Subgenomic RNA Detection in SARS-CoV-2 Assessing Replication and Inactivation Through Serial Passages, RT-qPCR, and Electron Microscopy
Abstract
1. Introduction
2. Results
2.1. Reducing sgRNA Levels in Inactivated SARS-CoV-2 Samples
2.2. SARS-CoV-2 Replication Kinetics
3. Discussion
4. Materials and Methods
4.1. Cell Cultivation
4.2. Viral Production and Inactivation
4.3. Blind Serial Passage
4.4. Replication Kinetics
4.5. Transmission Electron Microscopy (TEM)
4.6. Viral RNA Extraction
4.7. Reverse Transcription Followed by SARS-CoV-2 Genomic RNA Quantitative Polymerase Chain Reaction (RT-qPCR)
4.8. Reverse Transcription Followed by Quantitative Polymerase Chain Reaction (RT-qPCR) of Subgenomic RNA from the SARS-CoV-2 Envelope Region
4.9. Reverse Transcription Followed by Quantitative Polymerase Chain Reaction (RT-qPCR) of Negative-Polarity RNA from the SARS-CoV-2 Envelope Target
4.10. Standard Curve Construction
4.11. Results Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Strain | Variant | State | Municipality | GISAID ID * |
---|---|---|---|---|
Gama | P1 | AM | Manaus | EPI_ISL_1402431 |
Alfa | B1.1.7 | RJ | Rio de Janeiro | EPI_ISL_1402430 |
Zeta | P2 | AL | Maceió | EPI_ISL_792642 |
Whuan | - | RJ | Rio de Janeiro | EPI_ISL_414045 |
Ômicron | BA.1 | SC | Jaraguá do Sul | EPI_ISL_8430488 |
References
- Hasöksüz, M.; Kiliç, S.; Saraç, F. Coronaviruses and SARS-CoV-2. Turk. J. Med. Sci. 2020, 50 (Suppl. S1), 549–556. [Google Scholar] [CrossRef] [PubMed]
- V’kovski, P.; Kratzel, A.; Steiner, S.; Stalder, H.; Thiel, V. Coronavirus biology and replication: Implications for SARS-CoV-2. Nature reviews. Microbiology 2021, 19, 155–170. [Google Scholar] [CrossRef] [PubMed]
- Hu, B.; Guo, H.; Zhou, P.; Shi, Z.L. Characteristics of SARS-CoV-2 and COVID-19. Nature reviews. Microbiology 2021, 19, 141–154. [Google Scholar] [CrossRef] [PubMed]
- Brant, A.C.; Tian, W.; Majerciak, V.; Yang, W.; Zheng, Z.M. SARS-CoV-2: From its discovery to genome structure, transcription, and replication. Cell Biosci. 2021, 11, 136. [Google Scholar] [CrossRef] [PubMed]
- Long, S. SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives. Viruses 2021, 13, 1923. [Google Scholar] [CrossRef] [PubMed]
- Mina, M.J.; Parker, R.; Larremore, D.B. Rethinking Covid-19 Test Sensitivity—A Strategy for Containment. N. Engl. J. Med. 2020, 383, e120. [Google Scholar] [CrossRef] [PubMed]
- Dagotto, G.; Mercado, N.B.; Martinez, D.R.; Hou, Y.J.; Nkolola, J.P.; Carnahan, R.H.; Crowe, J.E., Jr.; Baric, R.S.; Barouch, D.H. Comparison of Subgenomic and Total RNA in SARS-CoV-2 Challenged Rhesus Macaques. J. Virol. 2021, 95, e02370-20. [Google Scholar] [CrossRef] [PubMed]
- Wölfel, R.; Corman, V.M.; Guggemos, W.; Seilmaier, M.; Zange, S.; Müller, M.A.; Niemeyer, D.; Jones, T.C.; Vollmar, P.; Rothe, C.; et al. Virological assessment of hospitalized patients with COVID-2019. Nature 2020, 581, 465–469. [Google Scholar] [CrossRef] [PubMed]
- Centers for Disease Control and Prevetion (CDC). CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel; Centers for Disease Control and Prevetion (CDC): Atlanta, GA, USA, 2020; pp. 1–48. [Google Scholar]
- Gomes, M.P.D.B.; Linhares, J.H.R.; Dos Santos, T.P.; Pereira, R.C.; Santos, R.T.; da Silva, S.A.; Souza, M.C.D.O.; da Silva, J.F.A.; Trindade, G.F.; Gomes, V.S.; et al. Inactivated and Immunogenic SARS-CoV-2 for Safe Use in Immunoassays and as an Immunization Control for Non-Clinical Trials. Viruses 2023, 15, 1486. [Google Scholar] [CrossRef]
- Dutta, D.; Naiyer, S.; Mansuri, S.; Soni, N.; Singh, V.; Bhat, K.H.; Singh, N.; Arora, G.; Mansuri, M.S. COVID-19 Diagnosis: A Comprehensive Review of the RT-qPCR Method for Detection of SARS-CoV-2. Diagnostics 2022, 12, 1503. [Google Scholar] [CrossRef] [PubMed]
- Brandolini, M.; Taddei, F.; Marino, M.M.; Grumiro, L.; Scalcione, A.; Turba, M.E.; Gentilini, F.; Fantini, M.; Zannoli, S.; Dirani, G.; et al. Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management. Viruses 2021, 13, 1022. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
- CDC Labs. Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19). Available online: https://www.cdc.gov/coronavirus/2019-ncov/lab/lab-biosafety-guidelines.html (accessed on 23 June 2023).
- de Castro Barbosa, E.; de Souza Andrade, A.; Duarte, M.M.; Faria, G.; de Melo Iani, F.C.; Ataide AC, Z.; Cunha, L.M.; Duarte, C.G.; Fialho, S.L.; Caldas, S. Influence of SARS-CoV-2 inactivation by different chemical reagents on the humoral response evaluated in a murine model. Mol. Immunol. 2022, 147, 199–208. [Google Scholar] [CrossRef] [PubMed]
- World Health Organization. Laboratory Biosafety Guidance Related to SARS-CoV-2 (COVID-19): Interim Guidance, 11 March 2024. Available online: https://www.who.int/publications/i/item/who-whe-epp-2024.3 (accessed on 12 October 2024).
- Vogels CB, F.; Brito, A.F.; Wyllie, A.L.; Fauver, J.R.; Ott, I.M.; Kalinich, C.C.; Petrone, M.E.; Casanovas-Massana, A.; Catherine Muenker, M.; Moore, A.J.; et al. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets. Nat. Microbiol. 2020, 5, 1299–1305. [Google Scholar] [CrossRef] [PubMed]
- Corman, V.M.; Landt, O.; Kaiser, M.; Molenkamp, R.; Meijer, A.; Chu, D.K.; Bleicker, T.; Brünink, S.; Schneider, J.; Schmidt, M.L.; et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. Bull. Eur. Sur Mal. Transm. Eur. Commun. Dis. Bull. 2020, 25, 2000045. [Google Scholar] [CrossRef] [PubMed]
- Alexandersen, S.; Chamings, A.; Bhatta, T.R. SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication. Nat. Commun. 2020, 11, 6059. [Google Scholar] [CrossRef] [PubMed]
- Wolff, G.; Limpens RW, A.L.; Zevenhoven-Dobbe, J.C.; Laugks, U.; Zheng, S.; de Jong AW, M.; Koning, R.I.; Agard, D.A.; Grünewald, K.; Koster, A.J.; et al. A molecular pore spans the double membrane of the coronavirus replication organelle. Science 2020, 369, 1395–1398. [Google Scholar] [CrossRef] [PubMed]
- Barth, O.M.; Silva, M.A.; Barreto-Vieira, D.F. Low impact to fixed cell processing aiming transmission electron microscopy. Mem. Inst. Oswaldo Cruz 2016, 111, 411–413. [Google Scholar] [CrossRef] [PubMed]
- Kim, J.Y.; Bae, J.Y.; Bae, S.; Cha, H.H.; Kwon, J.S.; Suh, M.H.; Lee, H.J.; Jung, J.; Kim, M.J.; Cui, C.; et al. Diagnostic usefulness of subgenomic RNA detection of viable SARS-CoV-2 in patients with COVID-19. Clin. Microbiol. Infect. 2022, 28, 101–106. [Google Scholar] [CrossRef] [PubMed]
- Oristo, S.; Lee, H.J.; Maunula, L. Performance of pre-RT-qPCR treatments to discriminate infectious human rotaviruses and noroviruses from heat-inactivated viruses: Applications of PMA/PMAxx, benzonase and RNase. J. Appl. Microbiol. 2018, 124, 1008–1016. [Google Scholar] [CrossRef]
- Mautner, L.; Hoyos, M.; Dangel, A.; Berger, C.; Ehrhardt, A.; Baiker, A. Replication kinetics and infectivity of SARS-CoV-2 variants of concern in common cell culture models. Virol. J. 2022, 19, 76. [Google Scholar] [CrossRef]
- Scutari, R.; Renica, S.; Cento, V.; Nava, A.; Sammartino, J.C.; Ferrari, A.; Pani, A.; Merli, M.; Fanti, D.; Vismara, C.; et al. Quantitative SARS-CoV-2 subgenomic RNA as a surrogate marker for viral infectivity: Comparison between culture isolation and direct sgRNA quantification. PLoS ONE 2023, 18, e0291120. [Google Scholar] [CrossRef]
- Roingeard, P.; Eymieux, S.; Burlaud-Gaillard, J.; Hourioux, C.; Patient, R.; Blanchard, E. The double-membrane vesicle (DMV): A virus-induced organelle dedicated to the replication of SARS-CoV-2 and other positive-sense single-stranded RNA viruses. Cell. Mol. Life Sci. CMLS 2022, 79, 425. [Google Scholar] [CrossRef] [PubMed]
- Koetzner, C.A.; Hurst-Hess, K.R.; Kuo, L.; Masters, P.S. Analysis of a crucial interaction between the coronavirus nucleocapsid protein and the major membrane-bound subunit of the viral replicase-transcriptase complex. Virology 2022, 567, 1–14. [Google Scholar] [CrossRef] [PubMed]
- Sabbaghi, A.; Miri, S.M.; Keshavarz, M.; Zargar, M.; Ghaemi, A. Inactivation methods for whole influenza vaccine production. Rev. Med. Virol. 2019, 29, e2074. [Google Scholar] [CrossRef] [PubMed]
- Barreto-Vieira, D.F.; da Silva MA, N.; de Almeida AL, T.; Rasinhas AD, C.; Monteiro, M.E.; Miranda, M.D.; Motta, F.C.; Siqueira, M.M.; Girard-Dias, W.; Archanjo, B.S.; et al. SARS-CoV-2: Ultrastructural Characterization of Morphogenesis in an In Vitro System. Viruses 2022, 14, 201. [Google Scholar] [CrossRef] [PubMed]
Gene | Oligonucleotide | Sense | Sequence 5′-3′ | Genome Position |
---|---|---|---|---|
Envelope (ENV) | E_Sarbeco_F | Forward | ACAGGTACGTTAATAGTTAATAGCGT | 26269–26294 |
E_Sarbeco_R | Reverse | ATATTGCAGCAGTACGCACACA | 26360–26381 | |
E_Sarbeco_P1 | Probe | FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1 | 26332–26357 | |
Subgenomic envelope (sgENV) | sgLeadSARSCoV2-F | Forward | CGATCTCTTGTAGATCTGTTCTC | 44–66 |
E_Sarbeco_R | Reverse | ATATTGCAGCAGTACGCACACA | 26360–26381 | |
E_Sarbeco_P1 | Probe | VIC-ACACTAGCCATCCTTACTGCGCTTCG-MGBNFQ | 26332–26357 | |
Nucleocapsid (N) | 2019-nCoV_N1-F | Forward | GACCCCAAAATCAGCGAAAT | 28287–20306 |
2019-nCoV_N1-R | Reverse | TCTGGTTACTGCCAGTTGAATCTG | 28335–28358 | |
2019-nCoV_N1-P | Probe | FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1 | 28309–28332 | |
2019-nCoV_N2-F | Forward | TTACAAACATTGGCCGCAAA | 29164–29183 | |
2019-nCoV_N2-R | Reverse | GCGCGACATTCCGAAGAA | 29213–29230 | |
2019-nCoV_N2-P | Probe | FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ1 | 29188–29210 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
França, T.d.S.; da Silva, J.F.A.; da Silva, G.C.N.; dos Santos, B.O.; Silva, S.A.; Linhares, J.H.R.; da Silva, M.A.N.; Barreto-Vieira, D.F.; de Paula, V.S.; de Morais, L.M.; et al. Subgenomic RNA Detection in SARS-CoV-2 Assessing Replication and Inactivation Through Serial Passages, RT-qPCR, and Electron Microscopy. Int. J. Mol. Sci. 2025, 26, 1281. https://doi.org/10.3390/ijms26031281
França TdS, da Silva JFA, da Silva GCN, dos Santos BO, Silva SA, Linhares JHR, da Silva MAN, Barreto-Vieira DF, de Paula VS, de Morais LM, et al. Subgenomic RNA Detection in SARS-CoV-2 Assessing Replication and Inactivation Through Serial Passages, RT-qPCR, and Electron Microscopy. International Journal of Molecular Sciences. 2025; 26(3):1281. https://doi.org/10.3390/ijms26031281
Chicago/Turabian StyleFrança, Talita da Silva, Juliana Fernandes Amorim da Silva, Gabriella Christine Neves da Silva, Barbara Oliveira dos Santos, Stephanie Almeida Silva, José Henrique Resende Linhares, Marcos Alexandre Nunes da Silva, Debora Ferreira Barreto-Vieira, Vanessa Salete de Paula, Liliane Monteiro de Morais, and et al. 2025. "Subgenomic RNA Detection in SARS-CoV-2 Assessing Replication and Inactivation Through Serial Passages, RT-qPCR, and Electron Microscopy" International Journal of Molecular Sciences 26, no. 3: 1281. https://doi.org/10.3390/ijms26031281
APA StyleFrança, T. d. S., da Silva, J. F. A., da Silva, G. C. N., dos Santos, B. O., Silva, S. A., Linhares, J. H. R., da Silva, M. A. N., Barreto-Vieira, D. F., de Paula, V. S., de Morais, L. M., Santos, R. T., & Trindade, G. F. (2025). Subgenomic RNA Detection in SARS-CoV-2 Assessing Replication and Inactivation Through Serial Passages, RT-qPCR, and Electron Microscopy. International Journal of Molecular Sciences, 26(3), 1281. https://doi.org/10.3390/ijms26031281