SARS-CoV-2 Variants Reveal Features Critical for Replication in Primary Human Cells

Abstract

Since entering the human population, SARS-CoV-2 (the causative agent of COVID-19) has spread across the world, causing >40 million infections and >1 million deaths. While large-scale sequencing efforts have identified numerous genetic mutations in SARS-CoV-2 during its circulation, it remains largely unclear whether these changes impact adaptation, replication or transmission of the virus in its new host. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all the major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, viral stocks and passaging experiments, with kinetic virus replication data from non-human Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 sequence features that associate with distinct phenotypes. Notably, naturally-occurring substitutions in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G substitution generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E, and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin-cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human respiratory cells. Overall, our study uncovers natural sequence features in the SARS-CoV-2 genome that determine efficient virus replication and tropism for the human respiratory epithelium.

Functional importance of the D614G mutation in the SARS-CoV-2 spike protein

“…SARS-CoV-2 acquired a furin cleavage site not found in SARS-CoV-1 or any other known sarbecovirus. Studies have shown that acquisition of this furin cleavage site could be beneficial to the virus, because mutations at this site decrease viral infectivity [ 4 , 14 , 21 , 58 ], but in other contexts or other mutations of the furin cleavage site, infectivity is increased [ 4 , 35 , 58 , 59 ]. We currently do not know whether viruses without the furin cleavage site would replicate more efficiently in vivo .…”

“…One caveat is that visualization of isogenic D614 and G614 variant SARS-CoV-2 by scanning EM and transmission EM did not reveal significant differences in spike density [ 48 ], which cannot be explained by the masking effect of contaminating S protein. Because many studies have observed deletion of the furin cleavage site in live SARS-CoV-2 in few as two to three passages in Vero cells [ [58] , [59] , [60] , [61] ], care must be taken to ensure that studies of live viruses are not impacted by artifacts of this adaptation. In line with our own observations on VLPs and MLV PVs, Turonova et al visualized live SARS-CoV-2 G614 virions by cryo-EM and observed much greater spike density than was reported in prior studies with D614 viruses [ 61 , 62 ].…”

SARS-CoV-2 one year on: evidence for ongoing viral adaptation

“…Vero E6 cells have been widely used for isolation and growth of SARS-CoV-2 stocks as they are readily available, easy to use and highly permissive to the virus [72]. However, during propagation of SARS-CoV-2 isolates in Vero cells, deletions incorporating, or flanking, the furin cleavage site between S1 and S2 spike subunits are often reported [43,[73][74][75][76][77][78]. Similar deletions have been detected in clinical samples at very low frequency, including from human autopsy samples [79][80][81].…”

“…In addition, several studies linked D614G containing viruses to lower Ct values in clinical SARS-CoV-2 diagnostic PCR tests, indicating the virus replicated more efficiently in the human respiratory tract, although without any link to higher pathogenicity or severe clinical outcomes [30,34,[38][39][40]. Several groups have also shown that D614G containing viruses (either recombinant or naturally occurring strains) had enhanced growth in primary human airway cells and replicated with greater efficiency in animal models such as hamster, ferret or the human ACE2-expressing mouse, and transmitted more efficiently in a hamster model, suggesting D614G alone is sufficient to confer this advantage in the absence of P323L [41][42][43][44][45].…”

A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents

“…We demonstrate that ex vivo models reconstituted from human tracheal or small airway epithelium are permissive for SARS-CoV-2 infection and robustly produces viral progenies from the apical side in longterm experiments (up to 14 days p.i.). Recent studies report on the effect of different SARS-CoV-2 isolates and incubation temperatures on virus replication kinetics [22][23][24][25] . We used two isolates, BavPat1 and GHB-03021, whereby the BavPat1 provedto be more readily infectious.…”