血清


定影液(丙酮和甲醇的1:1溶液)
50毫升丙酮


50毫升甲醇


0.1%牛血清白蛋白
将0.5 g BSA(4°C)添加到500 ml 1 × DPBS瓶中


猪红细胞的25%v / v悬浮液
加入9毫升红细胞沉淀和27毫升1 × DPBS,使25%的血液在1 × DPBS中


储存在4 °C标签上的红细胞的百分数和日期


70%乙醇
70毫升100%乙醇


30毫升dH 2 O


致谢


通过与美国国土安全部科学技术局的跨机构协议资助的Ma-104细胞的鉴定,编号为:70RSAT18KPM0000138。


通过美国能源部(DOE)与美国能源部(DOE)之间的跨机构协议,任命了橡树岭科学与教育学院(ORISE)管理的梅花岛动物疾病中心(PIADC)研究参与计划,这在一定程度上支持了这项研究。美国农业部(USDA)。ORISE由ORAU管理,DOE合同编号为DE-SC0014664。


利益争夺


作者Ayushi Rai,Manuel Borca和Douglas Gladue已申请使用MA-104细胞作为ASFV检测底物的专利。


参考


Borca,MV,Holinka,LG,Berggren,KA和Gladue,DP(2018)。CRISPR-Cas9,一种有效提高重组非洲猪瘟病毒发展的工具。科学代表8:3154。
Rai,A.,Pruitt,S.,Ramirez-Medina,E.,Vuono,EA,Silva,E.,Velazquez-Salinas,L.,Carrillo,C.,Borca,MV和Gladue,DP(2020)。连续稳定和可商购的细胞系的鉴定,用于鉴定临床样品中的非洲传染性猪瘟病毒。病毒12(8)。doi:10.3390 / v12080820。
里德(Reed,LJ)和穆安奇(Muench,H)(1938)一种估算百分之五十端点的简单方法。美国卫生学杂志27 (3):493-497。
Tulman,ER,Delhon,GA,Ku,BK和Rock,DL(2009)。非洲猪瘟病毒。于:鲜为人知的大型dsDNA病毒。施普林格出版社柏林海德堡卷。328页,第43-87页。
Whitaker,AM和Hayward,CJ(1985)。三种猴肾细胞系的表征。Dev Biol Stand 60:125-131。
吴,P 。,Lowe ,A 。d 。,罗德里格斯(Y. Rodriguez )。ÿ 。,Murgia ,M 。V 。,多德(Dodd),K 。一。,罗兰,R 。[R 。和贾,W. (2020 )。非洲猪瘟病毒磷蛋白P30的抗原区域。跨境紧急通讯地址:10.1111 / tbed.13533。
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引用:Rai, A., Pruitt, S., Ramirez-Medina, E., Vuono, E. A., Silva, E., Velazquez-Salinas, L., Carrillo, C., Borca, M. V. and Gladue, D. P. (2021). Detection and Quantification of African Swine Fever Virus in MA-104 Cells. Bio-protocol 11(6): e3955. DOI: 10.21769/BioProtoc.3955.
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