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Siew Ng
Chinese University of Hong Kong
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6850-4454
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Background: Coronavirus Disease 2019 (COVID-19) caused by the enveloped RNA virus SARS-CoV-2 primarily affects the respiratory and gastrointestinal tracts. SARS-CoV-2 was isolated from faecal samples and active viral replication was reported in human intestinal cells. The human gut also harbors an enormous amount of resident viruses (collectively known as the virome) that play a role in regulating host immunity and disease pathophysiology. Understanding gut virome perturbation that underlies SARS-CoV-2 infection and severity is an unmet need.
Methods: We enrolled 98 COVID-19 patients with varying disease severity (3 asymptomatic, 53 mild, 34 moderate, 5 severe, 3 critical) and 78 non-COVID-19 controls matched for gender and co-morbidities. All subjects had faecal specimens sampled at inclusion. Blood specimens were collected for COVID-19 patients at admission to test for inflammatory markers and white cell counts. Among COVID-19 cases, 37 (38%) patients had serial faecal samples collected 2 to 3 times per week from time of hospitalization until after discharge. Using shotgun metagenomics sequencing, we sequenced and profiled the faecal RNA and DNA virome. We investigated alterations and longitudinal dynamics of the gut virome in association with disease severity and blood parameters.
Results: Patients with COVID-19 showed underrepresentation of Pepper mild mottle virus (RNA virus) and multiple bacteriophage lineages (DNA viruses) and enrichment of environment-derived eukaryotic DNA viruses in faecal samples, compared to non-COVID-19 subjects. Such gut virome alterations persisted up to 30 days after disease resolution. Faecal virome in SARS-CoV-2 infection harboured more stress-, inflammation- and virulence-associated gene encoding capacities including those pertaining to bacteriophage integration, DNA repair, and metabolism and virulence associated with their bacterial host. Baseline fecal abundance of 10 virus species (1 RNA virus, Pepper chlorotic spot virus, and 9 DNA virus species) inversely correlated with disease COVID-19 severity. These viruses inversely correlated with blood levels of pro-inflammatory proteins, white cells and neutrophils. Among the 10 COVID-19 severity-associated DNA virus species, 4 showed inverse correlation with age; 5 showed persistent lower abundance both during disease course and after disease resolution relative to non-COVID-19 subjects.
Conclusions: Both enteric RNA and DNA virome in COVID-19 patients were different from non-COVID-19 subjects, which persisted after disease resolution of COVID-19. Gut virome may calibrate host immunity and regulate severity to SARS-CoV-2 infection. Our observation that gut viruses inversely correlated with both severity of COVID-19 and host age may partly explain that older subjects are prone to severe and worse COVID-19 outcomes. Altogether our data highlight the importance of human gut virome in severity and potentially therapeutics of COVID-19.
Keywords
RNA and DNA, Coronavirus Disease 2019, infection, chlorotic spot virus
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Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryTable1.Studysubjectmetadata.xlsx
Supplementary Table 1. Subject characteristics and blood measurements.
- SupplementaryTable2.Dietduringtimeofhospitalisation.xlsx
Supplementary Table 2. Diet regimen during time of hospitalization.
- SupplementaryTable3.viralspeciesabundance.xls
Supplementary Table 3. Viral abundance profile in faecal samples. Abundance is expressed in RPKM.
- SupplementaryFigure1.tif
Supplementary Figure 1. Schematic diagram of COVID-19 patient (n=37) follow-up, including disease onset, admission, stool sample collection, duration of hospital stay. “CoV” denotes patient with COVID-19. Stool specimens were serially collected for separate shotgun metagenomic sequencing of RNA and DNA virome; “SARS-CoV-2 PCR negative in nasopharyngeal test”: the first negative result for SARS-CoV-2 virus in two consecutive negative nasopharyngeal tests, upon which patient was then discharged.
- SupplementaryFigure2.tif
Supplementary Figure 2. Temporal changes of the RNA viruses, SARS-CoV-2 (A) and PMMoV (B), in the faecal RNA virome in each COVID-19 case. CPM, count per million reads.
- SupplementaryFigure3.tif
Supplementary Figure 3. SARS-CoV-2 viral load. Stool SARS-CoV-2 viral load in COVID-19 patients were detected by quantitative RT-PCR (A) and RNA virome shotgun metagenomics sequencing (B) respectively. Between-group comparison was conducted by one-way anova, **p<0.01, *p<0.05. (C) Comparison of SARS-CoV-2 viral loads between nasopharyngeal swab and fecal specimens. Statistical analysis was conducted by Mann-Whitney test. (D) Correlation of levels of SARS-CoV-2 viral load between nasopharyngeal swab and fecal specimens, calculated by Pearson correlation analysis.
- SupplementaryFigure4.tif
Supplementary Figure 4. Heatmap of correlations of COVID-19-enriched faecal viral functions and species. The color and intensity denote the spearman correlation direction and coefficient, where only the significant correlations were shown. Viruses labeled with asterisk were species enriched in COVID-19. Only the most abundant 80 species in faecal virome were plotted and ranked in descending order (from left to right) on the basis of the relative abundance.
- SupplementaryFigure5.tif
Supplementary Figure 5. Blood parameter levels in COVID-19 patients between non-severe and moderate/severe groups. Data are shown in mean±s.e. Statistical significance was performed by Mann-Whitney test.
- SupplementaryFigure6.tif
Supplementary Figure 6. The faecal DNA viruses at patient baseline correlated with blood parameters in COVID-19 patients. The color and intensity denote the spearman correlation direction and coefficient, where only the significant correlations with |correlation coefficient| > 0.3 were shown.
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CURRENT STATUS: Published
View the published article at Microbiome.
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Posted 20 Dec, 2020
No community comments so far
Editorial decision: Accept
On 10 Jan, 2021
Editor assigned
On 07 Dec, 2020
Submission checks complete
On 07 Dec, 2020
Editor invited
On 07 Dec, 2020
No comments provided
Review #3 received
Received 01 Nov, 2020
Editorial decision: Minor revision
On 01 Nov, 2020
Review #2 received
Received 20 Oct, 2020
Review #1 received
Received 18 Oct, 2020
Reviewer #3 agreed
On 12 Oct, 2020
Reviewer #2 agreed
On 23 Sep, 2020
Reviewer #1 agreed
On 22 Sep, 2020
Reviewers invited
Invitations sent on 14 Sep, 2020
Editor invited
On 02 Sep, 2020
Editor assigned
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A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
See the published version of this preprint at Microbiome.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Siew Ng
Chinese University of Hong Kong
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6850-4454
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Microbiome.
Version 2
Posted 20 Dec, 2020
No community comments so far
Editorial decision: Accept
On 10 Jan, 2021
Editor assigned
On 07 Dec, 2020
Submission checks complete
On 07 Dec, 2020
Editor invited
On 07 Dec, 2020
No comments provided
Review #3 received
Received 01 Nov, 2020
Editorial decision: Minor revision
On 01 Nov, 2020
Review #2 received
Received 20 Oct, 2020
Review #1 received
Received 18 Oct, 2020
Reviewer #3 agreed
On 12 Oct, 2020
Reviewer #2 agreed
On 23 Sep, 2020
Reviewer #1 agreed
On 22 Sep, 2020
Reviewers invited
Invitations sent on 14 Sep, 2020
Editor invited
On 02 Sep, 2020
Editor assigned
On 30 Aug, 2020
Submission checks complete
On 29 Aug, 2020
First submitted
On 28 Aug, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Microbiome.
Background: Coronavirus Disease 2019 (COVID-19) caused by the enveloped RNA virus SARS-CoV-2 primarily affects the respiratory and gastrointestinal tracts. SARS-CoV-2 was isolated from faecal samples and active viral replication was reported in human intestinal cells. The human gut also harbors an enormous amount of resident viruses (collectively known as the virome) that play a role in regulating host immunity and disease pathophysiology. Understanding gut virome perturbation that underlies SARS-CoV-2 infection and severity is an unmet need.
Methods: We enrolled 98 COVID-19 patients with varying disease severity (3 asymptomatic, 53 mild, 34 moderate, 5 severe, 3 critical) and 78 non-COVID-19 controls matched for gender and co-morbidities. All subjects had faecal specimens sampled at inclusion. Blood specimens were collected for COVID-19 patients at admission to test for inflammatory markers and white cell counts. Among COVID-19 cases, 37 (38%) patients had serial faecal samples collected 2 to 3 times per week from time of hospitalization until after discharge. Using shotgun metagenomics sequencing, we sequenced and profiled the faecal RNA and DNA virome. We investigated alterations and longitudinal dynamics of the gut virome in association with disease severity and blood parameters.
Results: Patients with COVID-19 showed underrepresentation of Pepper mild mottle virus (RNA virus) and multiple bacteriophage lineages (DNA viruses) and enrichment of environment-derived eukaryotic DNA viruses in faecal samples, compared to non-COVID-19 subjects. Such gut virome alterations persisted up to 30 days after disease resolution. Faecal virome in SARS-CoV-2 infection harboured more stress-, inflammation- and virulence-associated gene encoding capacities including those pertaining to bacteriophage integration, DNA repair, and metabolism and virulence associated with their bacterial host. Baseline fecal abundance of 10 virus species (1 RNA virus, Pepper chlorotic spot virus, and 9 DNA virus species) inversely correlated with disease COVID-19 severity. These viruses inversely correlated with blood levels of pro-inflammatory proteins, white cells and neutrophils. Among the 10 COVID-19 severity-associated DNA virus species, 4 showed inverse correlation with age; 5 showed persistent lower abundance both during disease course and after disease resolution relative to non-COVID-19 subjects.
Conclusions: Both enteric RNA and DNA virome in COVID-19 patients were different from non-COVID-19 subjects, which persisted after disease resolution of COVID-19. Gut virome may calibrate host immunity and regulate severity to SARS-CoV-2 infection. Our observation that gut viruses inversely correlated with both severity of COVID-19 and host age may partly explain that older subjects are prone to severe and worse COVID-19 outcomes. Altogether our data highlight the importance of human gut virome in severity and potentially therapeutics of COVID-19.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryTable1.Studysubjectmetadata.xlsx
Supplementary Table 1. Subject characteristics and blood measurements.
- SupplementaryTable2.Dietduringtimeofhospitalisation.xlsx
Supplementary Table 2. Diet regimen during time of hospitalization.
- SupplementaryTable3.viralspeciesabundance.xls
Supplementary Table 3. Viral abundance profile in faecal samples. Abundance is expressed in RPKM.
- SupplementaryFigure1.tif
Supplementary Figure 1. Schematic diagram of COVID-19 patient (n=37) follow-up, including disease onset, admission, stool sample collection, duration of hospital stay. “CoV” denotes patient with COVID-19. Stool specimens were serially collected for separate shotgun metagenomic sequencing of RNA and DNA virome; “SARS-CoV-2 PCR negative in nasopharyngeal test”: the first negative result for SARS-CoV-2 virus in two consecutive negative nasopharyngeal tests, upon which patient was then discharged.
- SupplementaryFigure2.tif
Supplementary Figure 2. Temporal changes of the RNA viruses, SARS-CoV-2 (A) and PMMoV (B), in the faecal RNA virome in each COVID-19 case. CPM, count per million reads.
- SupplementaryFigure3.tif
Supplementary Figure 3. SARS-CoV-2 viral load. Stool SARS-CoV-2 viral load in COVID-19 patients were detected by quantitative RT-PCR (A) and RNA virome shotgun metagenomics sequencing (B) respectively. Between-group comparison was conducted by one-way anova, **p<0.01, *p<0.05. (C) Comparison of SARS-CoV-2 viral loads between nasopharyngeal swab and fecal specimens. Statistical analysis was conducted by Mann-Whitney test. (D) Correlation of levels of SARS-CoV-2 viral load between nasopharyngeal swab and fecal specimens, calculated by Pearson correlation analysis.
- SupplementaryFigure4.tif
Supplementary Figure 4. Heatmap of correlations of COVID-19-enriched faecal viral functions and species. The color and intensity denote the spearman correlation direction and coefficient, where only the significant correlations were shown. Viruses labeled with asterisk were species enriched in COVID-19. Only the most abundant 80 species in faecal virome were plotted and ranked in descending order (from left to right) on the basis of the relative abundance.
- SupplementaryFigure5.tif
Supplementary Figure 5. Blood parameter levels in COVID-19 patients between non-severe and moderate/severe groups. Data are shown in mean±s.e. Statistical significance was performed by Mann-Whitney test.
- SupplementaryFigure6.tif
Supplementary Figure 6. The faecal DNA viruses at patient baseline correlated with blood parameters in COVID-19 patients. The color and intensity denote the spearman correlation direction and coefficient, where only the significant correlations with |correlation coefficient| > 0.3 were shown.
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