Production, quantification, and infection of Amazonian Phlebovirus (Bunyaviridae)

Materials and Reagents

1. Cell culture vessels, plates, and tubes

   1. 25-cm² cell culture flask (Corning, catalog number: CLS430639)

   2. 100-mm plates (SARSTEDT, catalog number: 82.1472.001)

   3. 6-well cell culture plates (Corning, catalog number: 83.3920)

   4. 24-well cell culture plates (Corning, catalog number: 83.3922)

   5. 15-ml and 50-ml Falcon tubes (BD Falcon)

   6. Cell counting slides with grids

   7. 1.5-ml Eppendorf tubes

   8. Eco Nitrile Gloves (SuperMax)

   9. Filter tips, low-retention: 1,000 µl, 200 µl, 20 µl, 10 µl (Axygen, catalog numbers: 31000LRS, 3200LRS, 320LRS, 310LRS, respectively)

   10. 5-ml serological pipettes (SARSTEDT, catalog number: 86.1253.001)

   11. 10-ml serological pipettes (SARSTEDT, catalog number: 86.1254.001)

   12. 0.22-µm cell culture medium filtration unit (Corning, catalog number: CLS430513-12EA)

       
       

2. Cells

   BHK-21 cells (baby hamster kidney cells, ATCC: CCL-10)

   Cells are maintained in 25-cm² cell culture flasks with complete DMEM (DMEM – high glucose and supplemented with 10% heat-inactivated FBS: see Recipe 1) at 37°C in a humidified atmosphere containing 5% CO₂. After reaching confluence, cells are subcultured to maintain cell viability. For this, remove the medium and rinse the cells with 3 ml warm PBS without calcium and magnesium. Dissociate the cells using 1 ml trypsin-EDTA solution for 1 min. Add 4 ml complete DMEM, transfer to a new 15-ml conical tube, and centrifuge the cells for 5 min at 300 × g. Resuspend the cell pellet in fresh complete medium and split 1:20 in new flasks.

   
   

3. Phlebovirus

   The Icoaraci Phlebovirus (BeAN 24262-ICOV) was obtained from Dr. Pedro Vasconcelos (Instituto Evandro Chagas/SVS/MS).

   The virus was isolated from the liver, spleen, kidney, and heart of Nectomys squamipes in Belém, Pará, Brazil, and subsequently inoculated intracerebrally in 2-day-old Swiss mice. The samples were lyophilized and stored at -80°C. Before use, reconstitute the samples in 0.5 ml Puck’s medium.

   
   

4. Cell culture reagents

   1. 0.25% trypsin-EDTA solution (Sigma-Aldrich, catalog number: T4049), stored at -20°C

   2. Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose and L-glutamine (Lonza, catalog number: 12-604Q)

   3. 100× penicillin-streptomycin solution (HyClone-GE, catalog number: SV30010)

   4. Puck’s medium (PAN Biotech, catalog number: P04-51100)

   5. Phosphate-buffered saline (PBS) (Lonza, catalog number: 17-516Q)

   6. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270106)

   7. UltraPure^TM DNase/RNase-free distilled water (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10977-035)

   8. Methylcellulose (Sigma-Aldrich, catalog number: M7027)

   9. 100-bp DNA ladder, 500 µg/ml (Promega, catalog number: G201A)

   10. 6× gel loading dye (Promega, catalog number: G190A)

   11. Standard agarose for routine gel electrophoresis (UniScience, catalog number: AGR-LE-100)

   12. Ethidium bromide (Sigma-Aldrich, catalog number: E7637)

   13. Tris-base (Promega, catalog number: H5131)

   14. Glacial acetic acid (Sigma-Aldrich, catalog number: A6283)

   15. UltraPure^TM 0.5 M EDTA (Ethylenediaminetetraacetic acid, disodium salt dihydrate), pH 8.0 (Thermo Fisher Scientific, catalog number: 15575020)

   16. Crystal violet (Sigma-Aldrich, catalog number: C0775)

   17. Sodium thioglycolate (Sigma-Aldrich, catalog number: T0632), stored at room temperature

   18. Direct-zol^TM RNA MiniPrep Plus kit (Zymo Research, catalog number: R2070)

   19. ImProm-II^TM Reverse Transcription System (Promega, catalog number: A3800)

   20. Random primers (Promega, catalog number: C1181)

   21. GoTaq^® DNA polymerase (Promega, catalog number: M3001)

   22. Complete DMEM (see Recipes)

   23. Semi-solid medium (see Recipes)

   24. Crystal violet solution (see Recipes)

   25. 10× TAE Buffer (1 L) (see Recipes)

   26. 1× TAE Buffer (1 L) (see Recipes)

   27. 1.5% agarose gel (see Recipes)

   28. 100-bp DNA ladder (50 µg/ml) (see Recipes)

   29. 3% sodium thioglycolate (see Recipes)

Equipment

1. P1000 (Gilson, catalog number: F123602)

2. P200 (Gilson, catalog number: F123601)

3. P20 (Gilson, catalog number: F123600)

4. P2 (Gilson, catalog number: F144801)

5. Lab refrigerator set at 4°C (Electrolux, model: IF55)

6. Lab freezer set at -20°C (Prosdocimo, model: F21)

7. Lab freezer set at -80°C (Panasonic, model: MDF-U56VC-PA)

8. Incubator at 37°C with 5% CO₂ (Sanyo inCU-Safe, model: MCO-17AC)

9. Laminar flow hood (Esco Class II BSC- AirStream)

10. Centrifuge 5418 (Eppendorf) at room temperature; set here at 24°C

11. Centrifuge (ThermoFisher Scientific, model: D-37520)

12. NanoDrop^TM ND-1000 Spectrophotometer (ThermoFisher Scientific)

13. Balance (BEL Analytical Equipment, model: S622)

14. Veriti 96-Well thermal cycler (Applied Biosystems PCR instruments, model: 9902)

15. Mini-gel migration system (Loccus Biotecnology, model: LCH-7X8)

16. Electrophoresis power supply (GIBCO BRL, Life Technologies, 250) and power cables

17. U.V. transilluminator (Fisher Scientific, model: 88A)

Procedure

Figure 1 illustrates the steps for virus production, quantitation, and infection. A detailed description of the procedures is found in the following items.

Figure 1. Different steps for the production, quantitation, and infection of Icoaraci Phlebovirus. Figure created in the Mind the Graph platform.

1. Production of the Icoaraci Phlebovirus

   1. Plate 4 × 10⁶ BHK-21 cells in 100-mm plates (7 ml/plate). In order to obtain a good virus concentration, seed approximately 30 plates. For this step, use BHK cells that have undergone no more than five passages.

   2. After exactly 24 h, wash the cells once with 2 ml warm PBS and proceed with viral absorption (next step).

   3. Dilute the virus in 1 ml DMEM without FBS [multiplicity of infection (MOI) 0.01] and absorb the inoculum for 2 h at room temperature. The inoculum volume is not sufficient to cover the whole plate surface, so please move the plate carefully every 20 min to allow the inoculum to move across the plate.

   4. Remove the inoculum, add 7 ml DMEM (supplemented with 10% FBS) (Recipe 1), and incubate at 37°C for 3 days.

   5. Collect the supernatant and centrifuge at 18,000 × g for 1 h at 4°C to concentrate the virus.

   6. Discard the supernatant and resuspend the pellet in 300 µl PBS. Split the volume between 3 cryovials and store at -80°C until the titration.

      
      

2. Quantitation of the Icoaraci Phlebovirus

   1. Viral titers are determined using a plaque assay. For this, plate 1.5 × 10⁶ BHK-21 cells/well in a 6-well plate. Cells should be used up to a maximum of passage 3.

   2. After 24 h, infect the cells (~80% confluence) with 200 µl 10-fold serial dilution of ICOV stock, as described in the previous section, and incubate for 2 h at room temperature. Again, the inoculum volume is not sufficient to cover the well surface, so please move the plate carefully every 20 min to allow the inoculum to move across the plate.

   3. Using a pipette, remove the inoculum completely and cover the monolayer with 2 ml semi-solid medium composed of complete DMEM plus 1% methylcellulose (Recipe 2). In this step, it is very important to ensure complete removal of the inoculum in order for the semi-solid medium to be evenly distributed throughout the cell monolayer.

   4. Three days post-infection, fix and stain the cells with 2 ml 0.1% crystal violet solution containing 10% formaldehyde (Recipe 3) under slight agitation for 12 h at room temperature.

   5. Wash the wells delicately with running water to remove the fixative/dye and allow the plate to dry upside down at room temperature for 24 h. Viral plaques can be counted on the white-light transilluminator, and titers are expressed as plaque-forming units (PFU)/ml (Figure 2).

      
      

      
      Figure 2. Quantitation of Icoaraci Phlebovirus. The supernatant from 48 h after infection was collected, diluted to three different concentrations (10^-2, 10^-3, and 10^-4), and titrated in a BHK-21 monolayer as described above. After 3 days, the cells were fixed and stained with crystal violet.

      
      

3. Obtaining and cultivating peritoneal macrophages

   Mouse colonies are maintained at the Department of Immunology, UFRJ in accordance with the rules established by the National Council for the Control of Animal Experimentation. The animal experimentation protocols used in this project were approved by the Animal Use Ethics Committee (Protocol No. 046/20).

   1. Peritoneal macrophages from 8-week-old C57 Black-6 (C57BL/6) mice are elicited with 2 ml 3% sodium thioglycolate (intraperitoneal injection, Recipe 8) for 4 days, and cells are obtained after euthanization by injecting 8 ml DMEM into the peritoneal cavity. The mice were inclined at a 45° angle with the head down, positioning the intestines cranially, away from the administration area. The needle was then inserted in the lower right quadrant of the abdomen at a 30-45° angle for either the inoculation of thioglycolate or to obtain macrophages.

   2. Centrifuge the cells at 400 × g for 10 min at 4°C.

   3. Resuspend the cells in 1 ml DMEM and count using a hemocytometer under the microscope (10× dilution in the Neubauer chamber with DMEM).

   4. Plate the macrophages on glass coverslips in 24-well plates (2 × 10⁵ cells per well) or 6-well plates (4 × 10⁶ cells per well).

   5. After a 1-h adherence at 37°C, wash the cells with PBS and add complete DMEM. The macrophages are kept for 1 day at 37°C in a 5% CO₂ atmosphere and subsequently subjected to infection.

      
      

4. Infection

   1. For peritoneal macrophage ICOV infections, adsorption is performed for 1 h at 37°C using an MOI of 1.

   2. Dilute the virus in DMEM without FBS and use 100 µl for 24-well plates and 1 ml for 6-well plates.

   3. Remove the inoculum with a pipette and add 1 ml (24-well plate) or 2 ml (6-well plate) complete DMEM.

   4. The subsequent treatment time, conditions, or other additional infections for which the experimental model is proposed may vary. For example, in Leishmania infections, we allow the parasite to interact for 4 h after viral adsorption and then lyse the cells for analysis of gene expression by qPCR.

      
      

5. Analysis of murine macrophage infection by semiquantitative PCR for viral detection

   To detect and confirm the viral infection and replication in peritoneal macrophages, a semiquantitative PCR must be performed.

   1. After 4, 24, or 48 h of infection, isolate and purify the total RNA using the Direct-zol^TM RNA MiniPrep Plus kit according to the manufacturer's instructions.

   2. Quantitate the RNA using the NanoDrop. The expected concentration should be 100-200 ng/µl, and the 260/280 ratio should be 1.8-2.0, ensuring good RNA quality.

   3. First-strand cDNA synthesis is performed in a reaction containing Improm-II Reverse Transcriptase, a mix of dNTPs, and random primers, as described in the manufacturer's instructions.

   4. RT-PCR is performed using 10 µM primers for the N protein gene of ICOV (sense 5’-AGGTGAGGCTGTAAATCTTG-3’ and antisense 5’-TCACATCATCCTTCCAAGTG-3’), 2.5 U GoTaq DNA polymerase, 1.5 mM MgCl₂, 10 mM dNTPs, 2 µl cDNA, and 1× appropriate buffer in a final volume of 50 µl. The PCR cycle is as follows: 95°C for 1 min, 48°C for 1 min, and 72°C for 1 min in 40 amplification cycles.

   5. PCR products are separated on a 1.2% agarose gel containing 0.5 µg/ml ethidium bromide in 1× TAE buffer (Recipe 5) at 80-100 V and photographed in a UV transilluminator. The product has 100 bp (Figure 3).

      
      

      
      Figure 3. Peritoneal macrophages from wild-type C57BL/6 mice were infected with stationary-phase promastigotes of L. (L.) amazonensis at a ratio of 5 parasites/cell, Icoaraci Phlebovirus (ICOV), or co-infected with both for 4 h, 24 h, or 48 h. Total RNA was extracted and analyzed by semiquantitative PCR for the Icoaraci N protein.

Recipes

1. Complete DMEM

   To make 500 ml complete DMEM:

   445 ml 1× DMEM

   50 ml 10% FBS (heat-Inactivated)

   5 ml 1× penicillin-streptomycin (100×)

2. Semi-solid medium

   Complete DMEM plus 1% methylcellulose

   1. Add 1 g methylcellulose to 100 ml distilled water in an autoclavable flask.

   2. Before autoclaving, add a stir bar in order to mix the solution and maintain sterility. For complete solubilization, store the solution overnight at 4°C and then leave the flask in a magnetic stirrer.

      Note: If a lumpy solution forms, cool the temperature of the solution and mix again. The procedure should be repeated until the solution is homogeneous.

   3. Add cold methylcellulose to the cold complete DMEM (v/v) and thoroughly mix to ensure a homogeneous mixture. Prior to use, wait until the bubbles dissipate and the temperature raises to room temperature.

3. Crystal violet solution

   0.1% crystal violet in PBS solution

   10% formaldehyde

4. 10× TAE Buffer (1 L)

   1. Weigh 48.5 g Tris-base, 11.4 ml glacial acetic acid and 20 ml 0.5 M EDTA pH 8.0

   2. Add distilled water up to 1 L

   3. Store at room temperature

5. 1× TAE Buffer (1 L)

   1. Add 100 ml 10× TAE (Recipe 5) to 900 ml distilled water

   2. Store at room temperature

6. 1.5% agarose gel

   1. Weigh agarose powder (1.5 g) in an autoclaved glass bottle

   2. Add 100 ml 1× TAE

   3. Heat in a microwave until the agarose is homogeneously dissolved

   4. Cool down at room temperature for a few minutes

7. 100-bp DNA Ladder (50 µg/ml)

   1. For 240 µl DNA Ladder in a 1.5-ml tube on ice, add 176 µl UltraPure^TM DNase/RNase-free distilled water

   2. Add 40 µl 6× gel loading dye and 24 μl 100-bp DNA ladder at 500 µg/ml

   3. Store at 4°C

8. 3% sodium thioglycolate

   1. Weigh sodium thioglycolate powder (3.0 g) in an autoclaved glass bottle

   2. Add water up to 100 ml

   3. Filter the solution with a 0.22-µm cell culture medium filtration unit

   4. Store at room temperature

Acknowledgments

We thank Professor Pedro Fernando da Costa Vasconcellos for providing the Phleboviruses, which are fundamental for carrying out this work. We thank Professor Clarissa Damaso for the support in the virology methodologies. We also thank all members of the Molecular Parasitology laboratory for helpful discussion. This research was supported by the National Council for Scientific and Technological Development (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments were performed in accordance with protocols approved by the National Council for Animal Experiment Control and Animal Use Ethics Committee (Protocol No. 046/20 – ID: 01200.001568/2013-87 Validity: 08/30/2022).

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