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. 2020 Jul 23;58(8):e01068-20.
doi: 10.1128/JCM.01068-20. Print 2020 Jul 23.

Culture-Based Virus Isolation To Evaluate Potential Infectivity of Clinical Specimens Tested for COVID-19

Chung-Guei Huang #  1   2   3 , Kuo-Ming Lee #  3 , Mei-Jen Hsiao  1 , Shu-Li Yang  1 , Peng-Nien Huang  3   4 , Yu-Nong Gong  1   3 , Tzu-Hsuan Hsieh  1 , Po-Wei Huang  1 , Ya-Jhu Lin  1 , Yi-Chun Liu  1 , Kuo-Chien Tsao  5   2   3 , Shin-Ru Shih  5   2   3   6
Affiliations

Culture-Based Virus Isolation To Evaluate Potential Infectivity of Clinical Specimens Tested for COVID-19

Chung-Guei Huang et al. J Clin Microbiol. .

Abstract

Real-time reverse transcription-PCR (RT-PCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19). However, the correlation between detectable viral RNA and culturable virus in clinical specimens remains unclear. Here, we performed virus culture for 60 specimens that were confirmed to be positive for SARS-CoV-2 RNA by real-time RT-PCR. The virus could be successfully isolated from 12 throat and nine nasopharyngeal swabs and two sputum specimens. The lowest copy number required for virus isolation was determined to be 5.4, 6.0, and 5.7 log10 genome copies/ml sample for detecting the nsp12, E, and N genes, respectively. We further examined the correlation of genome copy number and virus isolation in different regions of the viral genome, demonstrating that culturable specimens are characterized by high copy numbers with a linear correlation observed between copy numbers of amplicons targeting structural and nonstructural regions. Overall, these results indicate that in addition to the copy number, the integrity of the viral genome should be considered when evaluating the infectivity of clinical SARS-CoV-2 specimens.

Keywords: RT-PCR; SARS-CoV-2; culturability; genome copy; genome integrity.

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Figures

FIG 1
FIG 1
(A to C) Distribution of cycle threshold (CT) values and genome copies of the nsp12 (A), E (B), and N (C) SARS-CoV-2 genes in culturable (black) and nonculturable (blue) specimens. Data are presented as means ± SEM (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.1; ns, not significant). (D) A box plot shows the calculation of log10 genome copies/ml from the CT value, including the minimum to maximum values. The median is depicted as a line within the box.
FIG 2
FIG 2
Distributions of genome copies in SARS-CoV-2 nonstructural (nsp12) and structural (E and N) genes of culturable (A) and nonculturable (B) specimens. Correlations of nsp12 genome copies with those of the E (C) and N (D) genes in samples without a freeze-thaw cycle, and with E gene copy numbers in freeze-thawed samples (E), along with the respective regression equations and R2.
See this image and copyright information in PMC

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