Comparison of the Plaque Assay and 50% Cell Culture Infectious Dose Assay Methods for Quantification Measuring SARS-CoV-2 Wuhan-Hu-1 Strain Infectivity
Article Sidebar
Main Article Content
Abstract
Background: The Corona Virus Disease 2019 (COVID-19), which causes from the Severe Acute Respiratory Syndrome Corona Virus (SAR-CoV-2), is an emerging disease that several organizations have tried to develop vaccines against the viruses and therapeutic medicines, including specific anti-viral drugs. Therefore, viral cultivation and quantification are necessary for the laboratory testing. Most of common methods to quantify the viral titer are the Plaque and CCID50 assays.
Objective: To compare the quantity of the SAR-CoV-2 and the consumption resources test between the plaque and CCID50 assays (original strain).
Method: This study was an experimental research from March to June 2020 by comparing the quantification of the SAR-CoV-2 between both methods using the same virus set at 80 tests in the same period.
Result: The results showed that the geometric means of viral titers determined by the plaque and CCID50 assays were 5.97 log PFU/ml and 6.00 log CCID50/ml, respectively. The percentages of the coefficient of variance of the methods were 4.09 and 3.29, respectively. The statistical analysis by the Paired-t test showed no statistically significant difference between these two methods at a 95% confidence interval. In addition, the ratio of viral titers of the mean over the geometric mean ranged between 0.87 and 1.13. However, the CCID50 assay was faster by requiring only 4 days of testing and more cost-effective by using fewer materials, equipment, and medium.
Conclusion: The determination of SARS-CoV-2 virus counting by the plague counting method and the 50% cell culture infection test showed no difference in results, but testing for 50% infection in cell cultures was faster and more cost-effective. It revealed that both methods can be used to quantify the SARS-CoV-2 titer, which can be applied to detect immune responses in COVID-19 patients as well as in COVID-19 vaccine testing further in animals and humans.
Article Details
This work is licensed under a Creative Commons Attribution 4.0 International License.
References
Hui DS, I Azhar E, Madani TA, Ntoumi F, Kock R, Dar O, Ippolito G, Mchugh TD, Memish ZA, Drosten C, Zumla A, Petersen E. The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health - The latest 2019 novel coronavirus outbreak in Wuhan, China. Int J Infect Dis 2020;91:264-66.
Wong ACP, Li X, Lau SKP, Woo PCY. Global Epidemiology of Bat Coronaviruses. Viruses 2019 20;11(2):174.
Sexton NR, Smith EC, Blanc H, Vignuzzi M, Peersen OB, Denison MR. Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens. J Virol 2016;90(16):7415-7428.
World Health Organization. WHO announces COVID-19 outbreak a pandemic [Internet]. WHO; 2020 [cited 2020 Jun 12]. Available from: http://www.euro.who.int/en/health-topics/health-emergencies/coronavirus-covid-19/news/news/2020/3/who-announces-covid-19-outbreak-a-pandemic
Steenhuysen, Julie; Kelland, Kate. "With Wuhan virus genetic code in hand, scientists begin work on a vaccine". Thomson Reuters (25 January 2020). [cited 12 June 20]; [screens]. Available from: URL: Available from: https://www.reuters.com/article/us-china-health-vaccines/with-wuhan-virus-genetic-code-in-hand-scientists-begin-work-on-a-vaccine-idUSKBN1ZN2J8
Fukuda A, Sengün F, Sarpay HE, Konobe T, Saito S, Umino Y, Kohama T. Parameters for plaque formation in the potency assay of Japanese measles vaccines. J Virol Methods 1996;61(1-2):1-6.
World Health Organization. WHO. Requirements for varicella vaccine (live). Technical Report Series No. 848. 1994; Annex 1.
Husson-van Vliet J, Colinet G, Yane F, Lemoine P. A simplified plaque assay for varicella vaccine. J Virol Methods 1987;18(2-3):113-20.
Forcic D, Kosutić-Gulija T, Santak M, Jug R, Ivancic-Jelecki J, Markusic M, Mazuran R. Comparisons of mumps virus potency estimates obtained by 50% cell culture infective dose assay and plaque assay. Vaccine 2010;17;28(7):1887-92.
Covid 19 Vaccine tracker. Approved Vaccines; [update 2022 Oct 28; cite 2022 Oct 30]. Available from: https://covid19.trackvaccines.org/vaccines/approved/#vaccine-list
กระทรวงการอุดมศึกษา วิทยาศาสตร์ วิจัยและนวัตกรรม. อว. เผยความก้าวหน้าของการวิจัยวัคซีนโควิด 19 โดยนักวิจัยไทยความคาดหวังของประเทศ [อินเทอร์เน็ต]. 2564 [วันที่ปรับปรุง 15 เม.ย. 2564; เข้าถึงเมื่อ 30 ต.ค. 2565]. เข้าถึงได้จาก: https://www.mhesi.go.th/index.php/en/content_page/item/4228-168641.html
Recommendation for Japanese encephalitis vaccine (inactivated) for human use (revised 2007). WHO Expert Committee on Biological Standardization.2007; Annex 1
Guidelines for the production and control of Japanese Encephalitis vaccine (live) for human use. Technical Report Series No. 910. 2002; Annex 3.
Requirements for measles, mumps and rubella vaccines and combined vaccines (live). In: WHO Expert Committee on Biological Standardization. WHO Technical Report Series No. 840. 1994; 102-201.
Requirements for poliomyelitis vaccine (Oral). In: WHO Expert Committee on Biological Standardization. WHO Technical Report Series No. 800. 1990.
Cooper PD. The plaque assay of animal viruses. Adv Virus Res 1961;8:319-78.
Yan C, Cui J, Huang L, Du B, Chen L, Xue G, et al. Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay. Clin Microbiol Infect 2020;26(6):773-779.
Shurtleff AC, Bloomfield HA, Mort S, Orr SA, Audet B, Whitaker T, et al. Validation of the Filovirus Plaque Assay for Use in Preclinical Studies [Internet]. 2016 [cited 12 Jun 2020]; [screens]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848606/
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 Nov;193(2):565-71.
Freshey RI. Culture of animal cells: A Manual of Basic Technology (4th ed), Wiley-Liss, Inc 1994;290-96.
Low IE. Mycoplasma in tissue culture: Overview of detection methods. Health Lab Sci 1976; 13(2):129-36.
Chen TR. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res 1977;104(2): 255-62.
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 6;4(12):3511-30.
Ramakrishnan MA. Determination of 50% endpoint titer using a simple formula. World J Virol 2016;5(2):85-6.
Niroumand H, Zain MFM, Jamil M. Statistical methods for comparison of data sets of construction methods and building evaluation. Procedia Soc Behav Sci 2013;89:218-21.
Onen EA, Sonmez k, Yildirim F,Demirci EK, Gurel A. Development, analysis, and preclinical evaluation of inactivated vaccine candidate for prevention of Covid-19 disease. All Life 2022; 15(1):771-93.
Saadi J, Oueslati S, Bellanger L, Gallais F, Dortet L,Roque – Afonso AM, et al. Quantitative Assessment of SARS-CoV-2 Virus in Nasopharyngeal Swabs Stored in Transport Medium by a Straightforward LC-MS/MS Assay Targeting Nucleocapsid, Membrane, and Spike Proteins. J Proteome Res. 2021;20(2):1434–443.