Transplantation, INSERM Unité Mixte de Recherche Scientifique 1160, Paris, France

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1 1 SUPPLEMENTARY INFORMATION The eukaryotic gut virome in hematopoietic stem cell transplantation: new clues in enteric graft-versus-host disease Jérôme Legoff 1,2, Matthieu Resche-Rigon 3, Jerome Bouquet 2,4, Marie Robin 5, Samia N. Naccache 2,4, Séverine Mercier-Delarue 1, Scot Federman 2,4, Erik Samayoa 2,4, Clotilde Rousseau 5, Prescillia Piron 3, Nathalie Kapel 6, François Simon 1, Gérard Socié 7, Charles Y. Chiu 2,4,8 * 1 University of Paris Diderot, Sorbonne Paris Cité, Inserm U941, Microbiology laboratory, Hôpital Saint-Louis, APHP, Paris, France 2 UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, USA 3 University of Paris Diderot, Sorbonne Paris Cité, Inserm U1153, ECSTRA team, Biostatistics unit, APHP, Hôpital Saint-Louis, Paris, France 4 Department of Laboratory Medicine, University of California, San Francisco, CA, USA 5 University of Paris Descartes, EA4065, Microbiology laboratory, Hôpital Saint-Louis, APHP, Paris, France 6 Laboratoire de Coprologie Fonctionnelle, APHP, GH Pitié-Salpêtrière Charles Foix ; EA4065, Université Paris Descartes, Paris, France 7 University of Paris Diderot, Sorbonne Paris Cité, Paris, France; Hematology and Transplantation, INSERM Unité Mixte de Recherche Scientifique 1160, Paris, France 8 Department of Medicine, Division of Infectious Diseases, University of California, San Francisco, CA, USA Shared last authorship

2 SUPPLEMENTARY RESULTS Two hundred and one samples from 44 patients were analyzed. The distribution of samples according to enteric GVHD and time of sampling are detailed in Supplementary Figure 1. Metagenomic sequencing analysis using a viromespecific library preparation yielded a total of 5.9 x 10 9 raw reads, with a median 2.9 x 10 7 reads per sample (IQR: 2.5 x x 107). By SURPI analysis, the median percentages of human, bacterial, and viral reads were 0.33% (IQR: %), 16.9% (IQR: 0.12% %), and 0.22% (IQR: 0.001%-1.6%), respectively. Eukaryotic viral reads accounted for a median 1.76% of the viral fraction, although the proportion of eukaryotic viruses differed significantly between individuals (IQR: 0.16% %; range %). Eukaryotic viruses accounted for more than 10% of total viral reads in 32.6% and for more than 50% in 15.4% of stool samples. Eukaryotic viruses were mostly assigned to vertebrate taxa (86.9%), with plant viruses (12.1%) being the second most common Plant viruses A total of 149 species of plant viruses belonging to 31 genus and 14 families were identified. Absence of plant virus reads or detection with less than 15 reads per sample was observed in 53.5% (129 of 241) samples. In samples with more than 15 plant viral reads corresponding to a viral family, the median RPM per sample was 15.2 (IQR: range: ). Vigaviridae and Alphaflexiviridae were the most abundant viral families, representing 68.3% and 30.5% of the total number of plant viral reads. respectively. Virus species accounting for more than 1% of plant viral reads belonged to Alphaflexiviridae with Pepino mosaic virus (30.5%) and Virgaviridae (genus Tobamovirus) with Paprika mild mottle virus (23.2%). Tobacco 2

3 mild green mosaic virus (21.6%). Pepper mild mottle virus (10.5%. Bell pepper mottle virus (7.3%). Tomato mosaic virus (2.9%) and Tobacco mosaic virus (1.7%). We analyzed if plant viruses persist over time. Persistence was defined as detection of the same species in at least two successive samples. Persistence was seen in 9 patients (#5. #9. #11. #14. #17. #27. #31. #32. and #43). The most frequent genus was Tobamovirus (Virgaviridae) detected in 7 patients. Carlavirus (Betaflexiviridae) was found in 2 patients. Ilarvirus (Bromoviridae) and Foveavirus (Betaflexiviridae) in one patient each. Two patients had persistent infections with two viruses. Mean time of persistent detection was 10 days. with a range of 5 to 29 days. Persistent detection was attributed to either persistence of the virus in the digestive tract or repeated ingestion of food infected with the same viral species Vertebrate viruses. Among vertebrate viruses, a total of 20 families, 52 genera, and 347 species were identified. The most frequent viral families observed in stool samples from HSCT patients, in order of decreasing frequency, were Anelloviridae (37.8% of samples), Polyomaviridae (14.9%), Picobirnaviridae (13.9%), and Herpesviridae (10.9%) (Supplementary Figure 2; Supplementary Table 2). The frequency of detected viruses increased progressively in the weeks following transplantation (p=0.02 by using a linear model according to time) (Figure 3). The increase in detection of vertebrate viruses was only statistically significant when combining data from all patients, both with and without enteric GvHD Viral families associated with persistent (chronic) infections 3

4 There was an expansion over time of the number of persistent DNA viral families per sample (Anelloviridae, Herpesviridae, Papillomaviridae, Polyomaviridae) in patients with enteric GVHD (p<0.0001) and not in patients without (p=0.05) (Supplementary Table 5). There was also an increase in RPM of persistent DNA viruses (p=0.047) overall after HSCT without any specific difference in patients with (p=0.069) or without (p=0.65) enteric GvHD. Analysis of RPM counts according to viral family showed an increase in RPM for herpesviruses both in all patients (p=0.02) and in patients with digestive GVHD (p=0.009) Anelloviridae Thirty patients (68.2%) had a total of 77 stool samples (38.3%) positive for an anellovirus. with a median RPM of 27.0 (IQR: range: ). Eighteen patients of the 30 (60%) had persistent shedding. The vast majority of anelloviruses (95%) were viruses from the Alphatorquevirus genus. The diversity of anelloviruses was very high between patients (Supplementary Table 3), but was low in serial samples from the same patient Polyomaviridae Polyomavirus sequences were identified in 15 patients (34.1%). including 8 with persistent shedding. The median number of reads in positive samples was (IQR: range: ,241.1). The most frequent species were BK polyomavirus, detected in 27 of 201 (13.4%) samples. and JC virus, detected in 12 of 201 (6.0%) samples. Sequences matching Vervet monkey polyomavirus, Simian virus 40, WU polyomavirus, Simian virus 12, Cercopithecus erythrotis polyoma virus as reported by SURPI were actually found to be misclassified BK polyomavirus reads 4

5 upon manual confirmation using BLASTn alignment. Polyomavirus HPyV6 and Merkel cell polyomavirus were also identified. Among BK virus positive samples % (12) were co-infected with another polyomavirus Herpesviridae Twelve patients (27.3%) had reads matching to viruses belonging to the Herpesviridae family with a median RPM of 1.7 (IQR: range: ). Seven patient had samples with a RPM number of 5. Cytomegalovirus was the most frequent genus identified in 9 patients and accounted for 99% of Herpesviridae reads. Only two patients were positive for human herpesvirus 6 (HHV6) Papillomaviridae and Retroviridae Papillomavirus reads ( 15) were detected in 11 samples from 8 patients with a median RPM of 5.8 (IQR: range: ). For 3 patients. reads were detected in two samples. Thirteen samples from nine patients were positive for human endogenous retroviruses with a median RPM of 1.0 (IQR: range: ). Most of the retroviral reads derived from human endogenous retroviruses or murine retroviruses related to murine leukemia virus (Supplementary Table 4). Viruses related to murine leukemia virus were attributed to contamination from reagents or the environment 1-3. Other animal retroviruses belonging to the alpharetrovirus, betaretrovirus, or gammaretrovirus taxa and assigned to porcine, ovine, and avian hosts were detected in a minority of samples (<13% of all retroviral reads) and were attributed to dietary components. 5

6 Viral families associated with transient (acute) infections and/or acute gastroenteritis syndromes Picobirnaviridae Picobirnaviruses (PBV) are non-enveloped viruses with a bi-segmented double-stranded RNA (dsrna) genome 4,5. Segment 1 ( kb) encodes the capsid protein and segment 2 ( kb) encodes the viral RNA-dependent RNA polymerase (RdRp) 6. Based on the sequences of segment 1. two genogroups (I and II) have been described. Recently, a third genogroup (III) has been reported 7. PBV have frequently been found in human feces including during acute diarrheal outbreaks 8. However, their clinical significance impact remains to be fully elucidated. In this study, we first detected 37 samples with at least one PBV read and 27 with at least 15 reads. Given the frequency of PBV and the high divergence of PBV genomes, we then re-analyzed all PBV reads by SNAP at an edit distance of 12 and included all PBV divergent reads identified by RAPSearch 9 at an e-value of 1 rather than 0.5 used for the virome analysis to catch all PBV, we checked for their specificity and we confirmed their detection using specific single PCR and Sanger sequencing as described in methods. An infection with PBV was considered for a patient when PBV reads were detected over 15 reads at least in one sample over the follow-up as and if PBV detection was confirmed with specific RT-PCR and sequencing. Finally, 43 samples were found with at least one PBV read, including 38 samples with more than 15 reads and confirmed by virus-specific PCR testing. In total, eighteen patients (40.9%) had at least one stool sample positive for PBV after HSCT, of which 9 had between 5-50 RPM and 11 had 50 RPM (Supplementary 6

7 Table 7). Twelve patients were infected by a PBV in genogroup I (1, 2, 3, 4, 5, 15, 18, 25, 29, 30, 43, 44) and four by a PBV in genogroup II (7, 19, 24, 41). The number of PBV reads was insufficient to assign a genogroup in 2 patients (26, 35). Picobirnaviruses (PBV) were more common in stool samples from enteric GvHD patients (Figure 4A, 23.3% versus 11.1%, p=0.039). PBV were mainly detected before transplantation or within a week after transplantation (29.6%. 21 out of 71 samples) and less frequently after week 1 (13.8%. 17 out of 130 samples) (p=0.008). All but one (05 W0; patient #5. week 0) of the PBV sequences detected in the study clustered by phylogenetic analysis with other PBV sequences identified in human samples or wastewater. All viruses differed in sequence from one patient to another. These data rule out any cross contamination between samples from different patients. In individual patients for whom longitudinally collected samples were available with RdRP sequences (patients #3 (Pre-transplant and W0 samples), or #44 (W0 and W3 samples), viral sequences were closely related, suggesting that PBV shedding may persist in the gut mucosa and thus that PBV were adapted to replication in human cells (Figure 4 and Supplementary Table 8) Picornaviridae. Eleven patients were identified with picornaviruses reads at a median RPM of 5.2 (IQR: range: ). Four patients (#2, #5, #9, #41) had one sample at a single time point with reads matching to Melegrivirus A (RPM range: 0.8 4). Melegrivirus A was discovered in 2011 and is the probable cause of hepatitis in turkeys (Honkavuori et al ). The limited number of sequences detected and the lack of persistence over time likely reflect the ingestion of infected turkey meat, with no evidence supporting replication in the human digestive tract. Seven patients had stool samples containing rhinovirus sequences, with a median RPM of Median 9.0 7

8 (IQR: range: ), including 5 patients with rhinovirus detected in 2-3 longitudinally collected samples Parvoviridae Nine patients had stool samples with reads matching to the Parvoviridae family. Bovine parvovirus (genus Dependovirus) and porcine parvovirus (genus Parvovirus) were detected in 5 patients at a single time point, consistent with ingestion of bovine or pork meat infected with parvovirus at that time point. Three additional patients had sequences matching bufavirus. Patient #24 was positive only once at week 5, patient #3 had pregraft and W0 samples positive, while patient #31 was positive for bufavirus in all samples collected over 21 days from the onset of pregraft conditioning to week 2. For patient #31, the highest number of bufavirus reads (RPM = 428.6) was observed during the first week after HSCT. Bufavirus was first identified in feces of patients with acute diarrhea in Burkina Faso. More recently, it has also been found in patients with gastroenteritis in Finland. However, no diarrheal symptoms were recorded for patient #31 at any time following HSCT Astroviridae Human astroviruses are small positive single-stranded RNA viruses belonging to the Mamastrovirus genus. They are associated with gastroenteritis and encephalitis in humans. We found reads matching astrovirus (human astrovirus 1) in 9 of 241 (3.7%) samples collected from two patients (#24 and #42) at all time points. The number of astrovirus reads in patient #24 ranged from 0.39 to 7.68 RPM, while the pregraft, W0 and W1 samples from patient #42, each had more than

9 RPM. Despite seemingly high loads of astrovirus in stool, patient #42 did not present with any symptoms of gastroenteritis and did not develop enteric GvHD Adenoviridae. Only one patient (#18) had a single sample positive for a species C adenovirus (HAdV-1) (29.36 RPM) at week 5. Adenovirus species C is frequently detected in stool samples of pediatric HSCT recipients and rarely in adult HSCT patients. Adenovirus replication in HSCT recipients has been linked to enteric GvHD 10, and indeed, patient #18 presented with an enteric GvHD stage 1 beginning 11 days after engraftment that progressed to stage 4 17 days later. Adenovirus detection in patient #18 was confirmed by specific real-time PCR testing (data not shown) Caliciviridae Two patients had stool samples with reads matching to Caliciviridae. One patient (#12) had 4 consecutive samples with Calicivirdae reads from week 0 to week 3, of which only one had more than 15 reads, while the other patient (#9) had 4 consecutive samples from weeks 1 to 5 positive. All sequences mapped to the Norovirus genus corresponding to noroviruses, the most frequent etiology of viral gastroenteritis in adults 11. However, neither patient developed enteric GvHD Circoviridae Sixteen samples from 13 patients were found positive for the Circoviridae family. The number of reads per sample ranged from 0.43 to RPM, with a 9

10 median of 2.14 RPM. The Circovirus genus accounted for 40% of reads and was represented by two species: Duck circovirus and Porcine circovirus. The Gyrovirus genus accounted for 60% of reads and included Avian gyrovirus 2, chicken anemia virus, gyrovirus 4, gyrovirus 3 and Human gyrovirus 1. Three patients had two positive non-consecutive samples. Taken together, these data show that circoviruses were detected frequently at low levels but did not persist over time Coronaviridae Coronavirus reads were detected in three samples from the same patient (#12). Reads matched with the Betacoronavirus genus Microbiome analysis A slightly higher relative abundance of bacterial taxa at the genus level (p=0.047), but no difference in richness (p=0.53), was observed in patients with versus those without enteric GvHD. When looking at bacterial population dynamics over time in patients developing enteric GvHD following transplantation according to relative abundance, we found a significant increase in Lactobacillales (p=0.007), a significant decrease in Clostridiales, a non-significant increase in Bacillales (p=0.07), and no change in Enterobacteriales (p=0.85). In patients without enteric GvHD, there relative abundance of Enterobacteriales species increased over time (p=0.048) and that of Lactobacillales (p=0.54), Clostridiales (p=0.24) and Bacillales (p=0.59) remained stable. The significant changes in relative abundance of Clostridiales and Lactobacillales, however, were not predictive of the occurrence of enteric GVHD. Our results thus corroborate previously published analyses on microbiota shifts after HSCT in patients with GVHD and in patients with inflammatory bowel disease 12,13. 10

11 Indeed, Jenq et al. showed in mouse models of GVHD and in patients with gut GVHD expansion of Lactobacillales and loss of Clostridiales Phage analysis The analysis of phage dynamics over time after graft showed a difference of phage diversity between patients with or without enteric GVHD. The relative abundance was not different but richness was significantly decreased in patients with enteric GVHD compared to those without (p=0.44 and p=0.01, respectively). As already observed in other studies looking for the phage virome, the most abundant taxa were the Caudovirales order (Siphoviridae, Myoviridae and Podoviridae) and Microviridae and Inoviridae families. In a time dependent Cox model, none of them were predictive of the occurrence of enteric GVHD (p=0.68, p=0.57 and p=0.08 respectively). However, in patients with enteric GvHD, the relative abundance of Microviridae in patients who developed enteric GVHD was significantly higher before graft and the first week after than those without and decreased significantly over time after transplantation (p=0.03). No data on phages ecology and dynamics in stool of HSCT patients have been reported so far. One study specifically analyzed phage virome composition and evolution in patients with ulcerative colitis or Crohn s disease 14, showing an expansion of Caudovirales and fewer Microviridae in patients with ulcerative colitis and Crohn s disease than in controls. In our study, we found a progressive decrease in Microviridae in patients with enteric GVHD. Phage and microbiome analysis thus confirm a perturbation of viral and bacterial flora in patients with digestive GVHD Reactivation of CMV, EBV and HHV-6 in blood and association with GVHD. The association between systemic herpesvirus reactivation and GVHD has 11

12 been reported for viral detection in blood. However, the detection of herpesviruses in stool of HSCT patients is not frequent and is thought to have limited value in predicting CMV enteritis 15. The diagnosis of viral-induced enteritis relies mainly on histological analysis and specific quantitative PCR testing of intestinal biopsies. To assess the role of viral reactivation and GVHD, we measured titers of CMV, EBV, and HHV-6 in whole blood at 2 time points, within a week before and 8 weeks after transplant. A total of 543 whole blood samples were tested. CMV, EBV, and HHV-6 DNA was detected respectively in 26.5%, 21.0% and 11.3%. Median viral loads in positive samples were 2.94 log 10 IU/ml (IQR[2.30;3.96]) for CMV, 3.81 log 10 copies/ml (IQR[3.44;4.54]) for EBV and 3.59 log 10 copies/ml (IQR[3.33;4.86]) for HHV-6. There was no detected association between CMV or EBV reactivation in whole blood and enteric GvHD. However, CMV loads above 3.5 log 10 IU/ml were more frequently observed in patients with enteric GVHD (15/30; 50%) than in those without enteric GVHD (2/14, 14.3%) (p=0.04). HHV-6 reactivation at any level also occurred more frequently in patients who had enteric GVHD (p<0.001). Neither CMV nor HHV-6 reactivations, however, were predictive of enteric GVHD (p=0.68 and p=0.84 respectively). These results thus suggest that GVHD may facilitate herpesvirus reactivation rather than viral reactivation inducing GVHD, per se Assessment of enteric graft versus host disease (GvHD) Based on clinical criteria, 26 patients had enteric GvHD. In addition, histological analysis on intestinal biopsies were carried out when endoscopy was possible and the concentrations of fecal calprotectin and α1-antitrypsin were determined. Fecal calprotectin concentrations were obtained for all 23 patients with enteric GvHD, α1-antitrypsin concentrations for 16 patients, and intestinal biopsies for 20 patients. All of the results of histology and biomarkers testing are presented in 12

13 Supplementary Table 10 and summarized in Supplementary Table 11. Out of 23 patients with a clinical diagnosis of enteric GVHD and testing of fecal biomarkers, 14 had elevated levels of fecal calprotectin and/or α1-antitrypsin in stool samples. Of those 14 with elevated levels, the intestinal biopsies from 11 patients all showed epithelial inflammation, including 5 patients with epithelial abrasion and 8 patients with cell apoptosis (Tables S10 and S11). Among the nine patients with fecal calprotectin and α1-antitrypsin within the normal range, seven had enteric biopsies with mucosa inflammation, including 3 with cell apoptosis and 5 with epithelial abrasion (Tables S10 and S11). Only one biopsy showed inflammation without cell apoptosis or epithelial abrasion in patient #8 with a digestive stage 1 GvHD. Two patients without any data available fecal calprotectin and α1-antitrypsin had enteric biopsies with mucosa inflammation and cell apoptosis. Two patients had normal values of fecal calprotectin and α1-antitrypsin but had no endoscopy and were thus not evaluated for digestive mucosa histology. One patient had no testing for fecal calprotectin and α1-antitrypsin and no digestive biopsy. Overall, digestive mucosa inflammation as defined with quantification of fecal biomarkers or histological findings was confirmed in 23 patients out of 26 with a diagnosis of digestive GVHD. No viral induced cytopathic effect was reported in any biopsy. Results of fecal biomarkers and histological findings are summarized in Supplementary Table 11. We then compared values of calprotectin and α1- antitrypsin between patients who were found positive for Picobirnavirus and those with had no PBV reads. Values of calprotectin below normal values (50 µg/g) were observed in 81.8% of patients with no PBV infection and in 25.0% of patients with PBV infection (Fischer exact test, p=0.01). In patients with digestive GvHD, fecal calprotectin concentrations (mean=330.5 µg/g) were higher in patients with PBV reads than those without PBV (mean=40.3 µg/g) (Mann-Whitney U test, p=0.005) 13

14 (Figure 4). These findings corroborate previous observations showing in patients with inflammatory bowel disease that fecal calprotectin levels were higher in patients positive for a gastrointestinal virus than those without 16. Alpha-1 antitrypsin levels were also significantly higher in patients with PBV infection (mean=11.92 mg/g) than those without (4.43 mg/g) (Mann-Whitney U test, p=0.01) (Figure 4). These results suggest that the detection of Picobirnavirus in stool is associated with digestive mucosa inflammation and possibly to digestive antimicrobial response Predictive factors for GvHD No clinical characteristics, including sex, age, type of graft, total body irradiation, or human leukocyte antigen (HLA) matching, were found to be associated with the occurrence of GvHD of any type (data not shown). Age only was associated with the risk of severe enteric GvHD (p=0.005). Whole blood cytomegalovirus (CMV) loads of over 3.5 log 10 IU/mL and positive human herpesvirus 6 (HHV-6) detection in blood were significantly more frequent in patients with enteric GvHD than in patients without enteric GvHD (p=0.04 and p<0.0001, respectively), but herpesvirus reactivation was not predictive of subsequent development of enteric GvHD (p=0.68 and p=0.84, respectively) using a time-dependent Cox proportional hazards model. Similarly, the development of GvHD was not correlated with host type, except for viruses of plants, which were associated with a lower risk of GvHD of any type (Supplementary Table 9). Among all eukaryotic viruses, only the detection of PBV was predictive of the later development of GvHD, both enteric and non-enteric (hazard ratio [HR]=1.75; 95% confidential interval [CI]= ; p=0.02), with a stronger association for severe enteric GvHD of stage 2 or higher (HR=2.66; 95% CI= ; p=0.001) (Supplementary Table 9). 14

15 We performed a multivariate analysis for severe enteric GvHD by adjusting the impact of Picobirnaviridae on Age with a potential interaction term. Impact of Picobirnaviridae was still significant (p=0.03), as well as age (p=0.01) but not the interaction. Regarding correlation between clinical characteristics, only sex was associated with presence of Picobirnaviridae (PBV was identified in 2/13 (15.4%) of women and 16/31 (51.6%) of men, p=0.043). We also performed a multivariate analysis for severe enteric GvHD adjusted on sex. Picobirnavirus detection remained significant (p=0.007). The sample size and especially the number of events do not allow more complex statistic modeling without risks of overparameterization. Thus, the role of Picobirnaviridae will need confirmatory studies on specific clinical groups of patients. 15

16 Supplementary Table 1. Patient characteristics. Patient Sex Organ GVHD maximum GVHD Age Graft Myeloablative GVHD Acute GVHD Onset (days days Number of stool TBI stage resolution type regimen prevention max grade postgraft) postgraft samples Skin Liver Gut (all organs) 1 M 56 BM No No Yes III No 8 2 M 53 PB No No No IV No 4 3 M 59 PB No No Yes IV No 6 4 M 39 CB No Yes Yes II Yes F 57 PB No No Yes No GVHD 3 6 F 43 BM Yes No Yes No GVHD 4 7 M 55 PB No No Yes II No 6 8 F 63 PB No No Yes II Yes M 64 BM No No Yes No GVHD 6 10 M 60 PB No No Yes III No 5 11 F 28 PB Yes Yes Yes IV Yes M 54 PB No No Yes II Yes M 22 PB Yes No Yes I Yes M 54 PB No No Yes No GVHD 3 15 M 46 CB No Yes Yes II Yes M 40 PB Yes Yes Yes No GVHD 5 17 M 59 CB No Yes Yes II No 6 18 F 59 PB No No Yes III No 7 19 M 61 PB No No Yes III No 4 20 F 31 PB Yes Yes Yes No GVHD 3 21 F 60 PB No No Yes No GVHD 3 22 M 62 PB No No Yes II Yes F 39 CB No Yes Yes II Yes M 53 PB No No Yes III Yes M 63 PB No No Yes II Yes M 32 PB Yes No Yes No GVHD 7 27 F 38 PB No No Yes IV No 3 28 F 61 PB No No Yes IV No 3 29 M 62 PB No No Yes III Yes M 66 PB No No Yes II Yes M 33 PB Yes Yes Yes No GVHD 4 32 F 39 PB Yes Yes Yes I Yes M 18 BM No Yes Yes II Yes M 37 CB Yes Yes Yes No GVHD 6 35 M 29 BM Yes No Yes II Yes M 42 BM Yes Yes Yes I Yes M 28 PB Yes No Yes II Yes F 54 PB No No Yes No GVHD 3 39 M 55 PB No No Yes III No 4 40 M 23 PB No No Yes No GVHD 3 41 M 62 PB No No Yes IV No 4 42 F 33 BM Yes No Yes No GVHD 3 43 M 50 PB No No Yes II Yes M 32 PB Yes Yes Yes I Yes 30 3 TBI=Total body irradiation GVHD = graft versus host disease 16

17 Supplementary Table 2. Frequency of viral families in clinical samples and in patients. Proportion of samples and patients positive for each vertebrate viral family. Samples PBV positive Patient PBV positive Anelloviridae 38.3% 68.2% Polyomaviridae 14.9% 34.1% Picobirnaviridae 13.9% 36.4% Herpesviridae 11.4% 27.3% Picornaviridae 9.0% 25.0% Circoviridae 8.0% 29.5% Parvoviridae 6.5% 20.5% Retroviridae 6.5% 20.5% Papillomaviridae 5.5% 18.2% Astroviridae 3.0% 4.5% Caliciviridae 2.5% 4.5% Coronaviridae 1.5% 2.3% Adenoviridae 0.5% 2.3% 17

18 Supplementary Table 3. Diversity of anelloviruses detected in stool samples of HSCT patients. Virus identification, genus and total number of reads detected over the whole collection of samples tested are indicated. Virus Genus Total number of reads Micro_Torque_teno_virus 33 SEN_virus Small_anellovirus 1780 Torque_teno_midi_virus Torque_teno_virus TTV-like_mini_virus uncultured_anellovirus Torque_teno_virus_1 Alphatorquevirus 137 Torque_teno_virus_10 Alphatorquevirus Torque_teno_virus_11 Alphatorquevirus Torque_teno_virus_12 Alphatorquevirus 842 Torque_teno_virus_13 Alphatorquevirus Torque_teno_virus_14 Alphatorquevirus Torque_teno_virus_15 Alphatorquevirus 360 Torque_teno_virus_16 Alphatorquevirus Torque_teno_virus_19 Alphatorquevirus 1404 Torque_teno_virus_2 Alphatorquevirus 103 Torque_teno_virus_20 Alphatorquevirus 5961 Torque_teno_virus_21 Alphatorquevirus 4982 Torque_teno_virus_23 Alphatorquevirus 5069 Torque_teno_virus_24 Alphatorquevirus Torque_teno_virus_26 Alphatorquevirus 2 Torque_teno_virus_27 Alphatorquevirus 551 Torque_teno_virus_28 Alphatorquevirus 6988 Torque_teno_virus_29 Alphatorquevirus 8920 Torque_teno_virus_3 Alphatorquevirus 1605 Torque_teno_virus_4 Alphatorquevirus 3379 Torque_teno_virus_5 Alphatorquevirus Torque_teno_virus_6 Alphatorquevirus 840 Torque_teno_virus_7 Alphatorquevirus 277 Torque_teno_virus_8 Alphatorquevirus 2453 Torque_teno_mini_virus_1 Betatorquevirus Torque_teno_mini_virus_2 Betatorquevirus 136 Torque_teno_mini_virus_3 Betatorquevirus 1452 Torque_teno_mini_virus_4 Betatorquevirus 1217 Torque_teno_mini_virus_5 Betatorquevirus 3129 Torque_teno_mini_virus_6 Betatorquevirus 873 Torque_teno_mini_virus_7 Betatorquevirus 39 Torque_teno_mini_virus_8 Betatorquevirus 271 Torque_teno_mini_virus_9 Betatorquevirus 559 Torque_teno_midi_virus_1 Gammatorquevirus 105 Torque_teno_midi_virus_2 Gammatorquevirus 29 Torque_teno_sus_virus_1a Iotatorquevirus 5 Torque_teno_sus_virus_1b Iotatorquevirus 2 Torque_teno_sus_virus_k2 Kappatorquevirus 2 18

19 Supplementary Table 4. Diversity of retrovirus detected in stool samples of HSCT patients. Virus identification and total number of reads detected over the whole collection of samples tested are indicated. Virus Reads Human endogenous retrovirus 222 Alpharetrovirus (avian) 9 Beatretrovirus (goats, sheep) 29 Murine leukemia related retroviruses 188 Porcine type-c oncovirus 23 19

20 Supplementary Table 5. Increase in the mean number of persistent DNA families per stool sample after transplant. Longitudinal changes in the mean number of persistent DNA families (Anelloviridae, Herpesviridae, Papillomaviridae and Polyomaviridae) by week of collection following transplantation in all patients, patients with enteric GvHD, and patients without enteric GvHD. Pre- Tx W0 W1 W2 W3 W4 W5 P value All patients Enteric GVHD < No enteric GVHD

21 Supplementary Table 6. Picobirnavirus read-specific primers for RT-PCR Patient Week Forward primer Reverse primer 1 W6 AAATTGACCTCGCGAAGAAG TCGATATCTTTGAGACGTTCCTC 1 W5 GAGAGGTCAAGACGGTGGTC TCACTGAGAACGGAAACATCC 1 W0 TGAAACCCTTAGCCATCGAG ACCATCGTCTCCAAGACACA 2 W0 ACAGTGGCCCACCTTGATG AGGCAACATGACTGACATCG 3 W0 CCGTTTATGGATATGCTACAGAA TTGGTGTCAAACAATTTGGTG 3 PreT GCTGATATTGGTGGCAAGGA AACGTCCGAATTCTTTGGTC 4 PreT AACGCGGCAATCTCGTTC TCAGGTCGTTGTTACCCTTTG 4 W6 CGAACAGTGGGTCGCATAAC TAAGCTGAATCCATGGCGAAG 4 W3 GCTCGTAACATCTATGCATTCG TGAATCCATGGCGAAGAAAT 4 W4 TCACTTCTGCCAAACGAAGA AAGAACAAATCGCTCTCTCCA 5 W1 CATGGCGACTCGTTTGGTTG TCCACCAGGATCCAATCAAAA 5 W0 GTGCGTGGCAGCACTATAA TCGTCACCTTTTGTGTCGAA 5 W2 TCATGTCGTGTCCTATGGCG GCTTCTTCGATTTGTGCGCT 7 PreT CACAAAGGCACAATGTCGTC GAACATGGCTGTAATGCGTTT 15 W0 AGAACGATCTCGCGAAGAAG AAGCGTCTATGTCTTCCATACG 15 W2 CTCATTGAAAGCGCTCAACA TTTCTCGATCTACCATATCCATGC 15 W3 GATAATGAGGCGTCACGTTG CGCACCTTCCCATAATCGTA 15 W5 TGAAGAAGAGCACAAACTCTGG CAGTACGCTGTTTTGTTGACC 15 W4 TTGACGGGGTATGTGTAGGC CCAATTTTCCGGGTCATAAT 15 W1 TTGAGACGATGGCACCATAA GGAGGATACCTGGAGCCCTA 18 PreT CAGAAGCATATCGGCAGCAT AACTTGAAGTGAGCGTCCGTA 19 W0 AAAGACTAGGTTAGTATGGATGTTTCC ACTGCAGCGGTTGGAAGTAG 19 PreT TGTTTCCTATGTCTGCGAACC CCAGAAGCTCGCATTAGAATC 19 W2 TGTTTCCTATGTCTGCGAACC AGGACCAGAAGCTCGCATTA 24 W2 ACCGGGAGTAGCTCTTAGGG GCGACTGTTTCATCTGCATT 24 W1 GCTGCATTGAAGAAAAATGATG TGTCGTGAAGAGAAACTCTACCA 25 W0 ACAGCTACCTCGCTTTCCTT AGCGGTTGGTGTAGCTGTAC 25 PreT TCGATCTTGTCCAGGAATCCT ACAGCTACACCAACCGCTAC 26 W6 CCTCCAAGAACTACGTGTCTACC CGACCCATGCTGGTACTACTT 29 PreT AGCCAATGATTGAGGCAGCT TGGATTTCGCTGCAAGTTGC 29 W6 CCACCATCACTGGATTTGCG CAGCAGGATTTTGTGTGATTTGC 29 W0 GATTGAAGCCGCACAAAGAT GCTGTGATGCGTTTATCCAC 30 W0 CTGCTTGGGTTAGCATGGAT CGATGAGATCATCTTTGGCTTT 30 PreT TCGAAGAGCTAAGTGTCTATCAACC CTATCCACCGATTCCATGCT 35 PreT GGACTGCTCCAACTTTTGGT ATTAGCGTGGCGTACGAAAG 37 W1 TTTGATCAACACTTCAACAGTGA TGGATCTTTAGCGGCCATAC 41 W0 AATTCCGGGCATACGAATTA AGGCGCTTCAGCATAGCAT 41 W1 CGGGTTCGAGACACCTTG GATTGCACGCTCTTCGACTT 41 PreT GACAGTCGGGCAGGCTAAC CCAGAATTTGAGTTCAAGGTGTC 41 W4 TTTATTCGACATGCCAATTCC GTTGTCCATGGCCAGAAGAT 43 PreC TGCTGTTAACATTCAGGAGCTG GGACCAGATTGTGGTACTGGA 44 W0 GAACCGGTTTTACAAGGTTTCA AAACAGACGATGTTAGTGTGTCG 44 W1 GAACCGGTTTTACAAGGTTTCA AAACAGACGATGTTAGTGTGTCG 21

22 Supplementary Table 7. Detection of Picobirnavirus (PBV) in stool samples of HSCT patients. Only samples from patients with at least one sample positive for PBV (i.e ³15 reads) are presented. A patient was considered positive for PBV if reads were identified by SURPI and the presence of PBV RNA confirmed by readspecific RT-PCR assays (PBV PCR). PBV reads are expressed in reads per million of preprocessed reads (PBV RPM). Patient # Week a PBV RPM PBV PCR 1 PreT 0 1 W0 61,55 positive 1 W1 0 1 W2 0 1 W3 0 1 W4 0 1 W5 0,35 positive 1 W6 1,16 positive 2 PreT b 0 2 W0 147,46 positive 2 W1 0 2 W2 0 3 PreT 280,84 positive 3 W0 8,63 positive 3 W1 0 3 W2 0 3 W3 0 3 W6 0 4 PreT 0,53 positive 4 W0 0 4 W1 0 4 W2 0 4 W3 0,60 positive 4 W4 3,54 positive 4 W5 0 4 W6 621,10 positive 5 W0 6,54 positive 5 W1 0,15 negative 5 W2 0,21 negative 7 PreT 4,41 positive 7 W1 0 7 W3 0 7 W4 0 7 W5 0 7 W W0 8,55 positive 15 W1 0,10 positive 15 W2 0,07 positive 15 W3 0,48 positive 15 W4 0,47 positive 15 W5 1,39 positive 18 PreT 0,80 positive 18 W W W W W W PreT 90,85 positive 19 W0 3659,67 positive 19 W W2 2807,98 positive 24 W W1 7,67 positive 24 W2 3,87 positive 24 W W W PreT 2,82 negative 25 W0 0,15 positive 25 W W3 0 22

23 26 PreC 0 26 W W W W W W6 7,74 positive 29 PreT 1,19 positive 29 W0 102,04 positive 29 W W W W W W6 0,05 negative 30 PreT 31,33 positive 30 W0 2,69 positive 30 W W PreT 2,98 positive 35 W W W PreT 108,88 positive 41 W0 31,14 positive 41 W1 767,60 positive 41 W4 33,00 positive 43 PreC c 6,92 positive 43 W W W W0 114,32 positive 44 W1 4,60 positive 44 W2 0 a W=Week. W0 is the week following transplant b PreT. Samples were collected before the patient received HSCT. c PreC. Samples were collected before the patient received conditioning regimen. 23

24 Supplementary Table 8. Amino acid (aa) partial RNA-dependent RNA polymerase (RdRP) gene sequences Amino acid (aa) partial RNA-dependent RNA polymerase (RdRP) genes corresponding to aa of study stool samples positive for PBV and NCBI Genbank picobirnavirus reference sequences. The GenBank reference sequences selected for the phylogeny included those available in NCBI GenBank with coverage of the RdRP gene region corresponding to aa ,8, The designations C. D. M. P. R. S and W stand for cow. dog. mouse. porcine. rat. and snake hosts. or wastewater respectively. The letter H designates sequences previously detected in human samples. Genogroup 1 PBV sequences detected in the current study are highlighted in bold. Identity RdRP amino acid sequence (positions ) 01 W0 FAVNLCELQVYQPLIESAQHFNTVPAWVSMDAVDQEITHLFDTKGKDDLVVCTDFS 03 PRET FTVNLQELRVYQPFMDMLQKHKVVPAWVGLDEVDNKITKLFDTKGEDDVVICTDFS 03 W0 FTVNLQELRVYQPFMDMLQKHKVVPAWVGLDEVDNKITKLFDTKGEDDVVICTDFS 05 W0 FNTNVQELMAYNPLISAWQHYNINSAYISQRAVEEKITRLFDTKGDEYVVVTDFS 29 W0 YAVNIEELRVYQPLIEAAQRFNLVPAWVSMDAVDKRITAMFDTKADDDLIVCTDFS 15 W0 FAVNLCELQVYQPLIESAQRFNTVPAWVSMDMVDREITYLFDTKGKDDLVVCTDFS 30 PRET FAVNICELQVYQPLIECCQKLDLVPAWVSMDSVDRRITRMFDTKGVDDLVICTDFS 43 PRET YAVNIQELQVYQPLIEAFQYHNLVPAWVGMDAVDVEVTRLFDTKKPSDLVICTDFS 44 W0 FAVNIRELQVYQPLILTFQRLGLVPAWVSMEAVDRRITKMFDTKGPHDVVVCTDFS 44 W1 FAVNIRELQVYQPLILTFQRLGLVPAWVSMEAVDRRITKMFDTKGPHDVVVCTD GU H FTVNLQELRVYQPFMDMLQKHKIIPAWVGLEEVDNKITKLFDTKGEDDVVICTDFS GU H FTVNLQELRVYQPFMDMLQKHKVVPAWVGLDEVDNKITKLFDTKGEDDVVICTDFS AF H FAVNIQELRLYQPLIESCQKFNLVPAWVSMEAVDRRITQLFDTKGKDDDIICTDFS AB H FSVNIRELQVYQPLIESCQKFDLVPAWVSMESVDRRITKMFDSKGKDDVVICTDFS AB H FAVNIQELRLYQPLIESCQKFNLVPAWVSMESVDRRITQLFDTKGKDDDIICTDFS GU H FAVNIRELQVYQPLILTFQRLGLVPAWVSMEVVDRRITKMFDTKGPHDVVVCTDFS GU H YAVNIEELRVYQPLIEAAQRFNLVPAWVSMDAVDKRITAMFDTKADDDLVVCTDFS GU H YAVNIRELQVYQPLILTFQRLGLVPAWVSMEAVDRRITKMFDTKGPNDVVVCTDFS GU H YAVNIEELRVYQPLIEAAQRFRLVPAWVSMDEVDRRITRMFDTKRDDDLIVCTDFS GU H YAVNIAELQVYQPFIEAAQKFNIVPAWVSFDEVDKRITAMFDTKASDDLVVCTDFS GU H FAVNIEELRYYQPAIEAAQNFNLVPAWVSMESVDDRITRMFDTKGSDDLVVCTDFS AB H MALNIEELQFYQPAIEAIQKNGLIPAYASMDAVDDEVTALFATKGADDVVICTDFT GU H YAVNIEELRVYQPLIEAAQRFNLVPAWVSMDAVDKRITAMFDTKADDDLVVCTDFS GU H FSVNVQELQVYQPLIQAFQAHGSVPAWVGMEAVDAEITRMFDTKRPEDLVICTDFS GU H FAVNIQELRLYQPLIESCQRFDLVPAWVSMESVDRRITKMFDTKGKDDDIICTDFS AB H FGINVLELQLYQPLIEALQAHSDVPAWVGMDAVDLAITKLFDTKDSKDLVICTDFS AF H FGVNVKELQFYQPAIEIAQKRWITPAWIGMEAVDQRITKLFDTKAKSELVICTDFS AB H FAVNIRELQVYQPLILTFQRLGLVPAWVSMEAVDRRITKMFDTKGPRDVVVCTDFS AB H YAVNIRELQFYQPAIELAKRELLVPAWVGMDSVDVRITRLFDTKAKSDLIVCTDFS AB H FSVNICELQTYQPLIESCQNFNLVPAWVSMDAVDQRITKMFDTKASDDVVICTDFS EU W FAVNIQELRLYQPLIETCQKFNLVPAWVSMEAVDQRITKLFDTKSADDDIICTDFS EU W FAVNIRELQVYQPLIEVAQKKLVVPAWVSMEEVDKSITRMFDTKGVDDLVICTL 24

25 EU W FAVNIEELRYYQPAIEAAQNFNLVPAWVSMESVDDRITRMFDTKGSDDLVICTDFS EU W FGINICELQVYQPLIESCQKFNLVPAWVSMDAVDQRITDMFDTKGVDDVVICTDFS EU W FAVNIEELRYYQPAIEAAQNFNLVPAWVSMESVDDRITRMFDTKGSDDLVICTDFS EU W FAVNIEELRYYQPAIEAAQNFNLVPAWVSMESVDDRITRMFDTKGSDDLVICTDFS EU W YAVNIEELSVYQPLIEKVQSFNLVPAWVSMESVDRRITAMFDTKASNDLVVCTDFS EU W YAVNIEELRVYQPLIEAAQRFRLVPAWVSMDEVDRRITRMFDTKRDDDLIVCTDFS EU W FAVNICELQVYQSLIESAQRFNTVPAWVSMDAVDQEITNLFDTKGKDDLVVCTDFS EU W YAVNIEELRVYQPLIEAAQRFRLVPAWVSRDEVDRRITRMFDTKRADDLIVCTDFS EU W YAVNIEELRVYQPFIEAAQRFRLVPAWVSMDEVDRRITRMFDTKRDDDLIVCTDFS EU W YAVNTAELQLYQPFIEAAQRHNIIPAWVSFDEVDKRITAMFDTKASNDLVVCTDFS EU W FAVNIEELRYYQPAIEAAQDFNLVPAWVSMESVDDRITRMFDTKGSDDLVICTDFS EU W FAVNIRELQMYQPLIESCQRFNLVPAWVSMEAVDRRITDMFDTKGENDLVICTDFS EU W FAVNLAELQVYQPLIEAAQKLRLVPAWVSMYEVDRRITAMFDSKAPDDLVVCTDFS EU W YAVNIAELQLYQPFIEAAQRHNVVPAWVSFDEVDKRITAMFDTKADNDLVVCTDFS EU W YAVNIAELQLYQPFIEAAQGFNVIPAWVSMDAVDKRITDMFDTKSDSDLVVCTDFS EU W FGVNIRELQVYQPGIELAQQHNLVPAWIGMEAVDLAITKLFDTKGANDLIICTDFS EU W FAVNVSELQVYQPAIELAQQFELVPAWVSMEAVDIRVTKLFDTKGKHDLIICTDFS EU W FAVNIQELRLYQPLIETCQKFNLVPAWVSMEAVDRRITKLFDTKSADDDIICTDFS EU W YAVNIQELQVYQPLIEAFQYHNLVPAWVGMDAVDVEVTRLFDTKRPSDLVICTDFS EU W YAVNIRELQLYQPLIEACQRFDLVPAWVGMDAVDKRITDMFDTKSDSDLVVCTDFS EU W YAVNVKELQFYQPAIEQAQKFNLVPAWVGMDAVDARITKLFDTKAKRDLIVCTDFS EU W YAVNIAELQV*QPLIEGAQRFNLVPAWVGMEAVDVRITKLFDTKGRDDLIICTDFS EU W FGVNIAELQVYQPMIELAQKFDLVPAWIGSDAVDERITRLFDTKGKNDLVICTDFS EU W YAVNIAELQLYQPFIEAAQRHNIIPAWVSFDEVDKRITAMFDTKASNDLIVCTDFS AM P FAVNIAELRLYQPLIETCQRFDLVPAWVSMDSVDRRITRMFDTKDPKDDIVCTDFS AM P FAVNIAELRLYQPLIETCQRFNLVPAWVSMESVDQRITRMFDTKDPKGDIICTDFS AM P FTVNLQELRVYQPFMDMLQKHKIIPAWVGLEEVDNKITKLFDTKGEDDVVICTDFS AM P FAVNIRELQTYQPLIELCQRFELVPAWVSMESVDRRITKMFDTKAKDDLAICTDFS AM P FGVNIRELQVYQPLIESCQRFNLVPAWVSMDAVDQRITAMFDTKGVDDVVICTDFS AM P FGVNINELQVYQPLIECAQKFNLVPAWISMDEVDQRITRMFDTKGKDDVVICTDFS EU P FGINILEHQVYQPLTEAFKRFGYVPAWVSMDAVDLEITKLFDSKDDKDLVICTDFS AM P FAVNICELQVYQPLIEACQKFNLIPAWVSMESVDQRVTKMFDTKGTDDVVICTDFS AM_ P FAVNLCELQVYQPMIESCQRFNLVPAWVSREAVDQRITNMFDTKGVDDLVIWTDFS AM P FAVNIRELQMYQPLIECFQSFDLVPAWVSMESVDKRITRMFDTKGDDDDVVCTDFS AM P FAVNIEELRYYQPAIEAAQNFNLVPAWVSMESVDDRITRMFDTKGPDDLVICTDFS AM P FAVNIEELRAYQPLIEKVQSFNLVPAWVSMESVDDQVTKLFDTKGPDDLVVCTALH AM P FAVNVEELRVYQVLIETAQKFNLVPAWVSMEAVDAAITRLFDTKRPDDLIVCTDFS EU P YAVNIRELQFYQPAIELAQKHLLVPAWVGMDAVDVRITRLFDTKAKSDLIVCTDFS AM P FGVNIEGLRVYQVLIEVAQKFNKVPAWVSMEAVDAAITRLFDTKAPNDLIVCADFS AM P FGVNIRELQVYQPAIELAQRSNLVPAWVGMEAVDVAVTKLFETKGKNDLVICTDFE AM P FAVNVKELCFYQPAIEVAQRANIVPAWVSMDAVDRSITKLFDSKSKDLVVCTDFT AM P FAVNVAELQVYQPLIETAQRFNLVPAWISMDAVDREIAQLFDTKQPSDLVVCTDFS AM P FAVNVAELQVYQSLIETAQRFNLVPAWISMDAVDREITQLFDTKQPSDLVVCTDFS FJ D FAVNVRELQVYQPLIEAAQKFNLVPAWVSMDEVDKRITKLFDTKGQSDLVVCTDFS AB C FAISILELQMYIALIRACQRAEFNPAWISQDAVDMRMTQLMKTKGRNDEIVCTDFT JF M FAVNVAELQFYQPAIEYAQRFGLVPAWISNDEVDRVMTKLFDTKADNDLVVATDFT FJ R YAVNIAELQVYQPLIEAAQNTRLIPSWVSMDEVDRAITYLFDTKGRDDLIICTDF EU S FAVNIAELQVYQPMIEAAQRFDLVPAWVGMESVDRRITKLFDNKSSDDLVV 25

26 Supplementary Table 9. Risk of GVHD occurrence according to detection of eukaryotic viruses Any type of Acute GvHD Enteric GvHD stage 2 Virus host HR CI 95 p.value HR CI 95 p-value Plant ; ; Protozoa ; ; Vertebrate ; ; Plant Genera Unknown ; ; Carlavirus ; ; Closterovirus ; ; Polerovirus ; ; Potexvirus ; ; Tobamovirus ; ; Vertebrate Genera Tombusvirus ; ; Alphatorquevirus ; ; Betatorquevirus ; ; Enterovirus ; ; Gyrovirus ; ; Mamastrovirus ; ; Megrivirus ; ; Parvovirus ; ; Picobirnavirus ; ;4.86 <0.01 Polyomavirus ; ;

27 Supplementary Table 10. Histology of digestive biopsies and fecal biomarkers concentrations in patients with digestive GvHD. Patients with stool samples fpositive for picobirnavirus (as defined in methods) are indicated by PBV status 1 and those with stool samples negative for picobirnavirus by PBV status 0. Calprotectin levels are reported as µg/ml. Alpha-1 antitrypsin levels are reported as mg per g of dried stool sample. Patient Initial stage of digestive GVHD Maximum stage of digestive GVHD Resistance to corticosteroid treatment GVHD resolution Calprotectin (µg/g) AAT (mg/g) PBV status Digestive biopsy Apoptosis Chorion inflammation chorion oedema Cellular infiltrate Vascular damage Yes No Duodenum Yes Yes Yes Yes Yes Yes Yes No Yes No Duodenum Yes Yes No Yes No No No Yes Duodenum Yes Yes Yes Yes No Yes No No Duodenum Yes Yes Yes Yes No No No Yes < Duodenum No Yes No No No No Yes No Duodenum Yes Yes No Yes No Yes Yes Yes < No Yes No No Yes No Colon No Yes Yes No Yes Yes No Yes No Yes < Duodenum Yes Yes No Yes No Yes Yes Yes Duodenum No Yes Yes No Yes No No Yes Yes No 44 0 Duodenum No Yes No No No Yes Yes No 27 0 Duodenum No Yes Yes Yes Yes Yes Yes Yes < Duodenum No Yes No No No No No Yes 1 Duodenum Yes Yes Yes Yes No No No Yes 0 Duodenum Yes Yes No Yes No No No Yes <15 0 Duodenum Yes Yes Yes Yes No Yes No Yes Duodenum Yes Yes Yes Yes No No No Yes <15 1 Duodenum Yes Yes Yes Yes No No Yes No <15 0 Rectum No Yes No Yes No Yes Yes No Duodenum Yes Yes No Yes No Yes No Yes Rectum Yes Yes Yes No No No Epithelial abrasion 27

28 Supplementary Table 11. Summary of fecal biomarkers concentrations and histological findings in patients with digestive GVHD. Elevated fecal calprotectin or AAT* Fecal calprotectin and AAT* at normal values No testing of fecal calprotectin or AAT* Digestive biopsies Inflammation Apoptosis Abrasion No biopsy Total * AAT= α1-antitrypsin 28

29 SUPPLEMENTARY FIGURES # PreTx WO W1 W2 W3 W4 W5 W Supplementary Figure 1. Longitudinal collection of stool samples from HSCT patients before and after transplantation. All stool samples collected are indicated with grey boxes for every patient before transplantation (PreTx) and the weeks following transplantation (W0 to W6). 29

30 # of sool samples positive for virus family (>14 reads detected) Positive detection, <5 RPM Positive detection, >=5 RPM and <50 RPM Positive detection, >=50 RPM 0 Anelloviridae Herpesviridae Papillomaviridae Polyomaviridae Retroviridae Adenoviridae Astroviridae Caliciviridae Circoviridae Coronaviridae Parvoviridae Picobirnaviridae Picornaviridae Reoviridae Persistent infections Exogenous infections Supplementary Figure 2. Frequency of virus-positive samples according to vertebrate viral family obtained by metagenomic sequencing. The percentage of stool samples (out of 201 total samples) testing positive for detection of a virus with viral read counts per million of <5, 5 and <50, and 50 are indicated in green, red and blue, respectively. Only viral families meeting the pre-established detection threshold of 15 viral reads or more per family are shown. Viruses associated with putative chronic infections (persistent viruses) or transient infections (exogenous viruses) are indicated. 30

31 20 # of HSCT Patients # of Persistent Viruses Detected # of Exogenous Viruses Detected Supplementary Figure 3. Persistent (chronic) and exogenous (transient) viral infections in HSCT patients in the study. Shown plotted is the number of HSCT patients (z-axis) versus number of persistent infections (x-axis) and exogenous infections (y-axis) from different viral families detected in longitudinally collected stool samples. 31

32 A Persistent DNA viruses (p<0.0001) No enteric GvHD Enteric GvHD RPM Pre-T W0 W1 W2 W3 >W4 B Herpesviridae (p=0.009) 150 No enteric GvHD 100 Enteric GvHD RPM 50 0 Pre-T W0 W1 W2 W3 >W4 Supplementary Figure 4. Increase in read counts of persistent DNA viruses following transplantation. The mean and standard error of the mean of the viral reads per million (RPM) corresponding to (A) all DNA viral families (Anelloviridae, Herpesviridae, Papillomaviridae, and Polyomavirus) combined and (B) Herpesviridae alone are plotted by week of collection in patients with or without enteric GvHD. In 32

33 patients without enteric GvHD, there were no differences in levels of persistent DNA viral reads and/or Herpesviridae reads over time. In contrast, the mean number of reads assigned to persistent DNA viruses (p< by Kruskal-Wallis nonparametric oneway analysis of variance) and more specifically to Herpesviridae (p=0.009) changed significantly over time in patients with enteric GvHD, with pronounced increases in read counts after week 4. 33

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