An in vitro model for assessment of SARS-CoV-2 infectivity by defining the correlation between virus isolation and quantitative PCR value: isolation success of SARS-CoV-2 from oropharyngeal swabs correlates negatively with Cq value

Abstract

Background: At the beginning of the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), little was known about its actual rate of infectivity and any COVID-19 patient positive in laboratory testing was supposed to be highly infective and a public health risk factor.

Methods: One hundred oropharyngeal samples were obtained during routine work flow of testing symptomatic persons by quantitative polymerase chain reaction (qPCR) and were inoculated onto cell culture of VeroB4 cells to study the degree of infectivity of SARS-CoV-2 in vitro. Quantification by virus titration and an external standard using synthetic RNA gave the breaking point of infectivity in SARS-CoV-2 in vitro.

Results: A clear negative correlation (r = - 0.76; p < 0.05) could be asserted between the viral load in quantitative polymerase chain reaction (qPCR) and the probability of a successful isolation in serial isolation experiments of specific oropharyngeal samples positive in qPCR. Quantification by virus titration and an external standard using synthetic RNA indicate a Cq between 27 and 30 in E-gene screening PCR as a breaking point in vitro, where infectivity decreases significantly and isolations become less probable.

Conclusions: This study showed that only the 21% of samples with the highest viral load were infectious enough to transmit the virus in vitro and determined that the dispersion rate in vitro is surprisingly close to those calculated in large retrospective epidemiological studies for SARS-CoV-2. This raises the question of whether this simple in vitro model is suitable to give first insights in dispersion characters of novel or neglected viral pathogens. The statement that SARS-CoV-2 needs at least 40,000 copies to reliably induce infection in vitro is an indication of its transmissibility in Public Health decisions. Applying quantitative PCR systems in diagnosis of SARS-CoV2 can distinguish between patients providing a high risk of transmission and those, where the risk of transmission is probably limited to close and long-lasting contacts.

Keywords: Infectivity; Quantitation of viral infectivity; SARS-CoV-2; Transmission pattern in vitro.

Fig. 1

Success of isolation of SARS-CoV-2 in VeroB4 cells by Cq-values (%). Isolation success correlated negatively with the initial Cq value in the sample (r = − 0.76; p < 0.05). All oropharyngeal samples with an initial viral load lower than Cq 20 in E-gene screening PCR (n = 8), could be grown in vitro (100%). An initial Cq value between 20 and 24.9 led to a 72.7% chance of infection, which decreased to 25% at an initial viral load between 25 and 29.9 and to 7.1% at an initial load between 30 and 34.9. Samples with initial viral loads of Cq 35 or higher could not be isolated in our experiment

Fig. 2

The distribution of Cq values during the outbreak in a total data set of 371 samples (dark grey). The light grey bars show the comparable distribution of Cq values in our experimental sample collection and demonstrate that our sample set is representative for the distribution of initial viral loads during an outbreak of SARS-CoV-2

Fig. 3

Dilution series of plaque-forming units (PFU) of an average of our isolates compared to a commercially available external standard using synthetic RNA copies (COV019, CE, Exact Diagnostics, USA)