tetano
Editor, Senior Moderator
BMJ Open Respir Res
. 2021 Feb;8(1):e000830.
doi: 10.1136/bmjresp-2020-000830.
Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells
Souichi Yamada[SUP] 1 [/SUP], Shuetsu Fukushi[SUP] 2 [/SUP], Hitomi Kinoshita[SUP] 1 [/SUP], Makoto Ohnishi[SUP] 3 [/SUP], Tadaki Suzuki[SUP] 4 [/SUP], Tsuguto Fujimoto[SUP] 5 [/SUP], Masayuki Saijo[SUP] 1 [/SUP], Ken Maeda[SUP] 6 [/SUP], Virus Diagnosis Group (NIID Toyama)
Collaborators, Affiliations
Abstract
Background: An outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.
Methods: We developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.
Results: The SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.
Conclusion: In combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.
Keywords: COVID-19; viral infection.
. 2021 Feb;8(1):e000830.
doi: 10.1136/bmjresp-2020-000830.
Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells
Souichi Yamada[SUP] 1 [/SUP], Shuetsu Fukushi[SUP] 2 [/SUP], Hitomi Kinoshita[SUP] 1 [/SUP], Makoto Ohnishi[SUP] 3 [/SUP], Tadaki Suzuki[SUP] 4 [/SUP], Tsuguto Fujimoto[SUP] 5 [/SUP], Masayuki Saijo[SUP] 1 [/SUP], Ken Maeda[SUP] 6 [/SUP], Virus Diagnosis Group (NIID Toyama)
Collaborators, Affiliations
- PMID: 33627333
- DOI: 10.1136/bmjresp-2020-000830
Abstract
Background: An outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.
Methods: We developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.
Results: The SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.
Conclusion: In combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.
Keywords: COVID-19; viral infection.