Discovery and Genomic Characterization of a 382-Nucleotide Deletion in ORF7b and ORF8 during the Early Evolution of SARS-CoV-2

Abstract

To date, limited genetic changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described. Here, we report a 382-nucleotide (nt) deletion in SARS-CoV-2 that truncates open reading frame 7b (ORF7b) and ORF8, removing the ORF8 transcription regulatory sequence (TRS) and eliminating ORF8 transcription. The earliest 382-nt deletion variant was detected in Singapore on 29 January 2020, with the deletion viruses circulating in the country and accounting for 23.6% (45/191) of SARS-CoV-2 samples screened in this study. SARS-CoV-2 with the same deletion has since been detected in Taiwan, and other ORF7b/8 deletions of various lengths, ranging from 62 nt to 345 nt, have been observed in other geographic locations, including Australia, Bangladesh, and Spain. Mutations or deletions in ORF8 of SARS-CoV have been associated with reduced replicative fitness and virus attenuation. In contrast, the SARS-CoV-2 382-nt deletion viruses showed significantly higher replicative fitness in vitro than the wild type, while no difference was observed in patient viral load, indicating that the deletion variant viruses retained their replicative fitness. A robust antibody response to ORF8 has been observed in SARS-CoV-2 infection, suggesting that the emergence of ORF8 deletions may be due to immune-driven selection and that further deletion variants may emerge during the sustained transmission of SARS-CoV-2 in humans. IMPORTANCE During the SARS epidemic in 2003/2004, a number of deletions were observed in ORF8 of SARS-CoV, and eventually deletion variants became predominant, leading to the hypothesis that ORF8 was an evolutionary hot spot for adaptation of SARS-CoV to humans. However, due to the successful control of the SARS epidemic, the importance of these deletions for the epidemiological fitness of SARS-CoV in humans could not be established. The emergence of multiple SARS-CoV-2 strains with ORF8 deletions, combined with evidence of a robust immune response to ORF8, suggests that the lack of ORF8 may assist with host immune evasion. In addition to providing a key insight into the evolutionary behavior of SARS-CoV-2 as the virus adapts to its new human hosts, the emergence of ORF8 deletion variants may also impact vaccination strategies.

SARS-CoV-2 spike D614G variant exhibits highly efficient replication and transmission in hamsters

“…SARS-CoV-2 is of zoonotic origin and is currently in the process of becoming more adapted to humans as it circulates and acquires adaptative mutations. Different variants have already been identified among clinical specimens and in cultured isolates (Gong et al, 2020;Su et al, 2020). Some deletion or mutation variants may not be recognized by general sequencing protocols, for which the readout only shows the dominant population in clinical specimens or cell culture samples, but can be detected by more sensitive methods (Wong et al, 2020).…”

Structure of SARS-CoV-2 ORF8, a rapidly evolving coronavirus protein implicated in immune evasion

“…While ORF8 expression is not strictly essential for SARS-CoV and SARS-CoV-2 replication, a 29 nucleotide deletion (Δ29) that occurred early in human to human transmission of SARS-CoV, splitting ORF8 into ORF8a and 8b, is correlated with milder disease (8). A 382 nucleotide deletion (Δ382) in SARS-CoV-2 (9,10) was also found to correlate with milder disease and a lower incidence of hypoxia (11). SARS-CoV-2 ORF8 is a 121 amino acid protein consisting of an N-terminal signal sequence followed by a predicted Ig-like fold (12).…”

Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

“…In 2003, the SARS-like coronavirus characterized from civet cats and earlyoutbreak human SARS-CoV isolates contained a 29-bp sequence in the ORF8 sequence which was deleted following its subsequent circulation in humans 29 . Deletions in ORF7b and ORF8 have also been observed in SARS-CoV-2, although the significance of these alterations is currently unknown 30,31 . It remains to be seen if continued evolution of SARS-CoV-2 in humans will subsequently select for less pathogenic variants similar to Ca-DelMut, or with other mutations.…”