Rowe, L. A. | Search bioRxiv

bioRxiv2023

Real time analysis of SARS-CoV-2 induced cytolysis reveals distinct variant-specific replication profiles

The continuous evolution of new SARS-CoV-2 variants with enhanced immune evasion capacity suggests the entire population is and will continue to be potentially vulnerable to infection despite pre-existing immunity. The ability of each new variant to evade host humoral immunity is the focus of intense research across the globe. Each variant may also harbor unique replication capabilities relevant for disease and transmission. Here we demonstrate the utility of a new approach to assessing viral replication kinetics using Real Time Cell Analysis (RTCA). Virus induced cell death is measured in real time by the detection of electrical impedance through cell monolayers. Using this system, we quantified replication kinetics of five clinically important viral variants; USA WA1/2020 (an A1 ancestral lineage isolate), Delta, and Omicron subvariants BA.1, BA.4, and BA.5. We identified multiple kinetic measures that proved useful in variant replication comparisons including time (in hours) to the maximum rate of cell death at each log10 viral dilution and the slope at the maximum rate of cell death. We found that WA1/2020 and Delta were the most rapid but in distinct ways. While WA1/2020 induced cell death most rapidly after inoculation, Delta was slightly slower to reach cell death, it appeared to kill cells faster once cytotoxic effects began. Interestingly, BA.1, showed substantially reduced replication kinetics relative to all other variants. Together, these data show that real time analysis of cell death is a robust method to assess replicative capacity of any given SARS-CoV-2 variant rapidly and quantitatively, which may be useful in assessment of newly emerging variants.

microbiology↗

bioRxiv2021

Intra-host SARS-CoV-2 evolution in the gut of mucosally-infected Chlorocebus aethiops (African green monkeys)

In recent months, several SARS-CoV-2 variants have emerged that enhance transmissibility and escape host humoral immunity. Hence, the tracking of viral evolutionary trajectories is clearly of great importance. Little is known about SARS-CoV-2 evolution in nonhuman primate models used to test vaccines and therapies and to model human disease. Viral RNA was sequenced from rectal swabs from Chlorocebus aethiops (African green monkeys) after experimental respiratory SARS-CoV-2 infection. Two distinct patterns of viral evolution were identified that were shared between all collected samples. First, mutations in the furin cleavage site that were initially present in the virus as a consequence of VeroE6 cell culture adaptation were subsequently lost in virus recovered in rectal swabs, confirming the necessity of this motif for viral infection in vivo. Three amino acid changes were also identified; ORF 1a S2103F, and spike D215G and H655Y, that were detected in rectal swabs from all sampled animals. These findings are demonstrative of intra-host SARS-CoV-2 evolution unique to this nonhuman primate species and may identify a host-adapted variant of SARS-CoV-2 that would be useful in future development of primate disease models.

microbiology↗