Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER)

Materials and reagents

Biological materials

1. Bovine MDBK cells (ATCC, catalog number: CCL-22)

2. Lenti-X 293T cells (TAKARA BIO, catalog number: 632180)

3. Serum from cattle persistently infected with BVDV (BVDV-1b strain Shihoro/B_6, Hirose et al. [17])

Reagents

1. BVDV antibody-free FBS (Japan Bio Serum, catalog number: 621-00535)

2. DMEM (4.5 g/L glucose) with L-glutamine, without sodium pyruvate, liquid (Nacalai Tesque, catalog number: 08459-64)

3. Penicillin-streptomycin mixed solution (Nacalai Tesque, catalog number: 26253-84)

4. PrimeSTAR^® GXL DNA polymerase (TAKARA BIO, catalog number: 12292-04)

5. KOD One^® PCR master mix (Toyobo, catalog number: 18538-01)

6. 1 kb DNA ladder (TAKARA BIO, catalog number: 3412A)

7. SuperScript^TM IV VILO^TM master mix (Thermo Fisher Scientific, catalog number: 11756050)

8. Opti-MEM (Thermo Fisher Scientific, catalog number: 07088-54)

9. TransIT^®-LT1 transfection reagent (Mirus, catalog number: MIR2304)

10. Acetone (Nacalai Tesque, catalog number: 00309-35)

11. D-PBS (-) without Ca and Mg, liquid (Nacalai Tesque, catalog number: 14249-24)

12. Anti-pestiviral NS3 antibody (prepared in-house, see Kameyama et al. [18])

13. Goat anti-mouse IgG Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, catalog number: A32723)

14. PureLink^TM RNA Mini kit (Thermo Fisher Scientific, catalog number: 12183018A)

15. FastGene Gel/PCR Extraction kit (NIPPON Genetics, catalog number: FG-91202)

16. Primer sets (Fasmac; Table 1)

Table 1. Primer sets used for CPER of BVDV-1b

Solutions

1. Maintenance medium (see Recipes)

2. 2% FBS medium (see Recipes)

Recipes

1. Maintenance medium

2. 2% FBS medium

Laboratory supplies

1. Collagen-coated microplate 6-well with lid, collagen type I (IWAKI, catalog number: 4810-010N)

2. Collagen-coated microplate 24-well with lid, collagen type I (IWAKI, catalog number: 4820-010)

3. Flat bottom micro tube 1.5 mL (BIO-BIK, catalog number: CF-0150)

4. 0.2 mL 8-strip PCR tube caps with dome top, natural (WATSON BIO LAB, catalog number:137-432C)

Equipment

1. CO₂ incubator (PHCbi, model: MCO-170AICUVD)

2. NanoDrop Lite UV-Vis spectrophotometer ND-LITE (Thermo Fisher Scientific, catalog number: 32-1001)

3. Fluorescence microscope (KEYENCE, model: BZ-X810)

Software and datasets

1. Prism v. 10.2.2 (GraphPad, 7/31/2024)

Procedure

A. Preparation of viral RNA

1. Follow the manufacturer’s protocol to purify total RNA from the serum of cattle persistently infected with BVDV using the PureLink^TM RNA Mini kit (see General note 1). Use 100 μL of serum stored at -80 °C.

B. Generation of viral cDNA fragments

1. Reverse transcribe total RNA into cDNA using the SuperScript^TM IV VILO^TM master mix. Mix 16 μL of total RNA sample with 4 μL of SuperScript^TM IV VILO^TM master mix and heat at 25 °C for 10 min, 50 °C for 10 min, and 85 °C for 5 min.

2. Amplify a total of six viral gene fragments covering the entire viral genome and a linker fragment encoding a Pol-I terminator, spacer, and a Pol-I promoter by KOD One^® PCR master mix with gene-specific primer sets (primer sets in Table 1; the recipe of this reaction mixture is in Table 2; thermocycling conditions for the PCR reaction are in Table 3) [19].

Table 2. Reaction mixture

Table 3. Thermocycling conditions for PCR

3. Assess the size of the fragments by gel electrophoresis using a commercial 1 kb DNA ladder as a reference.

4. Follow the manufacturer’s protocol to purify the PCR fragments using the FastGene Gel/PCR Extraction kit.

5. Measure DNA concentration using NanoDrop.

6. Because CPER is based on assembly PCR, the correct joining of fragments via the overlapping regions is critical for making the complete circular cDNA assembly. Thus, use PCR assembly to connect neighboring fragments by PrimeSTAR^® GXL DNA polymerase (see Troubleshooting 1).

C. Circular polymerase extension reaction (CPER)

1. Mix each gene fragment of BVDV cDNA and the linker fragment at 0.1 pmol per fragment.

2. Assemble all fragments of equal molar amounts using PrimeSTAR^® GXL DNA polymerase (the recipe of this reaction mixture is in Table 4; thermocycling conditions for the CPER are detailed in Table 5).

Table 4. CPER mixture

Table 5. Thermocycling conditions for the CPER

D. Transfection

1. Prepare cells.

a. One day before transfection, co-culture MDBK and 293T cells in a 2:1 ratio (see General note 2) in maintenance medium (see Recipes) in a collagen-coated 6-well microplate. Seed cells by plating 7 × 10⁵ cells per well.

b. Upon 80% confluence, replace maintenance medium with 2% FBS medium (see Recipes).

2. Immediately after the CPER, add 25 μL of CPER product to 200 μL of Opti-MEM warmed to 37 °C and mix by pipetting.

3. Add 12 μL of TransIT-LT1^® transfection reagent and mix by slow pipetting.

4. Centrifuge at 5,000× g for several seconds and incubate for 10 min.

5. Add the whole mixture to the cells and culture in a CO₂ incubator at 37 °C in a humidified atmosphere with 5% CO₂.

6. After 4 days of transfection, collect 100 μL of culture supernatant in a flat bottom microtube and store at -80 °C.

E. Detection of viruses by immunofluorescence staining

1. Inoculate newly seeded MDBK cells at 80% confluence in a collagen-coated 24-well microplate with 100 μL of culture supernatant.

2. After 5 days of infection, wash with D-PBS and fix cells with acetone for 10 min at 4 °C. After washing with D-PBS three times, the plate can be stored at 4 °C.

3. Incubate with the indicated anti-pestiviral NS3 antibody (1:1,000 dilution) in D-PBS for 1 h at 37 °C in a humidified atmosphere.

4. After washing with D-PBS three times, incubate cells in the dark with goat anti-mouse IgG Alexa Fluor 488-conjugated (1:1,000 dilution) secondary antibody in D-PBS for 1 h at room temperature.

5. After washing with D-PBS four times, observe under the fluorescence microscope using the BX-Z filter GFP (Figure 1).

Figure 1. Image of immunofluorescence staining showing bovine viral diarrhea virus (BVDV)-positive cells. Antiviral NS3 immunofluorescence image of infected MDBK cells (A) and non-infectious condition (B). Scale bar, 100 μm.

Data analysis

Significant differences in viral titers between different ratios of co-cultures (Figure 2) were analyzed by student’s t-test using GraphPad Prism software.

Figure 2. Optimization of the co-culture of two cell lines for the production of recombinant bovine viral diarrhea virus (BVDV). The two cell lines employed in this study were bovine MDBK, which is highly permissive for BVDV infection, and 293T, which shows high transfection efficiency and protein expression. The infectious titers of the prepared working virus were measured by 50% tissue culture infectious dose (TCID₅₀) for BVDV. The value of TCID50/mL was calculated using the Reed–Muench method [20]. The ratios of cell numbers and infectious titers are shown as a bar graph. Asterisks indicate significant differences (*p < 0.05) between a pair.

Validation of protocol

This protocol, or parts of it, have been applied and validated in the following research article:

• Tamura et al. [13]. A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation (see Figure 2B, H, I).

General notes and troubleshooting

General notes

1. Viral RNA purified from infected cells or culture supernatants can also be used.

2. Because MDBK cells, which are difficult to transfect, are the primary choice for BVDV research, we employed a co-culture strategy with 293T cells to deliver sufficient viral cDNA into MDBK cells. The 293T monocultures were unable to produce BVDV, but recombinant BVDV was produced in transfected co-cultures with a ratio of 2:1 (MDBK:293T), which was the ratio demonstrating the greatest efficacy (Figure 2).

3. The protocol can also generate HCV, hepatitis A virus (HAV), and encephalomyocarditis virus (EMCV) using primer sets and cells appropriate for each virus.

Troubleshooting

Problem 1: Transfection is unsuccessful.

Possible cause: Neighboring fragments are not joined.

Solution: Change the overlapping region or length between neighboring fragments, use PCR assembly to connect neighboring fragments, and assess the size of joined fragments by gel electrophoresis.

Problem 2: PCR for amplifying a total of six viral gene fragments is unsuccessful.

Possible cause: The cDNA amount of the viral genome is insufficient for PCR.

Solution: Purify viral RNA from high-viral serum, infected cells, or culture supernatants.

Acknowledgments

We thank H. Kubo, M. Tetsuka, and S. Shimamura for their secretarial work and H. Maruyama, M. Hanazaki, T. Matsuoka, R. Kudo, M. Hommura, A. Imajoh, and K. Oyama for their technical assistance. We thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

This work was supported in part by AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP22fk0108617 to Takasuke Fukuhara); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005 to Takasuke Fukuhara); AMED CREST (JP22gm1610008 to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736 to Takasuke Fukuhara); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041 to Takasuke Fukuhara); JST SPRING (JPMJSP2119 to Hirotaka Yamamoto); Takeda Science Foundation (to Takasuke Fukuhara); Hokkaido University Support Program for Frontier Research (to Takasuke Fukuhara); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Grants-in-Aid for R&D of Young Scientist from Northern Advancement Center for Science & Technology (NOASTEC) of Hokkaido (T-1-26 to Tomokazu Tamura); and Kuribayashi Scholarship Academic Foundation (to Tomokazu Tamura).

This protocol was developed and used in [13].

Competing interests

The authors declare no competing interests.

References

1. Venter, J. C., Glass, J. I., Hutchison, C. A. and Vashee, S. (2022). Synthetic chromosomes, genomes, viruses, and cells. Cell. 185(15): 2708–2724. https://doi.org/10.1016/j.cell.2022.06.046
2. Almazán, F., DeDiego, M. L., Galán, C., Escors, D., Álvarez, E., Ortego, J., Sola, I., Zuñiga, S., Alonso, S., Moreno, J. L., et al. (2006). Construction of a Severe Acute Respiratory Syndrome Coronavirus Infectious cDNA Clone and a Replicon To Study Coronavirus RNA Synthesis. J Virol. 80(21): 10900–10906. https://doi.org/10.1128/jvi.00385-06
3. Yount, B., Curtis, K. M., Fritz, E. A., Hensley, L. E., Jahrling, P. B., Prentice, E., Denison, M. R., Geisbert, T. W. and Baric, R. S. (2003). Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci USA. 100(22): 12995–13000. https://doi.org/10.1073/pnas.1735582100
4. Scobey, T., Yount, B. L., Sims, A. C., Donaldson, E. F., Agnihothram, S. S., Menachery, V. D., Graham, R. L., Swanstrom, J., Bove, P. F., Kim, J. D., et al. (2013). Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. Proc Natl Acad Sci USA. 110(40): 16157–16162. https://doi.org/10.1073/pnas.1311542110
5. Terada, Y., Kuroda, Y., Morikawa, S., Matsuura, Y., Maeda, K. and Kamitani, W. (2019). Establishment of a Virulent Full-Length cDNA Clone for Type I Feline Coronavirus Strain C3663. J Virol. 93(21): e01208–19. https://doi.org/10.1128/jvi.01208-19
6. Edmonds, J., van Grinsven, E., Prow, N., Bosco-Lauth, A., Brault, A. C., Bowen, R. A., Hall, R. A. and Khromykh, A. A. (2013). A Novel Bacterium-Free Method for Generation of Flavivirus Infectious DNA by Circular Polymerase Extension Reaction Allows Accurate Recapitulation of Viral Heterogeneity. J Virol. 87(4): 2367–2372. https://doi.org/10.1128/jvi.03162-12
7. Tamura, T., Fukuhara, T., Uchida, T., Ono, C., Mori, H., Sato, A., Fauzyah, Y., Okamoto, T., Kurosu, T., Setoh, Y. X., et al. (2018). Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag. J Virol. 92(2): e01582–17. https://doi.org/10.1128/jvi.01582-17
8. Motozono, C., Toyoda, M., Zahradnik, J., Saito, A., Nasser, H., Tan, T. S., Ngare, I., Kimura, I., Uriu, K., Kosugi, Y., et al. (2021). SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity. Cell Host Microbe. 29(7): 1124–1136.e11. https://doi.org/10.1016/j.chom.2021.06.006
9. Saito, A., Irie, T., Suzuki, R., Maemura, T., Nasser, H., Uriu, K., Kosugi, Y., Shirakawa, K., Sadamasu, K., Kimura, I., et al. (2022). Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation. Nature. 602(7896): 300–306. https://doi.org/10.1038/s41586-021-04266-9
10. Tamura, T., Ito, J., Uriu, K., Zahradnik, J., Kida, I., Anraku, Y., Nasser, H., Shofa, M., Oda, Y., Lytras, S., et al. (2023). Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants. Nat Commun. 14(1): 2800. https://doi.org/10.1038/s41467-023-38435-3
11. Tamura, T., Irie, T., Deguchi, S., Yajima, H., Tsuda, M., Nasser, H., Mizuma, K., Plianchaisuk, A., Suzuki, S., Uriu, K., et al. (2024). Virological characteristics of the SARS-CoV-2 Omicron XBB.1.5 variant. Nat Commun. 15(1): 1176. https://doi.org/10.1038/s41467-024-45274-3
12. Tamura, T., Mizuma, K., Nasser, H., Deguchi, S., Padilla-Blanco, M., Oda, Y., Uriu, K., Tolentino, J. E., Tsujino, S., Suzuki, R., et al. (2024). Virological characteristics of the SARS-CoV-2 BA.2.86 variant. Cell Host Microbe. 32(2): 170–180.e12. https://doi.org/10.1016/j.chom.2024.01.001
13. Tamura, T., Yamamoto, H., Ogino, S., Morioka, Y., Tsujino, S., Suzuki, R., Hiono, T., Suzuki, S., Isoda, N., Sakoda, Y., et al. (2024). A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation. J Virol. 98(3): e01638–23. https://doi.org/10.1128/jvi.01638-23
14. Ruggli, N. and Rice, C. M. (1999). Functional cDNA Clones of The Flaviviridae: Strategies and Applications. Adv Virus Res. 53: 183–207. https://doi.org/10.1016/s0065-3527(08)60348-6
15. Nagai, M., Sakoda, Y., Mori, M., Hayashi, M., Kida, H. and Akashi, H. (2003). Insertion of cellular sequence and RNA recombination in the structural protein coding region of cytopathogenic bovine viral diarrhoea virus. J Gen Virol. 84(2): 447–452. https://doi.org/10.1099/vir.0.18773-0
16. Osorio, J. S. and Bionaz, M. (2017). Plasmid transfection in bovine cells: Optimization using a realtime monitoring of green fluorescent protein and effect on gene reporter assay. Gene. 626: 200–208. https://doi.org/10.1016/j.gene.2017.05.025
17. Hirose, S., Notsu, K., Ito, S., Sakoda, Y. and Isoda, N. (2021). Transmission Dynamics of Bovine Viral Diarrhea Virus in Hokkaido, Japan by Phylogenetic and Epidemiological Network Approaches. Pathogens. 10(8): 922. https://doi.org/10.3390/pathogens10080922
18. Kameyama, K., Sakoda, Y., Tamai, K., Igarashi, H., Tajima, M., Mochizuki, T., Namba, Y. and Kida, H. (2006). Development of an immunochromatographic test kit for rapid detection of bovine viral diarrhea virus antigen. J Virol Methods. 138: 140–146. https://doi.org/10.1016/j.jviromet.2006.08.005
19. Masaki, T., Suzuki, R., Saeed, M., Mori, K. I., Matsuda, M., Aizaki, H., Ishii, K., Maki, N., Miyamura, T., Matsuura, Y., et al. (2010). Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription. J Virol. 84(11): 5824–5835. https://doi.org/10.1128/jvi.02397-09
20. Reed, L. and Muench, H. (1938). A Simple Method Of Estimating Fifty Per Cent Endpoints12. Am J Epidemiol. 27(3): 93–497. https://doi.org/10.1093/oxfordjournals.aje.a118408