Viable SARS-CoV-2 Omicron sub-variants isolated from autopsy tissues

Abstract

Introduction: Pulmonary and extrapulmonary manifestations have been described after infection with SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). The virus is known to persist in multiple organs due to its tropism for several tissues. However, previous reports were unable to provide definitive information about whether the virus is viable and transmissible. It has been hypothesized that the persisting reservoirs of SARS-CoV-2 in tissues could be one of the multiple potentially overlapping causes of long COVID.

Methods: In the present study, we investigated autopsy materials obtained from 21 cadaveric donors with documented first infection or reinfection at the time of death. The cases studied included recipients of different formulations of COVID-19 vaccines. The aim was to find the presence of SARS-CoV-2 in the lungs, heart, liver, kidneys, and intestines. We used two technical approaches: the detection and quantification of viral genomic RNA using RT-qPCR, and virus infectivity using permissive in vitro Vero E6 culture.

Results: All tissues analyzed showed the presence of SARS-CoV-2 genomic RNA but at dissimilar levels ranging from 1.01 × 10² copies/mL to 1.14 × 10⁸ copies/mL, even among those cases who had been COVID-19 vaccinated. Importantly, different amounts of replication-competent virus were detected in the culture media from the studied tissues. The highest viral load were measured in the lung (≈1.4 × 10⁶ copies/mL) and heart (≈1.9 × 10⁶ copies/mL) samples. Additionally, based on partial Spike gene sequences, SARS-CoV-2 characterization revealed the presence of multiple Omicron sub-variants exhibiting a high level of nucleotide and amino acid identity among them.

Discussion: These findings highlight that SARS-CoV-2 can spread to multiple tissue locations such as the lungs, heart, liver, kidneys, and intestines, both after primary infection and after reinfections with the Omicron variant, contributing to extending knowledge about the pathogenesis of acute infection and understanding the sequelae of clinical manifestations that are observed during post-acute COVID-19.

Keywords: SARS-CoV-2; Vero E6 cell line; omicron variants; postmortem tissue; virus isolation.

Figure 1

(A) Heat map representing the SARS-CoV-2 viral load recorded for each autopsy case from different tissues (including its homogenate and infectivity measurement in cell culture). A grid of colored squares ranging from green (minimum) to red (maximum) is used to represent the values of the viral load (expressed as copies/mL). The gray cells indicate an undetectable viral load, while “NA” indicates that the sample was not available. (B) The SARS-CoV-2 viral load (RNA copies/mL) was measured directly from tissue extracts (TE) and after 7-day-cell culture virus isolation (VI). The ORF1ab and N viral genes were detected in different tissues (lung, heart, liver, kidney, and small intestine) extract and culture supernatants from cell culture virus isolation.

Figure 2

Phylogenetic analysis was performed on 602-base-pair-long nucleotide sequences of the SARS-CoV-2 Spike gene using the Neighbor Joining method with 1,000 bootstrap replicates. The lateral branches displayed the Omicron variant (as well as its sub-variants and recombinants represented by dashed lines), while the initially black branches represented the remaining non-Omicron variants. Each SARS-CoV-2 variant was accompanied by its reference sequence. The nucleotide sequences of SARS-CoV-2 isolates identified from autopsy materials were named according to the code provided in Table 1. Viral isolates from different tissues/cases were identified with a particular diamond color, while white diamonds represented unique viral isolates/cases. The numbers marked along the branches represented the bootstrap values higher than 0.7 out of 1,000 bootstrap resamplings. The tree scale depicted the number of substitutions/site.

Figure 3

WebLogo profiles of SARS-CoV-2 receptor binding domain partial amino acid sequences (aa 359–558) characterized in viral isolates from different tissues are shown above. The upper profile represents sequences from the pulmonary tissue, the center profile represents sequences from the heart tissue, and the lower profile represents sequences from other tissues, including kidney, liver, and intestine. The size of the letter (bits; Y axis) indicates the frequency of the amino acid substitutions at a certain residue position (X axis). Different residues at the same position are scaled according to their frequency and colored based on their amino acid characteristics. An arrow is used to indicate RBD amino acid positions with a distinctive but infrequent residue.