Shedding of Viable Virus in Asymptomatic SARS-CoV-2 Carriers

Abstract

Information regarding the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in asymptomatic carriers is scarce. In order to determine the duration of infectivity and its correlation with reverse transcription-PCR (RT-PCR) results and time since initial positive PCR test in this population, we evaluated SARS-CoV-2 cell infectivity in nasopharyngeal samples longitudinally obtained from asymptomatic carriers who disembarked from a cruise ship during a COVID-19 outbreak. Of 166 nasopharyngeal samples collected from 39 asymptomatic carriers every 48 h until two consecutive negative PCR test results were obtained, SARS-CoV-2 was successfully isolated from 9 PCR-positive samples which were obtained from 7 persons (18%; 7/39). Viable viruses were isolated predominantly within 7 days after the initial positive PCR test, except for one person who shed viable virus until day 15. The median crossing point (Cp) value of RT-PCR of culture-positive samples was 24.6 (interquartile range [IQR], 20.4 to 25.8; range, 17.9 to 30.3), and Cp values were significantly associated with isolation of viable virus (odds ratio, 0.496; 95% confidence interval [CI], 0.329 to 0.747; P value, 0.001), which was consistent with existing data for symptomatic patients. Genome sequence analysis of SARS-CoV-2 samples consecutively obtained from a person who shed viable virus for 15 days identified the emergence of two novel single nucleotide variants (C8626T transition and C18452T transition) in the sample collected on day 15, with the latter corresponding to an amino acid substitution in nonstructural protein 14. The impact of these mutations on prolonged viable-virus shedding is unclear. These findings underscore the potential role of asymptomatic carriers in transmission.IMPORTANCE A growing number of studies suggest the potential role of asymptomatic SARS-CoV-2 carriers as a major driver of the COVID-19 pandemic; however, virological assessment of asymptomatic infection has largely been limited to reverse transcription-PCR (RT-PCR), which can be persistently positive without necessarily indicating the presence of viable virus (e.g., replication-competent virus). Here, we evaluated the infectivity of asymptomatic SARS-CoV-2 carriers by detecting SARS-CoV-2-induced cytopathic effects on Vero cells using longitudinally obtained nasopharyngeal samples from asymptomatic carriers. We show that asymptomatic carriers can shed viable virus until 7 days after the initial positive PCR test, with one outlier shedding until day 15. The crossing point (Cp) value of RT-PCR was the leading predictive factor for virus viability. These findings provide additional insights into the role of asymptomatic carriers as a source of transmission and highlight the importance of universal source control measures, along with isolation policy for asymptomatic carriers.

Keywords: COVID-19; COVID-19 nucleic acid testing; SARS-CoV-2; asymptomatic infections; carrier state; cell culture techniques; cytopathogenic effect; infectivity; virus shedding; whole-genome sequencing.

FIG 1

Crossing point (Cp) values of RT-PCR and result of cell culture in asymptomatic SARS-CoV-2 carriers, according to number of days since SARS-CoV-2 infection diagnosis by PCR of nasopharyngeal swabs. Red dots indicate samples in which cytopathic effect (CPE) was observed, and black dots represent culture-negative samples. Triangles connected with a dotted line correspond to samples obtained from Carrier_1. With fluorescence-based real-time PCR, the number of cycles at which fluorescence signal from amplification exceeds the background fluorescence level is determined as the crossing point, threshold cycle (C_T), or other values by different instrument manufacturers. While there are differences in how they are calculated, they are considered biologically equivalent in that a lower value correlates with a higher copy number of the target nucleotide sequence.

FIG 2

Distribution and allele frequency of SNVs (single nucleotide variants) and intrahost single nucleotide variation (iSNV) identified in specimens obtained from asymptomatic SARS-CoV-2 carriers, compared with the reference genome. Each SNV or iSNV is colored based on the substituted bases (A, gray; G, blue; T, orange; C, green), with color gradients reflecting allele frequencies (in percent). The reference sequence was Wuhan Hu 1 (MN908947.3). SN, supernatant of cell culture; NP, nasopharyngeal swab.