Here, we enrolled 98 hospitalized patients with COVID-19 (mean age 33 years; 53% male) and 78 non-COVID-19 controls matched for gender and co-morbidities (mean age 48 years; 42% male, Table 1). All COVID-19 patients and non-COVID-19 controls had stool specimens sampled at inclusion. Blood specimens were additionally sampled for COVID-19 patients at admission to test for pro-inflammatory markers and white cell count (Supplementary Table 1). Amongst these COVID-19 patients, 37 (38%) had serial faecal samples collected from hospitalization until after discharge (Supplementary Figure 1). We enriched both faecal RNA and DNA virions from a total of 277 faecal samples and performed non-targeted shotgun metagenomic sequencing on the RNA virome (mostly eukaryotic viruses) and DNA virome (mostly prokaryotic bacteriophages). We reported gut virome profiles in association with SARS-CoV-2 infection, disease severity, and blood parameters.
Table1. Clinical Characteristics of COVID-19 patients and non-COVID-19 controls
Variables
|
COVID-19 cases
|
non-COVID-19 controls
|
Number
|
99
|
78
|
Male
|
52 (53%)
|
33 (42%)
|
Age, years (mean±s.e.)
|
37 (± 2)
|
45 (±2)
|
Co-morbidities
|
55 (56%)
|
24 (31%)
|
Symptoms at admission
|
|
|
Fever
|
38 (38%)
|
Gastrointestinal symptoms
|
|
Diarrhea
|
17 (17%)
|
Respiratory symptoms
|
|
Cough
|
40 (40%)
|
Sputum
|
18 (18%)
|
Rhinorrhea
|
19 (19%)
|
Shortness of breath
|
9 (9%)
|
Blood result
|
|
|
Lymphocyte counts (x109/L, normal range 1.1-2.9, median (IQR))
|
1.2 (1.0, 1.7)
|
Death
|
0 (0%)
|
|
Alterations in faecal RNA virome of COVID-19 patients
To understand whether SARS-CoV-2 infection influences the gut RNA virome, we compared faecal RNA virome of COVID-19 patients at baseline (Day 0, the first timepoint of stool collection after hospitalization) with that of non-COVID-19 controls. Among all host factors (SARS-CoV-2 infection, age, gender, medications, co-morbidities), SARS-CoV-2 infection showed the largest effect size on impacting composition of faecal RNA virome (permanova test p<0.01, R2=0.041, Figure 1a) followed by the chronic hepatitis B (HBV) infection and asthma. At the species level, SARS-CoV-2 was enriched in faecal samples of patients with COVID-19 compared with non-COVID-19 controls (via MaAsLin2 analysis with adjustment for chronic hepatitis B infection and asthma, FDR p<0.05, Figure 1b). In contrast, Pepper mild mottle virus (PMMoV), a plant virus known to be prevalent and abundant in human feces [17], was underrepresented in patients with COVID-19 (FDR p<0.05, Figure 1b&c). Seven (19%) of the 37 COVID-19 patients with longitudinal follow-up showed prolonged faecal SARS-CoV-2 shedding after nasopharyngeal clearance of the virus (Supplementary Figure 2a). PMMoV virus was persistently underrepresented both during hospitalization and after disease resolution in COVID-19 patients (Figure 1c, Supplementary Figure 2b). Overall, the faecal RNA virome composition of COVID-19 patients markedly differed from that of non-COVID-19 controls during the disease course and after disease resolution (Figure 1d&e). Among 16 COVID-19 patients who had nasopharyngeal clearance of SARS-CoV-2 virus (disease resolution as determined by negative PCR result for SARS-CoV-2 on nasopharyngeal swab and/or deep throat saliva), eight (50%) had persistently altered faecal RNA virome after disease resolution and two (13%) lasted up to 30 days (Figure 1d).
Alterations in faecal DNA virome of COVID-19 patients
We then investigated the effect of SARS-CoV-2 infection on faecal DNA virome composition at baseline and during disease course. At the community level, viromes of COVID-19 patients at baseline differed significantly from that of non-COVID-19 controls (permanova p<0.01, Figure 2a) and were more heterogeneous than that of non-COVID-19 controls (p<0.0001, Figure 2b). Among all host factors (SARS-CoV-2 infection, age, gender, medications, co-morbidities), SARS-CoV-2 infection again showed the largest effect size on impacting composition of faecal DNA virome (R2=0.018, Figure 2c) followed by hyperlipidemia and antiviral drug (Lopinavir-ritonavir). A total of 45 DNA virus species were found to be significantly different in faecal DNA virome of COVID-19 patients relative to non-COVID-19 controls (19 virus species enriched in COVID-19 patients versus 26 virus species enriched in non-COVID-19 controls, identified via DESeq while controlling for the factors Hyperlipidemia and Lopinavir-ritonavir, shown in Figure 2d). A majority (69%, 18 out of 26 virus species) of the DNA viruses enriched in feces of non-COVID-19 controls were prokaryotic viruses particularly bacteriophages (62%). In contrast, more eukaryotic viruses as opposed to prokaryotic viruses particularly environment-derived eukaryotic viruses with unknown host were enriched in feces of COVID-19 patients. The bacteriophages, Escherichia virus (phage) and Enterobacter phage, were also enriched in COVID-19 patients (Figure 2d). Expansion of these phages has been causally implicated in gut inflammation and host interferon response in mice and humans [14, 18]. These data highlight enteric virome alteration is likely a consequence of SARS-CoV-2 infection which may further contribute to the immunological and physiological changes in the host during the disease course.
The differentially enriched gut DNA virus species in COVID-19 patients showed substantial temporal variations during the disease course (Figure 2e) suggesting unstable gut DNA virome. Overall, faecal DNA virome composition of COVID-19 patients differed markedly from that of non-COVID-19 controls during the disease course and after clearance of SARS-CoV-2 (Figure 2f&g). Among COVID-19 patients who had follow-up after disease resolution, six (32%) showed marked more dissimilar faecal DNA virome to non-COVID-19 controls at the last follow-up (three patients lasted up to 20-30 days), compared to their dissimilarity to non-COVID-19 controls at baseline (Figure 2f).
Alterations in functionality of the enteric virome in COVID-19 patients
We next investigated functionality alterations of the gut virome using HUMAnN2 predication. More gene families were enriched in COVID-19 viromes at baseline than non-COVID-19 controls (28 versus 9 gene families, FDR p<0.05, Figure 3). We found significant enhancement in the functional capacity of gene mobilization and phage integration into the host in COVID-19 viromes (Figure 3). Features of viral integration (expansion of temperate virions) have been observed in the gut under inflammatory conditions in both humans and mice [18, 19]. In addition, functions involved in host stress/inflammation/virulence response (DNA repair, Arginine repressor, Hemolysin channel protein, DNA polymerase IV), bacterial metabolism and membrane transport were also enriched in the faecal virome of COVID-19 patients (Figure 3). These data highlight that a SARS-CoV-2 infection may induce a functionality shift of the gut virome to inflammation- and stress-responsive in association with their hosts (both the commensal bacteria and humans).
Faecal virome alterations correlated with disease severity of COVID-19
Based on COVID-19 disease symptoms and severity classification criteria [20], we stratified our patients into non-severe (N=56; asymptomatic/mild cases) and moderate/severe groups (N=62; moderate/severe/critical cases) (Figure 4a). Compared to non-severe cases, moderate/severe cases showed a significantly higher blood levels of LDH, neutrophil count, C-reactive protein (CRP), Alanine aminotransferase (ALT), and lower blood levels of Albumin at admission (all p<0.05, Figure 4b-f, Supplementary Figure 3). Our data are in line with recent reports highlighting that more severe cases had more pronounced systemic inflammatory responses [2, 21-24]. We then explored association between baseline faecal RNA and DNA virome profiles with COVID-19 severity and blood measurements at hospitalization. Abundance of the plant-derived RNA virus, Pepper chlorotic spot virus (PCSV) was higher in patients with non-severe than those with moderate/severe disease (p=0.013, Figure 4g). In addition, a high abundance of PCSV in feces was associated with low blood concentrations of the inflammation markers, LDH and CRP (correlation coefficient Rho=-0.269 and -0.276 respectively, Figure 4h&i). Similarly, abundance of 9 DNA virus species (Myxococcus phage, Rheinheimera phage, Microcystis virus, Bacteroides phage, Murmansk poxvirus, Saudi moumouvirus, Sphaerotilus phage, Tomelloso virus, and Ruegeria phage) in feces negatively correlated with COVID-19 severity (all FDR p<0.05, Figure 4j). In particular, 8 out of the 9 DNA virus species showed strong negative correlation with blood levels of the inflammation indicators LDH, neutrophil count, white cell count, or CRP (Figure 4k). Interestingly, Myxococcus phage, Bacteroides phage, Murmansk poxvirus, and Sphaerotilus phage also inversely correlated with host age (Figure 4k), which may partly account for the observation that elderly individuals were at higher risk for unfavorable severe COVID-19 outcomes[2, 25]. These data suggest that RNA and DNA viruses may counteract the effect of SARS-CoV-2 infection predisposing infected subjects to a less severe COVID-19 course. Five out of the 9 severity-associated DNA virus species showed persistent lower abundance in the feces of COVID-19 patients during disease course and after disease resolution compared to non-COVID-19 controls (all p<0.05, Figure 5), indicating an unfavorable effect of SARS-CoV-2 infection on these gut viruses. The cause or consequence of such associations needs to be further explored. In addition, a large amount of DNA virus species in feces (n=132) showed significant correlations with blood parameters in COVID-19 patients, most of which were negative correlations with blood LDH concentrations, neutrophil and white cell counts (Supplementary Figure 4). These data underscore the potential significance of gut DNA virome in calibrating host immunity and counteracting infection of SARS-CoV-2.