Isolation and Identification of a Rare Spike Gene Double-Deletion SARS-CoV-2 Variant From the Patient With High Cycle Threshold Value

Coronavirus disease 2019 (COVID-19) is an emerging life-threatening pulmonary disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, Hubei Province, China, in December 2019. COVID-19 develops after close contact via inhalation of respiratory droplets containing SARS-CoV-2 during talking, coughing, or sneezing by asymptomatic, presymptomatic, and symptomatic carriers. This virus evolved over time, and numerous genetic variants have been reported to have increased disease severity, mortality, and transmissibility. Variants have also developed resistance to antivirals and vaccination and can escape the immune response of humans. Reverse transcription polymerase chain reaction (RT-PCR) is the method of choice among diagnostic techniques, including nucleic acid amplification tests (NAATs), serological tests, and diagnostic imaging, such as computed tomography (CT). The limitation of RT-PCR is that it cannot distinguish fragmented RNA genomes from live transmissible viruses. Thus, SARS-CoV-2 isolation by using cell culture has been developed and makes important contributions in the field of diagnosis, development of antivirals, vaccines, and SARS-CoV-2 virology research. In this research, two SARS-CoV-2 strains were isolated from four RT-PCR-positive nasopharyngeal swabs using VERO E6 cell culture. One isolate was cultured successfully with a blind passage on day 3 post inoculation from a swab with a Ct > 35, while the cells did not develop cytopathic effects without a blind passage until day 14 post inoculation. Our results indicated that infectious SARS-CoV-2 virus particles existed, even with a Ct > 35. Cultivable viruses could provide additional consideration for releasing the patient from quarantine. The results of the whole genome sequencing and bioinformatic analysis suggested that these two isolates contain a spike 68-76del+spike 675-679del double-deletion variation. The double deletion was confirmed by amplification of the regions spanning the spike gene deletion using Sanger sequencing. Phylogenetic analysis revealed that this double-deletion variant was rare (one per million in public databases, including GenBank and GISAID). The impact of this double deletion in the spike gene on the SARS-CoV-2 virus itself as well as on cultured cells and/or humans remains to be further elucidated.

Automated summary

COVID-19 is a life-threatening pulmonary disease caused by SARS-CoV-2. The virus can be transmitted through respiratory droplets. The use of cell culture for isolating the virus plays an important role in diagnosis, research, and development of antivirals and vaccines.