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Isolation and Identification of a Rare Spike Gene Double-Deletion SARS-CoV-2 Variant From the Patient With High Cycle
Li-Teh Liu1, Jih-Jin Tsai2,3,4, Chun-Hong Chen5,6
1Department of Medical Laboratory Science and Biotechnology, College of Medical Technology, Chung-Hwa University of Medical Technology, Tainan, Taiwan.
Insights
Researchers isolated infectious SARS-CoV-2 virus particles from samples with high cycle threshold (Ct) values, indicating viable virus even when RT-PCR is inconclusive. This finding has implications for patient quarantine and understanding SARS-CoV-2 variants.
Area of Science:
- Virology
- Infectious Diseases
- Molecular Biology
Background:
- Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is a significant global health threat.
- Viral evolution has led to variants with increased transmissibility and immune evasion.
- Limitations of RT-PCR include inability to differentiate live virus from fragmented RNA.
Purpose of the Study:
- To isolate infectious SARS-CoV-2 from RT-PCR positive samples.
- To investigate the presence of viable virus in samples with high Ct values.
- To identify and characterize novel SARS-CoV-2 variants.
Main Methods:
- SARS-CoV-2 isolation using VERO E6 cell culture from nasopharyngeal swabs.
- Reverse transcription polymerase chain reaction (RT-PCR) for initial detection.
- Whole genome sequencing, Sanger sequencing, and phylogenetic analysis for variant characterization.
Main Results:
- Two SARS-CoV-2 strains were successfully isolated from RT-PCR positive samples.
- Infectious virus was detected even in samples with a Ct value greater than 35.
- A rare double-deletion variant in the spike gene (spike 68-76del+spike 675-679del) was identified.
Conclusions:
- Infectious SARS-CoV-2 can be present in samples with high Ct values, impacting quarantine guidelines.
- The identified double-deletion variant is rare and requires further study to understand its biological significance.
- Cell culture isolation remains crucial for virology research and diagnostic advancements.
Abstract:
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.
