OBJECTIVE: This study used next-generation sequencing (NGS) technologies to characterize the gut virome at the onset of islet autoimmunity. RESEARCH DESIGN AND METHODS: We conducted a case-control study nested within the Finnish Diabetes Prediction and Prevention (DIPP) cohort. The stool virome in 19 case children, who turned islet autoantibody positive before the age of 2 years and later developed clinical type 1 diabetes, and 19 tightly matched control subjects was analyzed using NGS performed from stool samples collected 3, 6, and 9 months before the onset of islet autoimmunity. Human virus findings were verified using real-time PCR. RESULTS: One or more human viruses were present in 10.4% and bacteriophages were in 54% of the samples. The virome composition showed no association with islet autoimmunity. NGS was less sensitive and specific than real-time PCR. CONCLUSIONS: The present data suggest no dramatic changes in the gut virome shortly before the emergence of islet autoimmunity and emphasize the need of verification of mass sequencing results when viral exposure is assessed in association studies.

• MeSH
• autoimunita imunologie   MeSH
• autoprotilátky metabolismus   MeSH
• diabetes mellitus 1. typu imunologie   virologie   MeSH
• dítě MeSH
• feces virologie   MeSH
• genotyp MeSH
• hyperglykemie imunologie   virologie   MeSH
• kojenec MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• Langerhansovy ostrůvky imunologie   MeSH
• lidé MeSH
• předškolní dítě MeSH
• střeva virologie   MeSH
• studie případů a kontrol MeSH
• viry izolace a purifikace   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVES: Studies of the fecal virome in type 1 diabetes (T1D) have been limited to populations of Europe and the United States. We therefore sought to characterize the stool virome in children after onset of T1D and in matched control subjects from four geographically distant African and Asian countries. METHODS: Samples of stool were collected from 73 children and adolescents shortly after T1D onset (Azerbaijan 19, Jordan 20, Nigeria 14, Sudan 20) and 105 matched control subjects of similar age and locale. Metagenomic sequencing of the DNA and RNA virome was performed, and virus positivity was defined as more than 0.001% of reads of the sample. Selected viruses were also quantified using real-time PCR. Conditional logistic regression was used to model associations with eukaryotic virus positivity. RESULTS: Signals of 387 different viral species were detected; at least one eukaryotic virus was detected in 71% case and 65% control samples. Neither of observed eukaryotic virus species or genera differed in frequency between children with T1D and controls. There was a suggestive association of the total count of different viral genera per sample between cases (1.45 genera) and controls (1.10 genera, OR 1.24, 95%CI 0.98-1.57), and an unplanned subanalysis suggested marginally more frequent endogenous retrovirus signal in cases (in 28.8% vs. in 8.6% controls, OR = 4.55, 95%CI 1.72-12). CONCLUSIONS: No clear and consistent association with T1D was observed in the fecal viromes from four distant non-European populations. The finding of borderline associations of human endogenous retroviruses merits further exploration.

• MeSH
• diabetes mellitus 1. typu diagnóza   virologie   MeSH
• dítě MeSH
• feces virologie   MeSH
• lidé MeSH
• mladiství MeSH
• střevní mikroflóra MeSH
• studie případů a kontrol MeSH
• virom MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Ázerbájdžán MeSH
• Jordánsko MeSH
• Nigérie MeSH
• Súdán MeSH

Next-generation sequencing has opened avenues to studying complex populations such as the bacteriome (all bacteria), mycobiome (all fungi), and virome (all viruses in a given sample). Viromes are less often investigated as compared to bacteriomes. The reasons are mostly methodological: because no common pan-viral sequence signature exists, metagenomic sequencing remains the only option. This brings about the need of laborious virus enrichment, multiple signal amplification steps with virtually no possibility of interim quality control, and complicated bioinformatic analysis of the ensuing sequence data. Nevertheless, over the past decade virome sequencing has been enormously successful in identifying new agents in human and animal diseases, and in characterizing viruses in various ecological niches. Recently, virome sequencing has been also employed in studies of non-infectious diseases, which has brought about new challenges of sensitivity, costs, and reproducibility in testing of large sets of samples. Here, we present a detailed protocol that has been utilized in virome studies where hundreds of samples had to be reliably tested in order to assess the association of the stool virome with susceptibility to type 1 diabetes, a non-infectious autoimmune disease.

• MeSH
• bakteriofágy klasifikace   genetika   izolace a purifikace   MeSH
• DNA virů genetika   izolace a purifikace   MeSH
• feces virologie   MeSH
• genová knihovna MeSH
• metagenom * MeSH
• metagenomika * metody   MeSH
• polymerázová řetězová reakce MeSH
• RNA virová genetika   izolace a purifikace   MeSH
• střevní mikroflóra * MeSH
• ultracentrifugace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

BACKGROUND: We set out to explore associations between the stool bacteriome profiles and early-onset islet autoimmunity, taking into account the interactions with the virus component of the microbiome. METHODS: Serial stool samples were longitudinally collected from 18 infants and toddlers with early-onset islet autoimmunity (median age 17.4 months) followed by type 1 diabetes, and 18 tightly matched controls from the Finnish Diabetes Prediction and Prevention (DIPP) cohort. Three stool samples were analyzed, taken 3, 6, and 9 months before the first detection of serum autoantibodies in the case child. The risk of islet autoimmunity was evaluated in relation to the composition of the bacteriome 16S rDNA profiles assessed by mass sequencing, and to the composition of DNA and RNA viromes. RESULTS: Four operational taxonomic units were significantly less abundant in children who later on developed islet autoimmunity as compared to controls-most markedly the species of Bacteroides vulgatus and Bifidobacterium bifidum. The alpha or beta diversity, or the taxonomic levels of bacterial phyla, classes or genera, showed no differences between cases and controls. A correlation analysis suggested a possible relation between CrAssphage signals and quantities of Bacteroides dorei. No apparent associations were seen between development of islet autoimmunity and sequences of yet unknown origin. CONCLUSIONS: The results confirm previous findings that an imbalance within the prevalent Bacteroides genus is associated with islet autoimmunity. The detected quantitative relation of the novel "orphan" bacteriophage CrAssphage with a prevalent species of the Bacteroides genus may exemplify possible modifiers of the bacteriome.

• MeSH
• autoimunita * MeSH
• autoimunitní nemoci krev   epidemiologie   etiologie   imunologie   MeSH
• Bacteroides klasifikace   imunologie   izolace a purifikace   virologie   MeSH
• bakteriální RNA chemie   metabolismus   MeSH
• bakteriofágy klasifikace   imunologie   izolace a purifikace   MeSH
• diabetes mellitus 1. typu krev   epidemiologie   etiologie   imunologie   MeSH
• dítě MeSH
• dysbióza imunologie   mikrobiologie   patofyziologie   virologie   MeSH
• feces mikrobiologie   virologie   MeSH
• fylogeneze MeSH
• kohortové studie MeSH
• Langerhansovy ostrůvky imunologie   MeSH
• lidé MeSH
• longitudinální studie MeSH
• molekulární typizace MeSH
• nemocnice univerzitní MeSH
• prospektivní studie MeSH
• riziko MeSH
• RNA ribozomální 16S chemie   metabolismus   MeSH
• RNA virová chemie   metabolismus   MeSH
• střevní mikroflóra imunologie   MeSH
• studie případů a kontrol MeSH
• výpočetní biologie MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Geografické názvy
• Finsko epidemiologie   MeSH

Despite their ecological importance, nothing is known about the diversity and abundance of RNA viruses in termites (Termitoidae). We used a metatranscriptomics approach to determine the RNA virome structure of 50 diverse species of termite that differ in both phylogenetic position and colony composition. From these samples, we identified 67 novel RNA viruses, characterized their genomes, quantified their abundance and inferred their evolutionary history. These viruses were found within or similar to those from the Togaviridae, Iflaviridae, Polycipiviridae, Flaviviridae, Leviviridae, Narnaviridae, Mitoviridae, Lispivirdae, Phasmaviridae, Picobirnaviridae and Partitiviridae. However, all viruses identified were novel and divergent, exhibiting only 20% to 45% amino acid identity to previously identified viruses. Our analysis suggested that 17 of the viruses identified were termite-infecting, with the remainder likely associated with the termite microbiome or diet. Unclassified sobemo-like and bunya-like viruses dominated termite viromes, while most of the phylogenetic diversity was provided by the picobirna- and mitovirus-like viruses. Of note was the identification of a novel flavi-like virus most closely related to those found in marine vertebrates and invertebrates. Notably, the sampling procedure had the strongest association with virome composition, with greater RNA virome diversity in libraries prepared from whole termite bodies than those that only sampled heads.

• MeSH
• genetická variace MeSH
• genom virový genetika   MeSH
• Isoptera virologie   MeSH
• RNA virová genetika   MeSH
• RNA-viry klasifikace   genetika   izolace a purifikace   MeSH
• virom genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Testování kandidátních virů ukázalo jejich roli při iniciaci ostrůvkové autoimunity vedoucí k diabetu 1. typu, ale dosud při výzkumu této problematiky nebylo použito sekvenování nové generace (NGS) všech virových populací (tzv. viromu) ve střevě. Naším cílem je zkoumat asociaci složení lidského střevního viromu s ostrůvkovou autoimunitou právě pomocí NGS kombinovaného se specifickými PCR testy. Finská studie DIPP a norská MIDIA poskytnou dohromady vzorky od 50 dětí s časnou ostrůvkovou autoimunitou a od dvou kontrol ke každému z případů. Střevní virom bude charakterizován pomocí NGS ze vzorků stolice odebraných v pěti časových bodech před ostrůvkovou autoimunitou; potvrzení hlavních nálezů bude provedeno pomocí PCR. Navíc budou testovány virové sekvenční signatury v nukleových kyselinách extrahovaných z krve odebrané ve třech časových bodech před ostrůvkovou autoimunitou. Data budou analyzována pomocí logistické regrese se smíšenými efekty. Použití dvou rozdílných vysokorizikových kohort považujeme za unikátní z hlediska robustnosti pro detekci případné kauzální asociace viru.; Candidate virus studies have indicated a role of viruses in initiating islet autoimmunity leading to type 1 diabetes, but no unbiased sequencing of all virus populations (virome) of the gut has been performed in this context. Our aim is to investigate the association of gut virome composition with islet autoimmunity using next-generation sequencing (NGS) followed by confirmation with specific PCR tests from stool and blood. The Finnish DIPP and Norwegian MIDIA studies will together provide samples from 50 young prediabetic children who developed early islet autoimmunity, and from two controls matched to each case. The virome will be characterized by NGS from stool samples collected at five time points before islet autoimmunity. The obtained virus signatures will be tested by a highly multiplexed set of specific PCR assays in stool and also in corresponding blood samples. Statistical analysis will utilize mixed effects logistic regression models. The use of two distinct high-risk cohorts renders the study a unique robustness for detection of causative virus associations.

• MeSH
• autoimunita MeSH
• biochemická analýza krve MeSH
• diabetes mellitus 1. typu MeSH
• kohortové studie MeSH
• novorozenec MeSH
• polymerázová řetězová reakce MeSH
• rizikové faktory MeSH
• střevní mikroflóra MeSH
• viry MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• novorozenec MeSH
• Geografické názvy
• Finsko MeSH
• Norsko MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• virologie
• alergologie a imunologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Hrbáček J, Hanáček V, Tláskal V, Saláková M, Čermák P, Tachezy R, Zachoval R. Detekce mikrobiomu horních močových cest u pacientů s urolitiázou: studie proveditelnosti. Cíl: Zjistit, zda existuje mikrobiální osídlení horních močových cest (HMC) a pokud ano, tak liší-li se od mikrobiomu dolních močových cest (DMC). Materiál a metoda: Soubor tvoří pacienti podstupující endoskopický operační zákrok v anestezii pro litiázu HMC. Moč z močového měchýře byla odebrána aseptickou katetrizací, z HMC cystoskopicky zavedeným ureterálním katétrem. Po izolaci DNA byla bakteriální 16S rDNA sekvenována na platformě Illumina MiSeq a získaná data zpracována příslušnými bioinformatickými nástroji. Část izolované DNA byla využita k detekci virových nukleových kyselin pomocí kvantitativní polymerázové řetězové reakce (qPCR). Párové vzorky vždy z HMC a DMC byly porovnány z hlediska struktury bakteriálního společenstva a přítomnosti vybraných DNA virů. Výsledky: Zařazeno bylo devět pacientů (8 mužů a 1 žena) průměrného věku 49,7 let, z nichž u osmi byla provedena sekvenace bakteriálních nukleových kyselin a u sedmi qPCR na přítomnost sedmi běžných DNA virů. Bohatost a diverzita mikrobiomu (alfa diverzita) HMC a DMC nebyla statisticky významně odlišná. Struktura mikrobiálních komunit z HMC a DMC (beta diverzita) vykázala větší podobnost v rámci jednoho pacienta, než byla podobnost všech vzorků HMC, resp. všech vzorků DMC. Virová DNA byla detekována jak v HMC, tak v DMC. Závěr: Výsledky naší pilotní studie poukazují na přítomnost bakteriálního a virového osídlení horních močových cest u pacientů s urolitiázou.

Hrbáček J, Hanáček V, Tláskal V, Saláková M, Čermák P, Tachezy R, Zachoval R. Profiling upper urinary tract microbiota: a feasibility study in patients with urinary stone disease. Aim: To investigate the putative existence of upper urinary tract microbiota and if present, to compare the microbial communities from the kidney with those in the urinary bladder. Patients and Methods: Patients were undergoing endoscopic procedures under anesthesia for upper urinary tract stones. Bladder urine was obtained by aseptic catheterisation, renal urine samples were collected via ureteric catheter inserted into the renal pelvis under cystoscopic and radiographic guidance. Pairwise comparison of urine samples from the same individual were made with regard to their bacteriome and virome. Results: A total of nine patients (8 males, 1 female) provided both samples for analysis. Next-generation sequencing of the V4 hypervariable region of the 16S rDNA using primers 515F and 806R was performed on eight of them and quantitative polymerase chain reaction for the presence of viral nucleic acids of seven common DNA viruses was performed on seven of them. There were no significant differences in alpha diversity measures (number of OTUs, iChao1, ACE, Shannon and Simpson indices) of samples from the kidney and bladder from the same individual. Likewise, microbial communities from the upper and lower urinary tract within the same individual were more similar to each other than samples within the respective group (e.g. all kidney samples). Viral nucleic acids were detected in the upper as well as lower urinary tract. Conclusion: Upper urinary tracts seem to be inhabited by microbial communities whose composition resembles that of the lower urinary tract.

• Klíčová slova
• Human microbiome, upper urinary tract, virome, kidney, urinary bladder, Lidský mikrobiom, horní močový trakt, virom, ledvina, močový měchýř,
• MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikrobiologické techniky MeSH
• mikrobiota * MeSH
• močové ústrojí * mikrobiologie   MeSH
• pilotní projekty MeSH
• polymerázová řetězová reakce MeSH
• sekvenční analýza MeSH
• studie proveditelnosti MeSH
• urolitiáza mikrobiologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH

The severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) dwells in the respiratory airways, however, its digestive tract presence, infectivity, mucosal attachment and penetrating mechanisms, enteric proliferation, stool shading, flashed bio-aerosol spreading and fecal-oral transmission is far from being understood. The present review opens some skylights to lighten the long, tortuous, dark and challenging tunnel of the gastrointestinal tract and his uninvited covid-19 viral new inhabitant.

• Klíčová slova
• Covid 19,
• MeSH
• angiotensin-konvertující enzym 2 škodlivé účinky   MeSH
• COVID-19 * etiologie   mortalita   patofyziologie   patologie   přenos   MeSH
• endoskopie škodlivé účinky   MeSH
• feces virologie   MeSH
• gastrointestinální trakt patofyziologie   patologie   virologie   MeSH
• lidé MeSH
• průjem etiologie   MeSH
• receptory koronavirů MeSH
• SARS-CoV-2 patogenita   MeSH
• střeva * virologie   MeSH
• virom MeSH
• vylučování virů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.

• MeSH
• Bacteroidetes klasifikace   genetika   virologie   MeSH
• bakteriofágy klasifikace   genetika   MeSH
• DNA virů genetika   MeSH
• feces virologie   MeSH
• fylogeneze MeSH
• fylogeografie MeSH
• genetická variace MeSH
• koevoluce * MeSH
• lidé MeSH
• primáti virologie   MeSH
• střevní mikroflóra * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH