Comparison among plaque assay, tissue culture infectious dose (TCID50) and real-time RT-PCR for SARS-CoV-2 variants quantification
-
Maria Isabel Zapata-Cardona
,
Lizdany Flórez-Álvarez
,
Diana Maryory Gómez-Gallego
,
María Juliana Moncada-Díaz
,
Juan Carlos Hernandez
,
Francisco Díaz
,
María Teresa Rugeles
,
Wbeimar Aguilar-Jiménez
,
Wildeman Zapata
Abstract
Background and Objectives: SARS-CoV-2 variants of concern (VOC) and interest (VOI) pose a significant threat to public health because the rapid change in the SARS-CoV-2 genome can alter viral phenotypes such as virulence, transmissibility and the ability to evade the host response. Hence, SARS-CoV-2 quantification techniques are essential for timely diagnosis and follow-up. Besides, they are vital to understanding viral pathogenesis, antiviral evaluation, and vaccine development.
Materials and Methods: Five isolates of SARS-CoV-2: D614G strain (B.1), three VOC (Alpha, Gamma and Delta), and one VOI (Mu) were used to compare three techniques for viral quantification, plaque assay, median tissue culture infectious dose (TCID50) and real-time RT-PCR.
Results: Plaque assay showed viral titers between 0.15 ± 0.01×107 and 1.95 ± 0.09×107 PFU/mL while viral titer by TCID50 assay was between 0.71 ± 0.01×106 to 4.94 ± 0.80×106 TCID50/mL for the five SARS-CoV-2 isolates. The PFU/mL titer obtained by plaque and the calculated from TCID50 assays differed by 0.61 log10, 0.59 log10, 0.59 log10 and 0.96 log10 for Alfa, Gamma, Delta, and Mu variants (p≤0.0007), respectively. No differences were observed for the D614G strain. Real-time PCR assay exhibited titers ranging from 0.39 ± 0.001×108 to 3.38 ± 0.04×108 RNA copies/µL for all variants. The relation between PFU/mL and RNA copies/mL was 1:29800 for D614G strain, 1:11700 for Alpha, 1:8930 for Gamma, 1:12500 for Delta, and 1:2950 for Mu.
Conclusion: TCID50 assay was comparable to plaque assay for D614G but not for others SARS-CoV-2 variants. Our data demonstrated a correlation among PFU/mL and E gene RNA copies/µL, units of measure commonly used to quantify the viral load in diagnostic and research fields. The results suggest that the proportion of infectious virions in vitro changes depending on the SARS-CoV-2 variant, being Mu, the variant reaching a higher viral titer with fewer viral copies.
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25. Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, et al. Tracking changes in SARS-CoV-2 Spike: Evidence that D614G increases infectivity of the COVID-19 virus. Cell 2020;182:812-827.e19.
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27. Richmond JY, McKinney RW (2009). Biosafety in microbiological and biomedical laboratories. 5th ed. U.S. Department of Health and Human Services Public. Health Service Centers for Disease Control and Prevention National Institutes of Health (CDC). U.S.
28. Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 2020;25:2000045.
29. Brandolini M, Taddei F, Marino MM, Grumiro L, Scalcione A, Turba ME, 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.
30. Uhteg K, Jarrett J, Richards M, Howard C, Morehead E, Geahr M, et al. Comparing the analytical performance of three SARS-CoV-2 molecular diagnostic assays. J Clin Virol 2020;127:104384.
31. Klimstra WB, Tilston-Lunel NL, Nambulli S, Boslett J, McMillen CM, Gilliland T, et al. SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients. J Gen Virol 2020;101:1156-1169.
32. Otto SP, Day T, Arino J, Colijn C, Dushoff J, Li M, et al. The origins and potential future of SARS-CoV-2 variants of concern in the evolving COVID-19 pandemic. Curr Biol 2021;31:R918-R929.
33. Jonsson N, Gullberg M, Lindberg AM. Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units. Microbiol Immunol 2009;53:149-154.
34. Abraham R, Manakkadan A, Mudaliar P, Joseph I, Sivakumar KC, Nair RR, et al. Correlation of phylogenetic clade diversification and in vitro infectivity differences among cosmopolitan genotype strains of Chikungunya virus. Infect Genet Evol 2016;37:174-184.
35. Baer A, Kehn-Hall K. Viral concentration determination through plaque assays: using traditional and novel overlay systems. J Vis Exp 2014;(93):e52065.
36. Grigorov B, Rabilloud J, Lawrence P, Gerlier D.Rapid titration of measles and other viruses: optimization with determination of replication cycle length. PLoS One 2011;6(9):e24135.
37. Smith DR, Singh C, Green J, Lueder MR, Arnold CE, Voegtly LJ, et al. Genomic and virological characterization of SARS-CoV-2 variants in a subset of unvaccinated and vaccinated U.S. military personnel. Front Med (Lausanne) 2022;8:836658.
38. Bae HG, Nitsche A, Teichmann A, Biel SS, Niedrig M. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay. J Virol Methods 2003;110:185-191.
39. Mallm JP, Bundschuh C, Kim H, Weidner N, Steiger S, Lander I, et al. Local emergence and decline of a SARS-CoV-2 variant with mutations L452R and N501Y in the spike protein. medRxiv 2021.04.27.21254849.
40. Richardson J, Molina-Cruz A, Salazar MI, Black W 4th. Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti. Am J Trop Med Hyg 2006;74:132-141.
41. Programa Nacional de Caracterización Genómica de SARS-CoV-2. Caracterización Genómica de SARS-CoV-2 por Muestreo Probabilístico en Colombia Segundo Muestreo. Instituto Nacional de Salud 2021; 1-10.
42. Pohl MO, Busnadiego I, Kufner V, Glas I, Karakus U, Schmutz S, et al. SARS-CoV-2 variants reveal features critical for replication in primary human cells. PLoS Biol 2021;19(3):e3001006.
43. Funnell SGP, Afrough B, Baczenas JJ, Berry N, Bewley KR, Bradford R, et al. A cautionary perspective regarding the isolation and serial propagation of SARS-CoV-2 in Vero cells. NPJ Vaccines 2021;6:83.
2. Laiton-Donato K, Franco-Muñoz C, Álvarez-Díaz DA, Ruiz-Moreno HA, Usme-Ciro JA, Prada DA, et al. Characterization of the emerging B.1.621 variant of interest of SARS-CoV-2. Infect Genet Evol 2021;95:105038.
3. WHO. Tracking SARS-CoV-2 variants. 2021 Available from: https://www.who.int/en/activities/tracking-SARS-CoV-2-variants
4. Singh J, Pandit P, McArthur AG, Banerjee A, Mossman K. Evolutionary trajectory of SARS-CoV-2 and emerging variants. Virol J 2021;18:166.
5. Faria NR, Mellan TA, Whittaker C, Claro IM, Candido DDS, Mishra S, et al. Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil. Science 2021;372:815-821.
6. Baral P, Bhattarai N, Hossen ML, Stebliankin V, Gerstman BS, Narasimhan G, et al. Mutation-induced changes in the receptor-binding interface of the SARS-CoV-2 Delta variant B.1.617.2 and implications for immune evasion. Biochem Biophys Res Commun 2021;574:14-19.
7. Planas D, Veyer D, Baidaliuk A, Staropoli I, Guivel-Benhassine F, Rajah MM, et al. Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization. Nature 2021;596:276-280.
8. WHO. Enhancing Readiness for Omicron (B.1.1.529): Technical Brief and Priority Actions for Member States. 2021 Available from: https://www.who.int/publications/m/item/enhancing-readiness-for-omicron-(b.1.1.529)-technical-brief-and-priority-actions-for-member-states
9. Uriu K, Kimura I, Shirakawa K, Takaori-Kondo A, Nakada TA, Kaneda A, et al. Neutralization of the SARS-CoV-2 Mu Variant by Convalescent and Vaccine Serum. N Engl J Med 2021;385:2397-2399.
10. Kaku Y, Kuwata T, Zahid HM, Hashiguchi T, Noda T, Kuramoto N, et al. Resistance of SARS-CoV-2 variants to neutralization by antibodies induced in convalescent patients with COVID-19. Cell Rep 2021;36:109385.
11. Mendoza EJ, Manguiat K, Wood H, Drebot M. Two detailed plaque assay protocols for the quantification of infectious SARS-CoV-2. Curr Protoc Microbiol 2020;57(1):ecpmc105.
12. Schutten M, Niesters HG. Clinical utility of viral quantification as a tool for disease monitoring. Expert Rev Mol Diagn 2001;1:153-162.
13. Cobo F. Application of molecular diagnostic techniques for viral testing. Open Virol J 2012;6:104-114.
14. Yaniv K, Ozer E, Kushmaro A. SARS-CoV-2 variants of concern, Gamma (P.1) and Delta (B.1.617), sensitive detection and quantification in wastewater employing direct RT-qPCR. medRxiv 2021;07.14.21260495.
15. Jureka AS, Silvas JA, Basler CF. Propagation, inactivation, and safety testing of SARS-CoV-2. Viruses 2020;12:622.
16. Hodinka RL. The clinical utility of viral quantitation using molecular methods. Clin Diagn Virol 1998;10:25-47.
17. Wulff NH, Tzatzaris M, Young PJ. Monte Carlo simulation of the Spearman-Kaerber TCID50. J Clin
Bioinforma 2012;2:5.
18. Keiser PT, Anantpadma M, Staples H, Carrion R, Davey RA.Automation of infectious focus assay for determination of filovirus titers and direct comparison to plaque and TCID(50) assays. Microorganisms 2021;9:156.
19. Shurtleff AC, Biggins JE, Keeney AE, Zumbrun EE, Bloomfield HA, Kuehne A, et al. Standardization of the filovirus plaque assay for use in preclinical studies. Viruses 2012;4:3511-3530.
20. Smither SJ, Lear-Rooney C, Biggins J, Pettitt J, Lever MS, Olinger GG Jr. Comparison of the plaque assay and 50% tissue culture infectious dose assay as methods for measuring filovirus infectivity. J Virol Methods 2013;193:565-571.
21. Yao H, Lu X, Chen Q, Xu K, Chen Y, Cheng M, et al. Patient-derived SARS-CoV-2 mutations impact viral replication dynamics and infectivity in vitro and with clinical implications in vivo. Cell Discov 2020;6:76.
22. Jeong GU, Yoon GY, Moon HW, Lee W, Hwang I, Kim H,et al. Comparison of plaque size, thermal stability, and replication rate among SARS-CoV-2 variants of concern. Viruses 2021;14:55.
23. Despres HW, Mills MG, Shirley DJ, Schmidt MM, Huang ML, Roychoudhury P, et al. Measuring infectious SARS-CoV-2 in clinical samples reveals a higher viral titer: RNA ratio for Delta and Epsilon vs. Alpha variants. Proc Natl Acad Sci U S A 2022;119(5):e2116518119.
24. Khateeb J, Li Y, Zhang H. Emerging SARS-CoV-2 variants of concern and potential intervention approaches. Crit Care 2021;25:244.
25. Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, et al. Tracking changes in SARS-CoV-2 Spike: Evidence that D614G increases infectivity of the COVID-19 virus. Cell 2020;182:812-827.e19.
26. Díaz FJ, Aguilar-Jiménez W, Flórez-Álvarez L, Valencia G, Laiton-Donato K, Franco-Muñoz C, et al. Isolation and characterization of an early SARS-CoV-2 isolate from the 2020 epidemic in Medellín, Colombia. Biomedica 2020;40(Supl. 2):148-158.
27. Richmond JY, McKinney RW (2009). Biosafety in microbiological and biomedical laboratories. 5th ed. U.S. Department of Health and Human Services Public. Health Service Centers for Disease Control and Prevention National Institutes of Health (CDC). U.S.
28. Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 2020;25:2000045.
29. Brandolini M, Taddei F, Marino MM, Grumiro L, Scalcione A, Turba ME, 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.
30. Uhteg K, Jarrett J, Richards M, Howard C, Morehead E, Geahr M, et al. Comparing the analytical performance of three SARS-CoV-2 molecular diagnostic assays. J Clin Virol 2020;127:104384.
31. Klimstra WB, Tilston-Lunel NL, Nambulli S, Boslett J, McMillen CM, Gilliland T, et al. SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients. J Gen Virol 2020;101:1156-1169.
32. Otto SP, Day T, Arino J, Colijn C, Dushoff J, Li M, et al. The origins and potential future of SARS-CoV-2 variants of concern in the evolving COVID-19 pandemic. Curr Biol 2021;31:R918-R929.
33. Jonsson N, Gullberg M, Lindberg AM. Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units. Microbiol Immunol 2009;53:149-154.
34. Abraham R, Manakkadan A, Mudaliar P, Joseph I, Sivakumar KC, Nair RR, et al. Correlation of phylogenetic clade diversification and in vitro infectivity differences among cosmopolitan genotype strains of Chikungunya virus. Infect Genet Evol 2016;37:174-184.
35. Baer A, Kehn-Hall K. Viral concentration determination through plaque assays: using traditional and novel overlay systems. J Vis Exp 2014;(93):e52065.
36. Grigorov B, Rabilloud J, Lawrence P, Gerlier D.Rapid titration of measles and other viruses: optimization with determination of replication cycle length. PLoS One 2011;6(9):e24135.
37. Smith DR, Singh C, Green J, Lueder MR, Arnold CE, Voegtly LJ, et al. Genomic and virological characterization of SARS-CoV-2 variants in a subset of unvaccinated and vaccinated U.S. military personnel. Front Med (Lausanne) 2022;8:836658.
38. Bae HG, Nitsche A, Teichmann A, Biel SS, Niedrig M. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay. J Virol Methods 2003;110:185-191.
39. Mallm JP, Bundschuh C, Kim H, Weidner N, Steiger S, Lander I, et al. Local emergence and decline of a SARS-CoV-2 variant with mutations L452R and N501Y in the spike protein. medRxiv 2021.04.27.21254849.
40. Richardson J, Molina-Cruz A, Salazar MI, Black W 4th. Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti. Am J Trop Med Hyg 2006;74:132-141.
41. Programa Nacional de Caracterización Genómica de SARS-CoV-2. Caracterización Genómica de SARS-CoV-2 por Muestreo Probabilístico en Colombia Segundo Muestreo. Instituto Nacional de Salud 2021; 1-10.
42. Pohl MO, Busnadiego I, Kufner V, Glas I, Karakus U, Schmutz S, et al. SARS-CoV-2 variants reveal features critical for replication in primary human cells. PLoS Biol 2021;19(3):e3001006.
43. Funnell SGP, Afrough B, Baczenas JJ, Berry N, Bewley KR, Bradford R, et al. A cautionary perspective regarding the isolation and serial propagation of SARS-CoV-2 in Vero cells. NPJ Vaccines 2021;6:83.
| Files | ||
| Issue | Vol 14 No 3 (2022) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v14i3.9758 | |
| Keywords | ||
| SARS-CoV-2 variants; Virus titer; Real-time reverse transcription-polymerase chain reaction; Plaque assay; Median tissue culture infectious dose assay | ||
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How to Cite
1.
Zapata-Cardona M, Flórez-Álvarez L, Gómez-Gallego D, Moncada-Díaz M, Hernandez J, Díaz F, Rugeles M, Aguilar-Jiménez W, Zapata W. Comparison among plaque assay, tissue culture infectious dose (TCID50) and real-time RT-PCR for SARS-CoV-2 variants quantification. Iran J Microbiol. 2022;14(3):291-299.
