Convergent Evolution of A-Lineage (Clade 19B) SARS-CoV-2 Spike Sequences with B-Lineage Variants of Concern Affects Virus Replication in a Temperature-Dependent Manner on Human Nasal Epithelial Cell Cultures

Abstract

The first three months of the COVID-19 pandemic was dominated by two SARS-CoV-2 lineages: A-lineages (Clade 19B) and B-lineages (Clade 19A). However, with the emergence of the Spike D614G substitution in B.1 lineages (Clade 20A), both early lineages were outcompeted and remained near-extinction from mid-2020 onwards. In early-2021, there was a re-emergence and persistence of novel A-lineage variants with substitutions in the Spike gene resembling those found in Variants of Concern (VOCs). An early A.3 variant (MD-HP00076/2020) and three A.2.5 variants (MD-HP02153/2021, MD-HP05922/2021 and CA-VRLC091/2021) were isolated and characterized for their genomic sequences, antibody neutralization, andin vitroreplication. All A.2.5 isolates had five Spike mutations relative to the A.3 variant sequence: D614G, L452R, Δ141-143, D215A, and ins215AGY. Plaque reduction neutralization assays demonstrated that A.2.5 isolates had a 2.5 to 5-fold reduction in neutralization using contemporaneous COVID-19 convalescent plasma when compared to A.3.In vitroviral characterization in VeroE6 cell lines revealed that the A.3 isolate grew faster and spread more than A.2.5. On VeroE6-TMPRSS2 cells, significant syncytia formation was also observed with the A.2.5 isolates, however Spike cleavage efficiency did not explain these differences. In human nasal epithelial cell (hNEC) cultures, the A.2.5 isolates grew significantly faster and to higher total infectious virus titers than A.3. All A.2.5 lineage isolates grew significantly faster at 37°C than at 33°C irrespective of cell type, and to higher peak titers except compared to A.3. This suggests A.2.5’s adapted to improve replication using similar mutations found in the B-lineage SARS-CoV-2 variants.ImportanceWhile both A- and B-lineage SARS-CoV-2 variants emerged and circulated together during the early months of the pandemic, the B-lineages that acquired Spike D614G eventually outcompeted all other variants. We show that the A-lineage variants eventually evolved mutations including Spike D614G and Spike L452R that improved their in vitro replication in human nasal epithelial cells in a temperature dependent manner, suggesting there are some highly selectable mutation landscapes that SARS-CoV-2 can acquire to adapt to replication and transmission in humans.

SARS-CoV-2 variants from long-term, persistently infected immunocompromised patients have altered syncytia formation, temperature-dependent replication, and serum neutralizing antibody escape

“…Omicron replication, but have not been previously used to investigate variants derived from immunocompromised individuals [49], [50]. The physiological relevance of polarized hNEC cultures can reveal virus fitness differences not apparent on widely used immortalized cell line models [26], [30], [51], [52], [53]. On hNECs, there were no differences in total virus production between the Patient 2 virus isolates at 33°C, though some timepoints displayed slight differences across the isolates (Fig.…”

“…We and others have previously shown that physiological temperature ranges can alter influenza A virus and live attenuated influenza A virus replication kinetics on immortalized and primary cell cultures, and influenza B virus hemagglutinin protein expression is increased at cooler temperatures corresponding to the upper airway [51], [52], [53], [72], [73]. SARS-CoV-2 replication on hNECs is also variable at 33°C versus 37°C, and early A-lineage SARS-CoV-2 isolates show temperature-dependent differences in replication on both Vero/TMPRSS2 cells and hNECs [30], [74]. Comparisons using pseudoviruses bearing the SARS-CoV-2 Spike protein versus other human coronavirus Spikes using indicate that Spike is a key driver of coronavirus temperature preferences, and psuedovirus infectivity was heightened at 33°C in the case of SARS-CoV-2 but not SARS-CoV Spike [47].…”

“…Vero/TMPRSS2 cells were grown on 24-well plates to 100% confluency, washed once with IM and infected at an MOI of 0.01 [30], [31]. Four replicate wells were infected per virus.…”

“…SARS-CoV-2 infectious virus titers were determined by TCID 50 [30], [31]. Vero/TMPRSS2 cells were grown on 96-well plates until 80% confluence.…”

“…All mutations are relative to the 2020 Wuhan-Hu-1 reference genome (NCBI Reference Sequence: NC_045512.2). Tissue Culture Infectious Dose (TCID50) Assay SARS-CoV-2 infectious virus titers were determined by TCID50 [30], [31]. Vero/TMPRSS2 cells were grown on 96-well plates until 80% confluence.…”