Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. Virus
2.3. Patient Samples
2.4. Virus Overlay Medium
2.5. Infectious Virus Particle Titration from Laboratory-Established Strains and Clinical Samples
2.6. Data Analysis
3. Results
3.1. Infectious Virus Titration Using Laboratory-Established Flavivirus Strains and SARS-CoV-2 Strains
3.2. Dengue Virus Titers in Serum Samples from Dengue Patients and SARS-CoV-2 Titers in Nasopharyngeal Swab Samples from COVID-19 Patients
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Log10 Virus Titer (PFU/mL) | |||||||||
---|---|---|---|---|---|---|---|---|---|
Cell Lines | BHK-21 | Vero | FcγR-Expressing BHK-21 | ||||||
Virus Strain | Av a | AvMc b | Mc c | Av | AvMc | Mc | Av | AvMc | Mc |
DENV-1 01-44-1 HuNIID | 6.4 | 6.6 | 4.1 | 3.3 | 4.4 | 3.8 | 6.5 | 6.1 | 5.7 |
DENV-1 99St12A | 4.4 | 5.5 | 2.5 | 4.5 | 4.7 | 4.8 | 4.9 | 6.1 | 4.6 |
DENV-2 DHF0663 | 5.4 | 5.1 | 4.4 | 5.4 | 5.6 | 5.0 | 5.9 | 5.8 | 5.2 |
DENV-2 TLC-30 | 5.0 | 4.8 | 4.5 | 5.0 | 4.7 | 5.0 | 4.5 | 4.8 | 4.0 |
DENV-2 08-77 | 4.9 | 4.3 | 5.2 | 5.6 | 5.4 | 5.6 | 6.1 | 5.8 | 5.2 |
DENV-2 00St22A | 4.7 | 5.6 | 5.7 | 6.3 | 6.0 | 6.0 | 6.6 | 5.6 | 5.8 |
DENV-3 CH53469 | 5.8 | 6.7 | 5.4 | 4.6 | 4.3 | 4.1 | 5.9 | 6.9 | 5.8 |
DENV-4 SLMC318 | 5.7 | 5.6 | 4.9 | 5.4 | 4.1 | 4.4 | 6.1 | 6.0 | 4.3 |
DENV-4 TVP360 | 4.7 | 5.1 | 3.4 | 4.8 | 4.2 | 4.6 | 4.9 | 5.0 | 3.9 |
ZIKV MR766 | 6.2 | 6.8 | 6.9 | 8.2 | 7.6 | 7.1 | 6.9 | 7.4 | 6.1 |
ZIKV PRVABC59 | 5.5 | 5.9 | 5.0 | 7.1 | 6.8 | 6.7 | 5.1 | 6.1 | 4.4 |
ZIKV MRS_OPY_Martinique_PaRi_2015 | 5.8 | 6.1 | 5.6 | 7.1 | 6.9 | 6.8 | 5.9 | 6.2 | 4.9 |
ZIKV H/PF/2013 | 6.5 | 6.7 | 5.9 | 7.4 | 7.2 | 6.2 | 6.8 | 6.7 | 5.7 |
JEV OH0566 | 7.8 | 7.8 | 7.9 | 7.6 | 7.7 | 7.5 | 7.9 | 7.7 | 7.7 |
YFV 17D | 6.6 | 6.5 | 5.2 | 5.8 | 6.3 | 6.5 | 6.9 | 6.6 | 4.9 |
Cell Lines | BHK-21 | Vero | FcγR-Expressing BHK | ||||||
---|---|---|---|---|---|---|---|---|---|
Virus Strain | Av a | AvMc b | Mc c | Av | AvMc | Mc | Av | AvMc | Mc |
DENV-1 01-44-1 HuNIID | 0.8 ± 0.2 | 0.8 ± 0.4 | 0.3 ± 0.1 | 0.3 ± 0.2 | 0.3 ± 0.2 | 0.3 ± 0.1 | 1.1 ± 0.3 | 0.4 ± 0.1 | 0.4 ± 0.1 |
DENV-1 99St12A | 1.0 ± 0.3 | 1.5 ± 0.3 | 0.4 ± 0.1 | 0.5 ± 0.2 | 0.5 ± 0.2 | 0.3 ± 0.1 | 0.9 ± 0.2 | 1.0 ± 0.2 | 0.2 ± 0.0 |
DENV-2 DHF0663 | 1.1 ± 0.3 | 1.1 ± 0.2 | 0.2 ± 0.1 | 1.1± 0.3 | 1.5 ± 0.3 | 0.7 ± 0.2 | 1.0 ± 0.4 | 1.1 ± 0.7 | 0.5 ± 0.1 |
DENV-2 TLC-30 | 1.7 ± 0.5 | 1.8 ± 0.6 | 0.5 ± 0.1 | 1.2 ± 0.3 | 1.0 ± 0.5 | 0.6 ± 0.1 | 0.8 ± 0.1 | 1.3 ± 0.1 | 0.3 ± 0.0 |
DENV-2 08-77 | 0.4 ± 0.1 | 0.6 ± 0.1 | 0.5 ± 0.1 | 1.0 ± 0.5 | 0.6 ± 0.2 | 0.4 ± 0.1 | 0.5 ± 0.1 | 1.4 ± 0.2 | 0.3 ± 0.0 |
DENV-2 00St22A | 0.6 ± 0.1 | 0.8 ± 0.2 | 0.3 ± 0.1 | 1.2 ± 0.4 | 0.9 ± 0.4 | 0.6 ± 0.2 | 0.7 ± 0.2 | 1.5 ± 0.3 | 0.3 ± 0.1 |
DENV-3 CH53469 | 1.5 ± 0.5 | 1.8 ± 0.7 | 0.5 ± 0.1 | 1.3 ± 0.5 | 0.6 ± 0.2 | 0.2± 0.0 | 1.9 ± 0.8 | 1.7 ± 0.8 | 0.4 ± 0.1 |
DENV-4 SLMC318 | 0.9 ± 0.2 | 0.7 ± 0.4 | 0.5 ± 0.1 | 0.6 ± 0.1 | 0.7 ± 0.3 | 0.4 ± 0.1 | 0.7 ± 0.1 | 0.4 ± 0.3 | 0.4 ± 0.1 |
DENV-4 TVP360 | 0.9 ± 0.2 | 1.8 ± 0.6 | 0.4 ± 0.1 | 0.9 ± 0.6 | 0.8 ± 0.2 | 0.8 ± 0.3 | 1.0 ± 0.3 | 1.6 ± 0.3 | 0.3 ± 0.1 |
ZIKV MR766 | 0.8 ± 0.2 | 1.1 ± 0.6 | 0.4 ± 0.1 | 2.2 ± 0.0 | 1.7 ± 0.4 | 1.1 ± 0.2 | 1.2 ± 0.4 | 1.5 ± 0.6 | 0.3 ± 0.0 |
ZIKV PRVABC59 | 0.4 ± 0.1 | 0.6 ± 0.2 | 0.5 ± 0.1 | 1.2 ± 0.5 | 1.3 ± 0.3 | 0.5 ± 0.1 | 0.4 ± 0.1 | 0.5 ± 0.1 | 0.4 ± 0.2 |
ZIKV MRS_OPY_Martinique_PaRi_2015 | 0.6 ± 0.1 | 0.8 ± 0.2 | 0.4 ± 0.1 | 1.3 ± 0.6 | 1.2 ± 0.4 | 0.5 ± 0.2 | 0.5 ± 0.2 | 0.6 ± 0.1 | 0.2 ± 0.1 |
ZIKV H/PF/2013 | 0.4 ± 0.1 | 0.8 ± 0.3 | 0.4 ± 0.1 | 1.4± 0.9 | 1.4 ± 0.6 | 0.9 ± 0.3 | 0.6 ± 0.2 | 0.8 ± 0.3 | 0.2 ± 0.0 |
JEV OH0566 | 0.2 ±0.0 | 0.6 ± 0.1 | 1.0 ± 0.3 | 1.4 ± 0.4 | 1.5 ± 0.4 | 0.8 ± 0.1 | 1.3 ± 0.9 | 0.7 ± 0.3 | 0.3 ± 0.1 |
YFV 17D | 1.2 ± 0.5 | 0.6 ± 0.1 | 0.3 ± 0.1 | 0.3 ± 0.1 | 0.7 ± 0.2 | 0.3 ± 0.1 | 1.4 ± 0.2 | 0.8 ± 0.3 | 0.4 ± 0.1 |
Virus Titer (Log10 PFU/mL) Vero | Plaque Size | |||
---|---|---|---|---|
SARS-CoV-2 Strain | AvMc a | Mc b | AvMc | Mc |
2019-nCoV/Japan/TY/WK-521/2020 | 4.9 | 4.8 | 1.4 ± 0.5 | 0.6 ± 0.1 |
NGS1B | 5.5 | 4.6 | 1.3 ± 0.5 | 0.6 ± 0.1 |
hCoV-19/Japan/QK002/2020 (Alpha) d | 3.7 | ND c | 0.7 ± 0.2 | ND |
hCoV-19/Japan/TY7-501/2021(Gamma) | 3.6 | ND | 0.8 ± 0.1 | ND |
hCoV-19/Japan/TY8-612/2021 (Beta) | 3.5 | 3.2 | 0.7 ± 0.1 | 0.3 ± 0.1 |
Sample Code | Days a | Log10 Genome Copies/mL | Dengue ELISA | Virus Titer (Log10 PFU/mL) | |||
---|---|---|---|---|---|---|---|
OD e-IgG | OD-IgM | IgM/IgG OD Ratio | AvMc c | Mc d | |||
Primary Infection | |||||||
01-TN-145 | 0 | 9.5 | 0.8 | 0.1 | 0.2 | 6.1 ± 0.03 | ND |
01-TN-087 | 1 | 10.2 | 0.5 | 0.1 | 0.2 | 5.4 ± 0.04 | ND |
29-HD-011 | 1 | 9.5 | 0.1 | 0.1 | 1.0 | 5.1 ± 0.04 | ND |
01-TN-067 | 2 | 7.6 | 0.5 | 0.1 | 0.2 | 6.4 ± 0.1 | 4.49 ± 0.03 |
01-TN-076 | 2 | 10.3 | 0.8 | 0.07 | 0.09 | 7.3 ± 0.1 | ND |
01-TN-078 | 2 | 9.2 | 0.9 | 0.06 | 0.06 | 6.2 ± 0.2 | ND |
01-TN-091 | 2 | 10.5 | 0.4 | 0.2 | 0.4 | 6.6 ± 0.02 | ND |
01-TN-040 | 3 | 8.1 | 0.5 | 0.2 | 0.3 | 6.0 ± 0.2 | ND |
01-TN-063 | 3 | 6.9 | 0.8 | 0.2 | 0.2 | 6.1 ± 0.07 | ND |
04-HD-010 | 7 | 5.2 | 1.8 | 3.5 | 2.0 | ND b | ND |
08-HD-001 | 8 | 7.6 | 1.0 | 2.6 | 2.5 | ND | ND |
09-HD-014 | 8 | 5.4 | 1.6 | 3.3 | 2.0 | ND | ND |
Secondary Infection | |||||||
01-TN-088 | 1 | 10.3 | 1.1 | 0.13 | 8.5 | 6.3 ± 0.2 | ND |
01-TN-077 | 1 | 10.4 | 1.0 | 0.05 | 22.1 | 5.0 ± 0.1 | ND |
01-TN-106 | 3 | 10.0 | 1.5 | 0.16 | 9.9 | 5.3 ± 0.06 | ND |
11-HD-006 | 3 | 7.7 | 2.3 | 0.45 | 5.2 | ND | ND |
11-HD-012 | 3 | 8.9 | 1.0 | 0.14 | 7.2 | 5.8 ± 0.1 | ND |
29-HD-001 | 3 | 8.9 | 1.4 | 0.30 | 4.7 | ND | ND |
01-TN-056 | 3 | 7.6 | 1.5 | 0.13 | 11.5 | 6.1 ± 0.1 | ND |
01-TN-061 | 3 | 6.8 | 1.3 | 0.13 | 10.0 | 6.7 ± 0.03 | ND |
01-TN-064 | 3 | 7.5 | 1.0 | 0.14 | 6.7 | 6.4 ± 0.08 | ND |
11-HD-002 | 4 | 8.3 | 1.1 | 0.16 | 0.2 | 6.0 ± 0.05 | 5.5 ± 0.03 |
01-TN-062 | 5 | 7.2 | 1.6 | 0.13 | 12.8 | 6.1 ± 0.1 | ND |
01-TN-068 | 5 | 6.5 | 2.0 | 0.39 | 5.1 | 5.8 ± 0.2 | ND |
01-PK-119 | 6 | 5.0 | 1.4 | 0.20 | 7.0 | ND | ND |
11-HD-004 | 6 | 6.1 | 1.9 | 2.0 | 1.0 | ND | ND |
03-HD-014 | 8 | 9.5 | 1.9 | 0.79 | 2.4 | 6.5 ± 0.01 | ND |
Sample No | Virus Load (log10 Genome Copies/mL) | Virus Titer (Log10 PFU/mL) | Plaque Size (Mean Diameters mm ± SD) | ||
---|---|---|---|---|---|
AvMc | Mc | AvMc | Mc | ||
1 | 10.9 | 4.3 | 4.2 | 0.87 ± 0.34 | 0.29 ± 0.1 |
2 | 10.2 | 3.4 | ND a | NT | NT |
3 | 9.9 | 5.1 | 4.9 | 1.0 ± 0.2 | 0.4 ± 0.07 |
4 | 9.7 | 3.8 | 3.6 | 1.3 ± 0.2 | 0.3 ± 0.09 |
5 | 9.7 | 3.2 | 3.1 | 1.4 ± 0.2 | 0.38 ± 0.1 |
6 | 9.5 | 2.6 | ND | NT b | NT |
7 | 9.4 | 4.1 | 3.94 | 0.96 ± 0.2 | 0.37 ± 0.1 |
8 | 9.21 | 4.0 | 3.81 | 1.1 ± 0.3 | 0.37 ± 0.1 |
9 | 9.03 | 3.7 | 4.11 | 1.7 ± 0.3 | 0.4 ± 0.1 |
10 | 8.51 | 3.3 | 3.20 | 0.7 ± 0.1 | 0.4 ± 0.1 |
11 | 8.44 | 2.9 | 3.23 | 1.4 ± 0.2 | 0.5 ± 0.1 |
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Fukuta, M.; Nguyen, C.T.; Nguyen, T.T.T.; Nguyen, T.T.N.; Vu, T.B.H.; Takemura, T.; Nguyen, L.K.H.; Inoue, S.; Morita, K.; Le, T.Q.M.; et al. Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment. Int. J. Environ. Res. Public Health 2021, 18, 9845. https://doi.org/10.3390/ijerph18189845
Fukuta M, Nguyen CT, Nguyen TTT, Nguyen TTN, Vu TBH, Takemura T, Nguyen LKH, Inoue S, Morita K, Le TQM, et al. Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment. International Journal of Environmental Research and Public Health. 2021; 18(18):9845. https://doi.org/10.3390/ijerph18189845
Chicago/Turabian StyleFukuta, Mizuki, Co Thach Nguyen, Thi Thu Thuy Nguyen, Thi Thanh Ngan Nguyen, Thi Bich Hau Vu, Taichiro Takemura, Le Khanh Hang Nguyen, Shingo Inoue, Kouichi Morita, Thi Quynh Mai Le, and et al. 2021. "Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment" International Journal of Environmental Research and Public Health 18, no. 18: 9845. https://doi.org/10.3390/ijerph18189845