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

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.

Keywords: COVID-19; Ct; RT–PCR; SARS-CoV-2; phylogenetic analysis; spike gene; variant; virus culture.

Figure 1

In-frame deletion and SNV in the spike gene in the SARS-CoV-2 genome. (A) WGS coverage and depths of KMUH-1 and KMUH-2. (B) Genomic regions of the spike gene deletion according to the genomic positions of the reference strain Wuhan-Hu-1/2019. (C) Results of Sanger sequencing of the regions spanning deletions in the spike gene in KMUH-1 and KMUH-2. (D) Deletions in the spike gene were verified by RT–PCR, which showed a reduced amplicon size. (E) Sanger sequencing of nt positions 23,014 and 25,002, which resulted in E484D and S1147 L aa substitutions in the spike protein. The Sanger sequencing results of KMUH-1 were used as a representative.

Figure 2

Phylogenetic analysis of 72 closely related SARS-CoV-2 genomes by the maximum likelihood method. The phylogenetic analysis was inferred by using the maximum likelihood method using TIM2+F+I as the best fitted model with the lowest Baysian information criterion (BIC) scores. An original tree was displayed using FigTree v1.4.4 with bootstrap values and a scale bar. The viruses are shown as the virus name (GISAIG) or accession number (GenBank)/sample collection year (date/pangolin_lineage/GISAID_clade/nextstrain_clade). •: Spike 68-76del; ■: Spike 675-679del; ♦ Spike 68-76del+Spike 675-679del double deletion.

Figure 3

Illustration of the spike gene variants in this study. The spike gene variations in the 72 closely related SARS-CoV-2 genomes were analyzed using Nextclade v1.7.3 using NC_045512.2 SARS-CoV-2 isolate Wuhan-Hu-1/2019 as a reference sequence. Box indicator: Red➔ The two isolates in this study, Orange➔ Phylogenetically closest strains with deletion(s) in the spike gene, which were the earliest recorded and deposited in the GISAID EpiCoV database, and the samples were collected between 2020-01-01 and 2020-11-30, Blue➔ Spike 68-76del, Green➔ Spike 675-679del, and the dotted line indicates the double-deletion variants. Other colorful bars indicate amino acid substitutions.

Figure 4

Phylogenetic analysis by using UShER. The phylogenetic tree was generated with UShER using maximum parsimony. UShER enables real-time phylogenetics for the SARS-CoV-2 pandemic using a phylogenetic tree version containing 5,035,953 genomes from GISAID, GenBank, COG-UK, and CNCB (2021-11-12). The phylogenetic tree data were visualized using Auspice v2.32.0 powered by Nextstrain (53). The 50 nearest neighboring GISAID and/or public sequences already in the UShER phylogenetic tree are shown.