@article{8784465b0c0646b3b087ef098daad99d,
title = "Characterizing SARS-CoV-2 Transcription of Subgenomic and Genomic RNAs During Early Human Infection Using Multiplexed Droplet Digital Polymerase Chain Reaction",
abstract = "Background: Control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission requires understanding SARS-CoV-2 replication dynamics. Methods: We developed a multiplexed droplet digital polymerase chain reaction (ddPCR) assay to quantify SARS-CoV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from genomic RNAs (gRNAs). We applied the assay to specimens from 144 people with single nasopharyngeal samples and 27 people with >1 sample. Results were compared to quantitative PCR (qPCR) and viral culture. Results: sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio sgRNA:gRNA was stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Daily testing of 6 persons revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. sgRNA:gRNA is constant during infection despite changes in viral culture. Conclusions: Ct values from qPCR correlate with active viral replication. More work is needed to understand why some cultures are negative despite presence of sgRNA.",
keywords = "COVID-19, SARS-CoV-2, droplet digital PCR, genomic RNA, multiplex ddPCR, replication dynamics, subgenomic RNA",
author = "Hwang, {Hyon S.} and Lo, {Che Min} and Michael Murphy and Tanner Grudda and Nicholas Gallagher and Luo, {Chun Huai} and Robinson, {Matthew L.} and Agha Mirza and Madison Conte and Abigail Conte and Ruifeng Zhou and Candelaria Vergara and Brooke, {Christopher B.} and Andrew Pekosz and Mostafa, {Heba H.} and Manabe, {Yukari C.} and Thio, {Chloe L.} and Ashwin Balagopal",
note = "Funding Information: Potential conflicts of interest. Y. C. M. has received research grant support to Johns Hopkins University from Hologic, Ceres, Cepheid, Roche, ChemBio, Becton Dickinson, miDiagnostics, and Yukon; and has provided consultative support to Abbott. All other authors report no potential conflicts. Funding Information: Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases, National Heart, Lung and Blood Institute, National Institute on Drug Abuse, National Institute of Mental Health, and Office of AIDS Research of the National Institutes of Health, Department of Health and Human Services (grant numbers UM1 AI068613 to the HIV Prevention Trials Network, R01 AI138810 to A. B. and C. L. T., R01AI148049 to C. V. U5411090366 to Y. C. M., U54EB007958-14 to Y. C. M. and M. L. R., U54EB007958-12S1 to Y. C. M., and U54HL143541-02S2 to Y. C. M., M. L. R., N. G., C. H. L., A. M., M. C., R. Z., C. B. B., and H. H. M. under the RADx-Tech program); the Johns Hopkins University COVID-19 Research and Response Program Fund; the Sherrilyn and Ken Fisher Center for Environmental Infectious Diseases Discovery Program; and the Henry Jackson Foundation. Publisher Copyright: {\textcopyright} 2022 The Author(s).",
year = "2023",
month = apr,
day = "15",
doi = "10.1093/infdis/jiac472",
language = "English (US)",
volume = "227",
pages = "981--992",
journal = "Journal of Infectious Diseases",
issn = "0022-1899",
publisher = "Oxford University Press",
number = "8",
}