Depicting SARS-CoV-2 faecal viral activity in association with gut microbiota composition in patients with COVID-19

Abstract

Objective: Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was detected in faeces of patients with COVID-19, the activity and infectivity of the virus in the GI tract during disease course is largely unknown. We investigated temporal transcriptional activity of SARS-CoV-2 and its association with longitudinal faecal microbiome alterations in patients with COVID-19.

Design: We performed RNA shotgun metagenomics sequencing on serial faecal viral extractions from 15 hospitalised patients with COVID-19. Sequencing coverage of the SARS-CoV-2 genome was quantified. We assessed faecal microbiome composition and microbiome functionality in association with signatures of faecal SARS-CoV-2 infectivity.

Results: Seven (46.7%) of 15 patients with COVID-19 had stool positivity for SARS-CoV-2 by viral RNA metagenomic sequencing. Even in the absence of GI manifestations, all seven patients showed strikingly higher coverage (p=0.0261) and density (p=0.0094) of the 3' vs 5' end of SARS-CoV-2 genome in their faecal viral metagenome profile. Faecal viral metagenome of three patients continued to display active viral infection signature (higher 3' vs 5' end coverage) up to 6 days after clearance of SARS-CoV-2 from respiratory samples. Faecal samples with signature of high SARS-CoV-2 infectivity had higher abundances of bacterial species Collinsella aerofaciens, Collinsella tanakaei, Streptococcus infantis, Morganella morganii, and higher functional capacity for nucleotide de novo biosynthesis, amino acid biosynthesis and glycolysis, whereas faecal samples with signature of low-to-none SARS-CoV-2 infectivity had higher abundances of short-chain fatty acid producing bacteria, Parabacteroides merdae, Bacteroides stercoris, Alistipes onderdonkii and Lachnospiraceae bacterium 1_1_57FAA.

Conclusion: This pilot study provides evidence for active and prolonged 'quiescent' GI infection even in the absence of GI manifestations and after recovery from respiratory infection of SARS-CoV-2. Gut microbiota of patients with active SARS-CoV-2 GI infection was characterised by enrichment of opportunistic pathogens, loss of salutary bacteria and increased functional capacity for nucleotide and amino acid biosynthesis and carbohydrate metabolism.

Keywords: diagnostic virology; gut inflammation; infectious disease.

Figure 1

Timeline of patient symptom onset, hospitalisation, throat (nasopharyngeal) swab clearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and discharge, through the course of disease for 15 patients hospitalised with COVID-19.

Figure 2

Hypothetical scenarios for the fecal viral RNA metagenomic profile of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in association with its infectivity in gut. (A) Schematic presentation of the full-length genome, transcribed subgenomic RNAs (sgRNA) and the virion structure of SARS-CoV-2 virus. (B) Three scenarios hypothesised for the presence and infectivity of SARS-CoV-2 in the gut of patients with COVID-19, and the detection of SARS-CoV-2 virus by faecal viral RNA metegenomics sequencing. If SARS-CoV-2 virus infects the host cells in the gut, its genomic and sgRNAs should be highly expressed and released into the gut lumen on cytolysis, where the 3’ end of SARS-CoV-2 genome should be highly covered by faecal viral RNA metagenomics sequencing.

Figure 3

Depiction of the viral infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in serial faeces of patients with COVID-19. Infectivity of SARS-CoV-2 virus in gut was investigated by faecal viral RNA metagenomics coverage profile of SARS-CoV-2 genome. (A) The subset of patients who manifested higher 3’ vs 5’ end coverage of the SARS-CoV-2 genome (signature of active SARS-CoV-2 infectivity) before throat swab turned negative for SARS-CoV-2. (B) The subset of patients who manifested higher 3’ vs 5’ end coverage of the SARS-CoV-2 genome (signature of SARS-CoV-2 infectivity) but gradually lost this signature over time of hospitalisation before throat swab turned negative for SARS-CoV-2. (C) The subset of patients who manifested higher 3’ vs 5’ end coverage of the SARS-CoV-2 genome (signature of SARS-CoV-2 infectivity) after throat swab turned negative for SARS-CoV-2. ‘Day 0’ is defined as the date when throat swab turned negative for SARS-CoV-2, as measured by RT-PCR. (D) The coverage and density of the 5’ and 3’ ends of the SARS-CoV-2 genome in COVID-19 faecal viral RNA metagenome. The baseline (the date of first stool collection after hospitalisation) faecal viral RNA metagenomes of the seven patients who were detected faecal positive for SARS-CoV-2 were plotted and subject to comparison. Coverage was defined as the number of shotgun reads mapped to a given genomic region of SARS-CoV-2 genome. Density was defined as the frequency of sequenced sites in a given genomic region of SARS-CoV-2 genome.

Figure 4

Differential bacterial species and functional capacities between faeces with high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity and faeces with low-to-none SARS-CoV-2 infectivity. Differential bacterial species (A) and functionality (B) were identified via LefSE analysis across all time-point stools of 15 patients with COVID-19. Only species and functional modules with LDA effect size >2 and FDR-corrected p value <0.05 were plotted. High SARS-CoV-2 infectivity was defined as higher 3’ vs 5’ end coverage of SARS-CoV-2 genome in faecal viral RNA metagenome. Low-to-none SARS-CoV-2 infectivity was defined as similar 3’ and 5’ end coverage or no coverage of the SARS-CoV-2 genome in faecal viral RNA metagenome.

Figure 5

Longitudal changes in the faecal microbiome of patients with COVID-19 in association with faecal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity. Patients 3 and 7 had serial stools displaying positive to negative faecal SARS-CoV-2 infectivity during follow-up, while patients 11, 12 and 15 had serial stools constantly displaying a signature high viral infectivity during follow-up. Only the most abundant 20 species were plotted and shown in relative abundance.