Emerging SARS-CoV-2 Genotypes Show Different Replication Patterns in Human Pulmonary and Intestinal Epithelial Cells

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) quickly spread worldwide following its emergence in Wuhan, China, and hit pandemic levels. Its tremendous incidence favoured the emergence of viral variants. The current genome diversity of SARS-CoV-2 has a clear impact on epidemiology and clinical practice, especially regarding transmission rates and the effectiveness of vaccines. In this study, we evaluated the replication of different SARS-CoV-2 isolates representing different virus genotypes which have been isolated throughout the pandemic. We used three distinct cell lines, including Vero E6 cells originating from monkeys; Caco-2 cells, an intestinal epithelium cell line originating from humans; and Calu-3 cells, a pulmonary epithelium cell line also originating from humans. We used RT-qPCR to replicate different SARS-CoV-2 genotypes by quantifying the virus released in the culture supernatant of infected cells. We found that the different viral isolates replicate similarly in Caco-2 cells, but show very different replicative capacities in Calu-3 cells. This was especially highlighted for the lineages B.1.1.7, B.1.351 and P.1, which are considered to be variants of concern. These results underscore the importance of the evaluation and characterisation of each SARS-CoV-2 isolate in order to establish the replication patterns before performing tests, and of the consideration of the ideal SARS-CoV-2 genotype-cell type pair for each assay.

Keywords: COVID-19; Caco-2 cells; Calu-3 cells; SARS-CoV-2; Vero E6 cells; genotype; isolate; variants; viral culture.

Figure 1

Replication rate of different SARS-CoV-2 lineages isolated in distinct cell lines. The replication rate of 36 SARS-CoV-2 isolates is expressed in delta CT (cycle threshold), three days post-infection, in cells derived from monkey kidney epithelium (Vero E6 in green), human intestines (Caco-2 in pink) and human lungs (Calu-3 in blue). Each isolate was classified according to its lineage, based on the PANGO classification, and viruses from the same lineage were grouped together.

Figure 2

Replication levels of different SARS-CoV-2 lineages in three distinct cell lines. The replication levels of the SARS-CoV-2 lineages (PANGO) are expressed in delta CT (cycle threshold), three days post-infection, in three distinct cell lines: (a) Vero E6, (b) Caco-2, and (c) Calu-3. The values with different superscript letters in a column are significantly different (p < 0.05). (d) Comparison of the replication of a SARS-CoV-2 isolate from each viral lineage inoculated on different cells: Vero E6 (Green), Caco-2 (Pink) and Calu-3 (Blue).

Figure 3

Genetic diversity of the SARS-CoV-2 isolates used. (a) Phylogenetic analysis of isolated SARS-CoV-2 presented through a maximum likelihood tree, with a bootstrap analysis based on the values of 1,000 replicates. The isolates were grouped according to the PANGO classification, based on the analysis previously obtained from the Nextclade tool. (b) Distribution of the mutations that generate amino acid exchange in structural proteins of SARS-CoV-2 among the isolates used in the study. Arrows link the isolates to their identified mutations; the size of the nodes represents the gradient incoming (the most shared mutations are in bigger circles). Legend: E, envelope protein; M, membrane protein; N, nucleocapsid protein; S, spike protein.