1、letters1080VOLUME 23|NUMBER 9|SEPTEMBER 2017 nature medicineMuch attention has been focused on the role of the bacterial microbiome in human health,but the virome is understudied.Although previously investigated in individuals with inflammatory bowel diseases or solid-organ transplants1,2,virome dyn
2、amics in allogeneic hematopoietic stem cell transplantation(HSCT)and enteric graft-versus-host disease(GVHD)remain unexplored.Here we characterize the longitudinal gut virome in 44 recipients of HSCT using metagenomics.A viral bloom was identified,and significant increases were demonstrated in the o
3、verall proportion of vertebrate viral sequences following transplantation (P=0.02).Increases in both the rates of detection (P 0.0001)and number of sequences(P=0.047)of persistent DNA viruses(anelloviruses,herpesviruses,papillomaviruses and polyomaviruses)over time were observed in individuals with
4、enteric GVHD relative to those without,a finding accompanied by a reduced phage richness(P=0.01).Picobirnaviruses were detected in 18 individuals(40.9%),more frequently before or within a week after transplant than at later time points(P=0.008).In a time-dependent Cox proportional-hazards model,pico
5、birnaviruses were predictive of the occurrence of severe enteric GVHD(hazard ratio,2.66;95%confidence interval(CI)=1.464.86;P=0.001),and correlated with higher fecal levels of two GVHD severity markers,calprotectin and a1-antitrypsin.These results reveal a progressive expansion of vertebrate viral i
6、nfections over time following HSCT,and they suggest an unexpected association of picobirnaviruses with early post-transplant GVHD.Viruses,arising mainly from the Herpesviridae and Adenoviridae families,have been reported to be frequently linked to enteric GVHD35.These viruses are associated with lat
7、ent infections that can be reactivated in the setting of host immunosuppression from HSCT.Active viral replication from reactivation,resulting in dynamic shifts in gut viral loads,may trigger intestinal inflammation and dysregula-tion of the restored immune response,which then leads to tissue dam-ag
8、e and enhanced risk of GVHD.HSCT induces profound changes in bacterial microbiota diversity and may potentially impact the gut virome in a parallel fashion6.Eukaryotic viruses are typically minor components of the gut as compared to phage populations7,but acute infections with exogenous viruses or c
9、hronic infections resulting from the reactivation of persist-ent viruses can give rise to high titers in individuals who are immuno-compromised3,8.Dynamic changes in the composition and diversity of viral populations following transplantation may have clinical sig-nificance,such as a recently descri
10、bed association between persistent viral infections and protective immunity during gut inflammation9.Broad surveillance approaches are needed to capture these changes,given the high degree of viral-sequence diversity and continual dis-covery of novel viruses in human stool by metagenomics1012.In thi
11、s study,we characterized the profile and longitudinal evolu-tion of the human eukaryotic gut virome in individuals before and after HSCT to investigate potential links between changing viral dynamics and the development of enteric GVHD(Fig.1).Here we focused on serial collection and analysis of stoo
12、l samples,because the detection of human viruses in feces suggests active viral replication and shedding in the gut.In parallel,the bacterial microbiome and phage virome were also analyzed.A total of 201 stool samples were collected from 44 individuals(Fig.1),with a median of four samples per patien
13、t(range:38 sam-ples)(Supplementary Fig.1).31 individuals(70.5%)developed acute GVHD,26(83.9%)of whom experienced enteric GVHD,16 of which(46.2%)were severe cases(stage 2 or higher)(Fig.1a and Supplementary Table 1).For each week,the numbers of collected The eukaryotic gut virome in hematopoietic ste
14、m cell transplantation:new clues in enteric graft-versus-host diseaseJrme Legoff1,2,Matthieu Resche-Rigon3,Jerome Bouquet2,4,Marie Robin5,Samia N Naccache2,4,Sverine Mercier-Delarue1,Scot Federman2,4,Erik Samayoa2,4,Clotilde Rousseau5,Prescillia Piron3,Nathalie Kapel6,Franois Simon1,Grard Soci7,9&Ch
15、arles Y Chiu2,4,8,9 1University of Paris Diderot,Sorbonne Paris Cit,Inserm U941,Microbiology Laboratory,Hpital Saint-Louis,APHP,Paris,France.2UCSF-Abbott Viral Diagnostics and Discovery Center,San Francisco,California,USA.3University of Paris Diderot,Sorbonne Paris Cit,Inserm U1153,ECSTRA Team,Biost
16、atistics Unit,APHP,Hpital Saint-Louis,Paris,France.4Department of Laboratory Medicine,University of California,San Francisco,California,USA.5University of Paris Descartes,EA4065,Microbiology Laboratory,Hpital Saint-Louis,APHP,Paris,France.6Laboratoire de Coprologie Fonctionnelle,APHP,GH Piti-Salptri
17、re Charles Foix,EA4065,Universit Paris Descartes,Paris,France.7University of Paris Diderot,Sorbonne Paris Cit,Paris,France,and Hematology and Transplantation,INSERM Unit Mixte de Recherche Scientifique 1160,Paris,France.8Department of Medicine,Division of Infectious Diseases,University of California
18、,San Francisco,California,USA.9These authors contributed equally to this work.Correspondence should be addressed to C.Y.C.(charles.chiuucsf.edu).Received 12 September 2016;accepted 29 June 2017;published online 31 July 2017;doi:10.1038/nm.4380 2017 Nature America,Inc.,part of Springer Nature.All rig
19、hts reserved.lettersnature medicine VOLUME 23|NUMBER 9|SEPTEMBER 2017 1081stool samples from patients with enteric GVHD were comparable(Fig.1b),although proportionally fewer samples from individuals without enteric GVHD(heretofore defined as both patients without GVHD and those few with GVHD of anot
20、her type)were available during later weeks(weeks 35).Acute GVHDof any typedevel-oped a median of 27.7 d after HSCT(interquartile range(IQR),1438 d)(Supplementary Table 1).Metagenomic sequencing analysis using a virome-specific library preparation(Fig.1c and Supplementary Results)yielded a total of 5
21、.9 billion raw sequence reads,with a median of 29 million reads per sample(IQR,25 million33 million).Reads were analyzed using the SURPI computational pipeline for pathogen identifica-tion13,which detects and taxonomically classifies microbial reads by nucleotide(NT)alignment with those in the Natio
22、nal Center for Biotechnology Information(NCBI)NT database,including the entirety of GenBank.Parallel stool-microbiome analysis by sequencing of the V3V4 region of the bacterial 16S ribosomal RNA(rRNA)gene showed that the most significant changes in relative abundance of bacterial taxa were an increa
23、se in Lactobacillales spp.(P=0.007)and a concomi-tant decrease in Clostridiales spp.(P=0.02)over time in individuals with enteric GVHD(Fig.2a and Supplementary Results).Overall,there was no difference in richness(P=0.53),but a slightly higher relative abundance(P=0.047),of bacterial taxa at the genu
24、s level in patients with,relative to those without,enteric GVHD(Fig.2b,c).Phage analysis of the metagenomic sequencing data showed that indi-viduals with enteric GVHD had both decreased richness(P=0.01)and a higher abundance of Microviridae prior to and during the first few weeks following transplan
25、tation(P=0.03)(Fig.2).By SURPI analysis,the median percentage of viral reads overall was 0.22%(IQR,0.0011.6%).Eukaryotic viral reads accounted for a median of 1.76%of the viral fraction,although the proportion differed between individuals(IQR,0.1621.3%;range,099.9%).Eukaryotic viruses accounted for
26、more than 10%of total viral reads in 32.6%of stool stamples,and more than 50%of total viral reads in 15.4%of stool samples.Eukaryotic viruses were assigned mostly to vertebrate taxa(86.9%),and plant viruses(12.1%)were the second most common(Supplementary Results).Among vertebrate viruses,a total of
27、20 families,52 genera and 347 species were identified.The most frequent viral families observed in stool samples from recipients of HSCT,in order of decreasing frequency,were Anelloviridae(37.8%of samples),Polyomaviridae(14.9%),Picobirnaviridae(13.9%)and Herpesviridae(10.9%)(Supplementary Fig.2 and
28、Supplementary Tables 24).Persistent DNA viruses(Anelloviridae,Herpesviridae,Papillomaviridae and Polyomaviridae)were detected in 100 of 201 samples(49.8%),whereas exogenous viruses were detected in 72 samples(35.8%).Repeat detec-tion of the same viral family at least twice following transplantation
29、was observed in 14 of 44 individuals(31.8%)harboring persistent viruses,four patients(9.1%)harboring exogenous viruses and nine(18.2%)harboring both types of virus(Supplementary Fig.3).In general,persistent viruses were detected more frequently and yielded higher read numbers than exogenous viruses.
30、The overall proportion of vertebrate viral sequences in the guts of all HSCT recipients increased progressively during the weeks following transplantation(P=0.02 for patients with or without enteric GVHD combined)(Fig.3a).No difference in vertebrate viral diversity was observed between individuals w
31、ith or without enteric GVHD(P=0.98),but decreased richness at the species level was observed during the first few weeks following transplantation in individuals with enteric GVHD(P=0.007)(Fig.3b,c).Notably,increases in both the rates of detection(Fig.3d and Supplementary Table 5;P=0.047)and the numb
32、er of reads(Supplementary Fig.4;P 3.5 log10 IU/ml)and human her-pesvirus 6(HHV-6)in blood from individuals with enteric GVHD (P=0.04 and P 3.5 log10 UI/ml)were also more frequent in individuals with enteric GVHD than in those without(P W0025507500PreG W0 W1 W2 W3 W4=WNo enteric GVHDEnteric GvHDabAlg
33、aeFish/amphibianFungusInsectPlantProtozoaUnknownVertebrate%of patients%of patients%of patients%of patientsdNo enteric GVHDEnteric GVHD%of reads assigned to viral family%of reads assigned to putative hostPolyomaviridaeRetroviridaePicobirnaviridaePicornaviridaePapillomaviridaeParvoviridae0255075100025
34、507510002550751000255075100HerpesviridaeOtherAnelloviridaeCircoviridaePolyomaviridaePicobirnaviridaePapillomaviridaeHerpesviridaeAnelloviridaeRetroviridaePicornaviridaeParvoviridaeOtherCircoviridaecShannon diversity indexChao richness scoreNo enteric GVHDEnteric GVHDP=0.007P=0.980123PreTxW0W1W2W3W4W
35、5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5010203040AnelloviridaeCircoviridaeHerpesviridaeOtherParvoviridaePicornaviridaePolyomaviridaeRetroviridaePicorbirnaviridaePapillomaviridae1007550250PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W
36、5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5PreTxW0W1W2W3W4W5Figure 3 Evolution of the enteric virome following transplantation.(a)Distribution of viral reads across putative eukaryotic hosts.The percentage of reads corresponding to each host is shown for ea
37、ch time point.(b)Distribution of viral reads across different viral families.The percentage of viral reads corresponding to each family is shown for each time point.(c)Viral alpha diversity(Shannon Diversity Index,P=0.98,linear random-effects model and Wald test for joint significance of regression
38、coefficients)and richness(Chao Richness Score,P=0.007,linear random-effects model and Wald test)of vertebrate viruses at the species level by week of collection in patients with or without enteric GVHD.(d)For each viral family,the proportion of patients positive for sequence reads from that family i
39、s plotted longitudinally over time.The color scheme is the same as in b.Summary data are stratified according to the presence or absence of enteric GVHD.A positive viral detection is defined as 15 reads,and viral families detected at a frequency lower than 5%are classified as“other”.2017 Nature Amer
40、ica,Inc.,part of Springer Nature.All rights reserved.letters1084VOLUME 23|NUMBER 9|SEPTEMBER 2017 nature medicinewith corticosteroid therapy.These findings suggest that the onset of GVHD might have triggered DNA viral reactivation,rather than reactivation constituting a risk factor for GVHD,per se.I
41、ndeed,the reactivation of persistent DNA viruses in the gut during weeks 13 after transplantation was seen mainly in individuals without enteric GVHD.This suggests a potential protective role for persistent DNA viral replication in the regulation of gut inflammation predisposing to enteric GVHD,as p
42、reviously postulated in a mouse model of IBD9.Surprisingly,the presence of plant viruses was associated with a reduced risk of GVHD of any type.It is possible that HSCT recipients without GVHD have an increased likelihood of advancing to a more robust diet.Previous reports show that a specific diet
43、modifies the gut microbiome7,and that these alterations in the gut microbiota can mitigate the severity of enteric GVHD15,16.A parallel mechanism may be present with respect to the plant virome,given that dietary antigens,including those derived from viruses,are known to have a role in intestinal im
44、munity17.Notably,we found that evidence of stool PBV infection was predic-tive of later development of GVHD.Because the Cox proportional-hazards model used here takes into account all time points,we cannot pinpoint a precise time point corresponding to maximal risk,although the proportion of individ
45、uals with enteric GVHD found to be positive for PBV(Fig.3b)suggests that it might fall near the time of transplan-tation.Data on the pathogenicity of Picobirnaviridae remain limited and inconclusive18.PBV surveillance efforts have been hampered by the high genetic variability of the virus1921,limiti
46、ng accurate rates of detection in clinical samples.Phylogenetic analyses showed that PBV sequences in HSCT recipients clustered broadly with other PBV sequences identified mostly in human or wastewater samples(Fig.4).These results suggest that the detection of PBV represents bona fide human infectio
47、ns from these viruses.In addition,PBV strains from individual patients were very different from each other according to phylogenetic analysis,excluding inadvertent cross-contamination between samples as an explanation for the observed strain diversity.Repeat detection of PBV was observed in longitud
48、inally collected samples from individuals with GVHD.Although chronic excretion of PBV for several months has been previously described in patients with HIV infection experiencing diarrhea22,our finding that serial PBV sequences from the same patient are closely related underscores the capacity of in
49、dividual PBV strains to cause persistent infection,which potentially promotes GVHD development by maintaining an inflam-matory milieu in the gut.Whereas biomarkers associated with acute GVHD and GVHD severity have been described in the literature,EU938871 WEU938806 WEU938908 WEU938717 WGU968929 HAM7
50、06366 PEU938815 WAM706392 PEU814971 SAM706358 PGU968938 HGU968927 HAB186898 H44 W044 W1AM706385 PAM706389 PAM706371 PAM706378 PGU968937 HAF245701 HAB517739 HEU938844 WEU938862 W30 PreTxEU938707 WEU938811 WAM706367 PAB517732 HAM706382 PAM706375 PEU938877 WAM 706363PAB517736 HEU938865 WAM706368 PFJ164
