Prolonged SARS-CoV-2 Infection and Intra-Patient Viral Evolution in an Immunodeficient Child

Abstract

Background: With the progression of the Coronavirus disease pandemic, the number of mutations in the viral genome has increased, showing the adaptive evolution of severe acute respiratory syndrome coronavirus 2 in humans and intensification in transmissibility. Long-term infections also allow the development of viral diversity. In this study, we report the case of a child with severe combined immu presenting a prolonged severe acute respiratory syndrome coronavirus 2 infection. We aimed to analyze 3 naso-oropharyngeal swab samples collected between August and December 2021 to describe the amino acid changes present in the sequence reads that may have a role in the emergence of new viral variants.

Methods: The whole genome from clinical samples was sequenced through high throughput sequencing and analyzed using a workflow to map reads and then find variations/single-nucleotide polymorphisms. In addition, the samples were isolated in cell culture, and a plaque forming units assay was performed, which indicates the presence of viable viral particles.

Results: The results obtained showed that the virus present in all samples is infectious. Also, there were 20 common mutations among the 3 sequence reads, found in the ORF1ab and ORF10 proteins. As well, a considerable number of uncommon mutations were found.

Conclusions: In conclusion, we emphasize that genomic surveillance can be a useful tool to assess possible evolution signals in long-term patients.

FIGURE 1.

Samples inoculation in cell culture. A: Cytopathic effect on cultures of samples 1, 2 and 3, observed 4 days after infection. PC= positive control (Gamma variant stock inoculated at MOI* of 0.01); CN= negative control. Photos obtained from the 20× objective, 400 µm scale. B: Figure showing viral titer in the supernatant of samples inoculated into Vero cell culture (PFU/mL), and results of viral RNA quantification in successive passages in culture, obtained by Quantitative reverse transcription Polymerase Chain Reaction. *MOI = Multiplicity of infection; multiplicity of infection or ratio of viral particles to each cell.

FIGURE 2.

Phylogenetic tree based on the SARS-CoV-2 complete genomes of the samples, pointing to the microevolution occurred in the course of infection.

FIGURE 3.

Profile of common mutations among the sequences reads and their frequency. Below, it is possible to see the distribution of the amino acid changes in the specific genes of these SARS-CoV-2 samples.