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Abstract Adenoid cystic carcinoma (ACC) is a rare, usually slow-growing yet aggressive head and neck malignancy. Despite its clinical significance, our understanding of the cellular evolution and microenvironment in ACC remains limited. We investigated the intratumoral microbiomes of 50 ACC tumor tissues and 33 adjacent normal tissues using 16S rRNA gene sequencing. This allowed us to characterize the bacterial communities within the ACC and explore potential associations between the bacterial community structure, patient clinical characteristics, and tumor molecular features obtained through RNA sequencing. The bacterial composition in the ACC was significantly different from that in adjacent normal salivary tissue, and the ACC exhibited diverse levels of species richness. We identified two main microbial subtypes within the ACC: oral-like and gut-like. Oral-like microbiomes, characterized by increased diversity and abundance of Neisseria, Leptotrichia, Actinomyces, Streptococcus, Rothia, and Veillonella (commonly found in healthy oral cavities), were associated with a less aggressive ACC-II molecular subtype and improved patient outcomes. Notably, we identified the same oral genera in oral cancer and head and neck squamous cell carcinomas. In both cancers, they were part of shared oral communities associated with a more diverse microbiome, less aggressive tumor phenotype, and better survival that reveal the genera as potential pancancer biomarkers for favorable microbiomes in ACC and other head and neck cancers. Conversely, gut-like intratumoral microbiomes, which feature low diversity and colonization by gut mucus layer-degrading species, such as Bacteroides, Akkermansia, Blautia, Bifidobacterium, and Enterococcus, were associated with poorer outcomes. Elevated levels of Bacteroides thetaiotaomicron were independently associated with significantly worse survival and positively correlated with tumor cell biosynthesis of glycan-based cell membrane components.

ABSTRACT Mexican Americans are disproportionally affected by metabolic dysfunction-associated steatotic liver disease (MASLD), which often co-occurs with diabetes. Despite extensive evidence on the causative role of the gut microbiome in MASLD, studies determining the involvement of the gut phageome are scarce. In this cross-sectional study, we characterized the gut phageome in Mexican Americans of South Texas by stool shotgun metagenomic sequencing of 340 subjects, concurrently screened for liver steatosis by transient elastography. Inter-individual variations in the phageome were associated with gender, country of birth, diabetes, and liver steatosis. The phage signatures for diabetes and liver steatosis were subsequently determined. Enrichment of Inoviridae was associated with both diabetes and liver steatosis. Diabetes was further associated with the enrichment of predominantly temperate Escherichia phages, some of which possessed virulence factors. Liver steatosis was associated with the depletion of Lactococcus phages r1t and BK5-T, and enrichment of the globally prevalent Crassvirales phages, including members of genus cluster IX (Burzaovirus coli, Burzaovirus faecalis) and VI (Kahnovirus oralis). The Lactococcus phages showed strong correlations and co-occurrence with Lactococcus lactis, while the Crassvirales phages, B. coli, B. faecalis, and UAG-readthrough crAss clade correlated and co-occurred with Prevotella copri. In conclusion, we identified the gut phageome signatures for two closely linked metabolic diseases with significant global burden. These phage signatures may have utility in risk modeling and disease prevention in this high-risk population, and identification of potential bacterial targets for phage therapy.IMPORTANCEPhages influence human health and disease by shaping the gut bacterial community. Using stool samples from a high-risk Mexican American population, we provide insights into the gut phageome changes associated with diabetes and liver steatosis, two closely linked metabolic diseases with significant global burden. Common to both diseases was an enrichment of Inoviridae, a group of phages that infect bacterial hosts chronically without lysis, allowing them to significantly influence bacterial growth, virulence, motility, biofilm formation, and horizontal gene transfer. Diabetes was additionally associated with the enrichment of Escherichia coli-infecting phages, some of which contained virulence factors. Liver steatosis was additionally associated with the depletion of Lactococcus lactis-infecting phages, and enrichment of Crassvirales phages, a group of virulent phages with high global prevalence and persistence across generations. These phageome signatures may have utility in risk modeling, as well as identify potential bacterial targets for phage therapy.

Patients undergoing immune effector cell therapy (IECT) are at high risk for infections. We assessed seropositivity against pneumococcus, tetanus, and diphtheria in patients before and after IECT and the patients’ response to vaccination. We enrolled patients who underwent IECT from January 2020 to March 2022. Antibody levels for diphtheria, tetanus, and pneumococcus were measured before IECT, at 1 month, and 3–6 months after. Eligible patients were vaccinated after IECT. In non-seroprotected patients, we discontinued testing. Before IECT, most patients had seroprotective antibody levels against tetanus (68/69, 99%) and diphtheria (65/69, 94%), but fewer did against pneumococcus (24/67, 36%). After IECT, all patients had seroprotective antibody levels for tetanus at 1 month (68/68) and 3–6 months (56/56). For diphtheria, 65/65 patients (100%) had seroprotective antibody levels at 1 month, and 48/53 (91%) did at 3–6 months. For pneumococcus, seroprotective antibody levels were identified in 91% (21/23) of patients at 1 month and 79% (15/19) at 3–6 months following IECT. Fifteen patients received a pneumococcal vaccine after IECT, but none achieved seroprotective response. One patient received the tetanus-diphtheria vaccine and had a seroprotective antibody response. Because some patients experience loss of immunity after IECT, studies evaluating vaccination strategies post-IECT are needed.

Abstract Although the genetic basis and pathogenesis of type 1 diabetes have been studied extensively, how host responses to environmental factors might contribute to autoantibody development remains largely unknown. Here, we use longitudinal blood transcriptome sequencing data to characterize host responses in children within 12 months prior to the appearance of type 1 diabetes-linked islet autoantibodies, as well as matched control children. We report that children who present with insulin-specific autoantibodies first have distinct transcriptional profiles from those who develop GADA autoantibodies first. In particular, gene dosage-driven expression of GSTM1 is associated with GADA autoantibody positivity. Moreover, compared with controls, we observe increased monocyte and decreased B cell proportions 9-12 months prior to autoantibody positivity, especially in children who developed antibodies against insulin first. Lastly, we show that control children present transcriptional signatures consistent with robust immune responses to enterovirus infection, whereas children who later developed islet autoimmunity do not. These findings highlight distinct immune-related transcriptomic differences between case and control children prior to case progression to islet autoimmunity and uncover deficient antiviral response in children who later develop islet autoimmunity.

Here, via metagenomics and ITS2 sequencing analysis of children's stool samples from three months to four years, the authors show that the fungal composition changes and relative abundance increases at weaning, but unlike bacteria, the overall levels of fungal diversity do not change substantially over time.

BackgroundSquamous cell carcinoma of the anus (SCCA) is a rare gastrointestinal cancer. Factors associated with progression of HPV infection to anal dysplasia and cancer are unclear and screening guidelines and approaches for anal dysplasia are less clear than for cervical dysplasia. One potential contributing factor is the anorectal microbiome. In this study, we aimed to identify differences in anal microbiome composition in the settings of HPV infection, anal dysplasia, and anal cancer in this rare disease.MethodsPatients were enrolled in two prospective studies. Patients with anal dysplasia were part of a cross-sectional cohort that enrolled women with high-grade lower genital tract dysplasia. Anorectal tumor swabs were prospectively collected from patients with biopsy-confirmed locally advanced SCCA prior to receiving standard-of-care chemoradiotherapy (CRT). Patients with high-grade lower genital tract dysplasia without anal dysplasia were considered high-risk (HR Normal). 16S V4 rRNA Microbiome sequencing was performed for anal swabs. Alpha and Beta Diversity and composition were compared for HR Normal, anal dysplasia, and anal cancer.Results60 patients with high-grade lower genital tract dysplasia were initially enrolled. Seven patients had concurrent anal dysplasia and 44 patients were considered HR Normal. Anorectal swabs from 21 patients with localized SCCA were included, sequenced, and analyzed in the study. Analysis of weighted and unweighted UniFrac distances demonstrated significant differences in microbial community composition between anal cancer and HR normal (p=0.018). LEfSe identified that all three groups exhibited differential enrichment of specific taxa. Peptoniphilus (p=0.028), Fusobacteria (p=0.0295), Porphyromonas (p=0.034), and Prevotella (p=0.029) were enriched in anal cancer specimens when compared to HR normal.ConclusionAlthough alpha diversity was similar between HR Normal, dysplasia and cancer patients, composition differed significantly between the three groups. Increased anorectal Peptoniphilus, Fusobacteria, Porphyromonas, and Prevotella abundance were associated with anal cancer. These organisms have been reported in various gastrointestinal cancers with roles in facilitating the proinflammatory microenvironment and neoplasia progression. Future work should investigate a potential role of microbiome analysis in screening for anal dysplasia and investigation into potential mechanisms of how these microbial imbalances influence the immune system and anal carcinogenesis.

Granulomatous/sarcoid-like lesions have been reported in patients treated with immune checkpoint inhibitors (ICIs). Here the authors report the occurrence of “nodal immune flare”, an apparent radiological cancer progression in the nodes characterized by the absence of cancer and the presence of non-caseating granulomas, in patients with non-small cell lung cancer following neoadjuvant ICI treatment.

We examined the association between the colonic adherent microbiota and nocturnal sleep duration in humans. In a cross-sectional study, 63 polyp-free adults underwent a colonoscopy and donated 206 mucosal biopsies. The gut microbiota was profiled using the 16S rRNA gene sequencing targeting the V4 region. The sequence reads were processed using UPARSE and DADA2, respectively. Lifestyle factors, including sleep habits, were obtained using an interviewer-administered questionnaire. We categorized the participants into short sleepers (n = 16) and normal sleepers (6–8 h per night; n = 47) based on self-reported data. Differences in bacterial biodiversity and the taxonomic relative abundance were compared between short vs. normal sleepers, followed by multivariable analysis. A false discovery rate-adjusted p value (q value) < 0.05 indicated statistical significance. The bacterial community composition differed in short and normal sleepers. The relative abundance of Sutterella was significantly lower (0.38% vs. 1.25%) and that of Pseudomonas was significantly higher (0.14% vs. 0.08%) in short sleepers than in normal sleepers (q values < 0.01). The difference was confirmed in the multivariable analysis. Nocturnal sleep duration was associated with the bacterial community composition and structure in the colonic gut microbiota in adults.

The family Tymoviridae comprises positive-sense RNA viruses, which mainly infect plants. Recently, a few Tymoviridae-like viruses have been found in mosquitoes, which feed on vertebrate sources. We describe a novel Tymoviridae-like virus, putatively named, Guachaca virus (GUAV), isolated from Culex pipiens and Culex quinquefasciatus species of mosquitoes and collected in the rural area of Santa Marta, Colombia. After a cytopathic effect was observed in C6/36 cells, RNA was extracted and processed through the NetoVIR next-generation sequencing protocol, and data were analyzed through the VirMAP pipeline. Molecular and phenotypic characterization of the GUAV was achieved using a 5′/3′ RACE, transmission electron microscopy, amplification in vertebrate cells, and phylogenetic analysis. A cytopathic effect was observed in C6/36 cells three days post-infection. The GUAV genome was successfully assembled, and its polyadenylated 3′ end was corroborated. GUAV shared only 54.9% amino acid identity with its closest relative, Ek Balam virus, and was grouped with the latter and other unclassified insect-associated tymoviruses in a phylogenetic analysis. GUAV is a new member of a family previously described as comprising plant-infecting viruses, which seem to infect and replicate in mosquitoes. The sugar- and blood-feeding behavior of the Culex spp., implies a sustained contact with plants and vertebrates and justifies further studies to unravel the ecological scenario for transmission.

Travis Sims and Molly El Alam et al. show that diversity of gut microbiota is associated with a favorable response to chemoradiation for cervical cancer and use flow cytometry to show that patients with high microbiome diversity had increased tumor infiltration of CD4+ lymphocytes as well as activated subsets of CD4 cells expressing ki67+ and CD69+ throughout radiation therapy. These results reveal how modulation of the gut microbiota could potentially be used to improve treatment efficacy and outcome.

Coronavirus disease (COVID-19) in Colombia was first diagnosed in a traveler arriving from Italy on February 26, 2020. However, limited data are available on the origins and number of introductions of COVID-19 into the country. We sequenced the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from 43 clinical samples we collected, along with another 79 genome sequences available from Colombia. We investigated the emergence and importation routes for SARS-CoV-2 into Colombia by using epidemiologic, historical air travel, and phylogenetic observations. Our study provides evidence of multiple introductions, mostly from Europe, and documents >12 lineages. Phylogenetic findings validate the lineage diversity, support multiple importation events, and demonstrate the evolutionary relationship of epidemiologically linked transmission chains. Our results reconstruct the early evolutionary history of SARS-CoV-2 in Colombia and highlight the advantages of genome sequencing to complement COVID-19 outbreak investigations.

Acute malnutrition, or wasting, is implicated in over half of all deaths in children under five and increases risk of infectious disease. Studies in humans and preclinical models have demonstrated that malnutrition is linked to an immature intestinal microbiota characterized by increased prevalence of Enterobacteriaceae. Observational studies in children with moderate acute malnutrition (MAM) have also observed heightened systemic inflammation and increased circulating bacterial lipopolysaccharides (LPS; endotoxin). However, the mechanisms that underpin the systemic inflammatory state and endotoxemia, and their pathophysiological consequences, remain uncertain. Understanding these pathophysiological mechanisms is necessary to design targeted treatments that will improve the unacceptable rate of failure or relapse that plague current approaches. Here we use a mouse model of MAM to investigate the mechanisms that promote inflammation in the malnourished host. We found that mice with MAM exhibited increased systemic inflammation at baseline, increased translocation of bacteria and bacterial LPS, and an exaggerated response to inflammatory stimuli. An exaggerated response to bacterial LPS was associated with increased acute weight loss. Remarkably, intestinal inflammation and barrier dysfunction was found in the cecum and colon. The cecum showed a dysbiotic microbiota with expansion of Gammaproteobacteria and some Firmicutes, and contraction of Bacteroidetes. These changes were paralleled by an increase in fecal LPS bioactivity. The inflammatory phenotype and weight loss was modulated by oral administration of non-absorbable antibiotics that altered the proportion of cecal Gammaproteobacteria. We propose that the heightened inflammation of acute malnutrition is the result of changes in the intestinal microbiota, intestinal barrier dysfunction in the cecum and colon, and increased systemic exposure to LPS.

Multiple studies have identified changes within the gut microbiome in response to diarrheal-inducing bacterial pathogens. However, examination of the microbiome in response to viral pathogens remains understudied. Compounding this, many studies use fecal samples to assess microbiome composition; which may not accurately mirror changes within the small intestine, the primary site for most enteric virus infections. As a result, the functional significance of small intestinal microbiome shifts during infection is not well defined. To address these gaps, rotavirus-infected neonatal mice were examined for changes in bacterial community dynamics, host gene expression, and tissue recovery during infection. Profiling bacterial communities using 16S rRNA sequencing suggested significant and distinct changes in ileal communities in response to rotavirus infection, with no significant changes for other gastrointestinal (GI) compartments. At 1-d post-infection, we observed a loss in Lactobacillus species from the ileum, but an increase in Bacteroides and Akkermansia, both of which exhibit mucin-digesting capabilities. Concomitant with the bacterial community shifts, we observed a loss of mucin-filled goblet cells in the small intestine at d 1, with recovery occurring by d 3. Rotavirus infection of mucin-producing cell lines and human intestinal enteroids (HIEs) stimulated release of stored mucin granules, similar to in vivo findings. In vitro, incubation of mucins with Bacteroides or Akkermansia members resulted in significant glycan degradation, which altered the binding capacity of rotavirus in silico and in vitro. Taken together, these data suggest that the response to and recovery from rotavirus-diarrhea is unique between sub-compartments of the GI tract and may be influenced by mucin-degrading microbes.

Dietary fibers and SCFAs can exert a protective effect against respiratory syncytial virus (RSV). Here, the authors report that microbiota-derived acetate protects mice against RSV infection via GPR43- mediated type 1 interferon response induction in the lungs.

Abstract Dengue is hyperendemic in Colombia, where a cyclic behavior of serotype replacement leading to periodic epidemics has been observed for decades. This level of endemicity favors accumulation of dengue virus genetic diversity and could be linked to disease outcome. To assess the genetic diversity of dengue virus type 2 in Colombia, we sequenced the envelope gene of 24 virus isolates from acute cases of dengue or severe dengue fever during the period 2013–2016. The phylogenetic analysis revealed the circulation of the Asian-American genotype of dengue virus type 2 in Colombia during that period, the intra-genotype variability leading to divergence in two recently circulating lineages with differential geographic distribution, as well as the presence of nonsynonymous substitutions accompanying their emergence and diversification.

Studies have demonstrated that purported biofield therapy emitted from humans can inhibit the proliferation of cancer cells and suppress tumor growth in various cancers. We explored the effects of biofield therapy on tumor growth in the Lewis lung carcinoma and expanded mechanistic outcomes. We found biofield therapy did not inhibit tumor growth. However, the experimental (Ex) condition exposed tumors had a significantly higher percentage of necrosis (24.4 ± 6.8%) compared with that of the Control condition (6.5 ± 2.7%; P < .02) and cleaved caspase-3 positive cells were almost 2.3-fold higher ( P < .05). Similarly, tumor-infiltrating lymphocytes profiling showed that CD8+/CD45+ immune cell population was significantly increased by 2.7-fold in Ex condition ( P < .01) whereas the number of intratumoral FoxP3+/CD4+ (T-reg cells) was 30.4% lower than that of the Control group ( P = .01), leading to a significant 3.1-fold increase in the ratio of CD8+/T-reg cells ( P < .01). Additionally, there was a 51% lower level of strongly stained CD68+ cells ( P < .01), 57.9% lower level of F4/80high/CD206+ (M2 macrophages; P < .02) and a significant 1.8-fold increase of the ratio of M1/M2 macrophages ( P < .02). Furthermore, Ex exposure resulted in a 15% reduction of stem cell marker CD44 and a significant 33% reduction of SOX2 compared with that of the Controls ( P < .02). The Ex group also engaged in almost 50% less movement throughout the session than the Controls. These findings suggest that exposure to purported biofields from a human is capable of enhancing cancer cell death, in part mediated through modification of the tumor microenvironment and stemness of tumor cells in mouse Lewis lung carcinoma model. Future research should focus on defining the optimal treatment duration, replication with different biofield therapists, and exploring the mechanisms of action.

Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the chlorovirus model system; where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype chlorovirus, PBCV-1, using single-molecule real time (SMRT) sequencing (aka PacBio). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives.

ABSTRACT Although the term “microbiome” refers to all microorganisms, the majority of microbiome studies focus on the bacteriome. Here, we characterize the oral mycobiome, including mycobiome-bacteriome interactions, in the setting of remission-induction chemotherapy (RIC) for acute myeloid leukemia (AML). Oral samples (n = 299) were prospectively collected twice weekly from 39 AML patients during RIC until neutrophil recovery. Illumina MiSeq 16S rRNA gene (V4) and internal transcribed spacer 2 (ITS2) sequencing were used to determine bacterial and fungal diversity and community composition. Intrakingdom and interkingdom network connectivity at baseline (T1) and at midpoint (T3) and a later time point (T6) were assessed via SPIEC-EASI (sparse inverse covariance estimation for ecological association inference). In this exploratory study, mycobiome α-diversity was not significantly associated with antibiotic or antifungal receipt. However, postchemotherapy mycobiome α-diversity was lower in subjects receiving high-intensity chemotherapy. Additionally, greater decreases in Malassezia levels were seen over time among patients on high-intensity RIC compared to low-intensity RIC (P = 0.003). A significantly higher relative abundance of Candida was found among patients who had infection (P = 0.008), while a significantly higher relative abundance of Fusarium was found among patients who did not get an infection (P = 0.03). Analyses of intrakingdom and interkingdom relationships at T1, T3, and T6 indicated that interkingdom connectivity increased over the course of IC as bacterial α-diversity diminished. In (to our knowledge) the first longitudinal mycobiome study performed during AML RIC, we found that mycobiome-bacteriome interactions are highly dynamic. Our study data suggest that inclusion of mycobiome analysis in the design of microbiome studies may be necessary to optimally understand the ecological and functional role of microbial communities in clinical outcomes. IMPORTANCE This report highlights the importance of longitudinal, parallel characterization of oral fungi and bacteria in order to better elucidate the dynamic changes in microbial community structure and interkingdom functional interactions during the injury of chemotherapy and antibiotic exposure as well as the clinical consequences of these interrelated alterations.

Neonatal rotavirus infections are associated with gastrointestinal symptoms in some settings, but the role of host factors in clinical presentation is unclear. Here, Ramani et al. show that human milk oligosaccharides and microbiome are associated with symptomatic infection with neonatal strain G10P[11].

Abstract Recent epidemiological evidence suggests that exposure to antibiotics in early‐to‐middle adulthood is associated with an increased risk of colorectal adenoma. However, mechanistic studies in established preclinical cancer to examine these claims are extremely limited. Therefore, we investigated the effect of long‐term exposure of an antibiotic cocktail composed of Vancomycin, Neomycin, and Streptomycin, on tumor development and progression in the ApcMin/+ mouse, an established genetic model for familial adenomatous polyposis. Clinical pathologies related to tumor development as well as intestinal and colon tissue histopathology were studied at ages 8, 12, and 16 weeks of age, which correspond to the approximate ages of development of neoplasia, gut inflammation with polyposis, and cancer progression, respectively, in this animal model. We show that the antibiotics significantly increase the severity of clinical symptoms, including effects on intestinal histology and goblet cell numbers. In addition, they promote small intestinal polyposis. Finally, metagenomic analysis of fecal samples demonstrated that antibiotic exposure is associated with a significant but nonuniform depletion of the animal's natural gut flora. Overall, these findings support the premise that long‐term antibiotic exposure mediates the selected depletion of gut microbial communities and the concomitant thinning of the protective mucus layer, resulting in an increase in tumor development.

Abstract Background The airway microbiome is a subject of great interest for the study of respiratory disease. Anterior nare samples are more accessible than samples from deeper within the nasopharynx. However, the correlation between the microbiota found in the anterior nares and the microbiota found within the nasopharynx is unknown. We assessed the anterior nares and nasopharyngeal microbiota to determine (1) the relation of the microbiota from these two upper airway sites and (2) if associations were maintained between the microbiota from these two sites and two bronchiolitis severity outcomes. Results Among 815 infants hospitalized at 17 US centers for bronchiolitis with optimal 16S rRNA gene sequence reads from both nasal swab and nasopharyngeal aspirate samples, there were strong intra-individual correlations in the microbial communities between the two sample types, especially relating to Haemophilus and Moraxella genera. By contrast, we found a high abundance of Staphylococcus genus in the nasal swabs—a pattern not found in the nasopharyngeal samples and not informative when predicting the dominant nasopharyngeal genera. While these disparities may have been due to sample processing differences (i.e., nasal swabs were mailed at ambient temperature to emulate processing of future parent collected swabs while nasopharyngeal aspirates were mailed on dry ice), a previously reported association between Haemophilus-dominant nasopharyngeal microbiota and the increased severity of bronchiolitis was replicated utilizing the nasal swab microbiota and the same outcome measures: intensive care use (adjusted OR 6.43; 95% CI 2.25–20.51; P

Abstract Background Most studies describing the human gut microbiome in healthy and diseased states have emphasized the bacterial component, but the fungal microbiome (i.e., the mycobiome) is beginning to gain recognition as a fundamental part of our microbiome. To date, human gut mycobiome studies have primarily been disease centric or in small cohorts of healthy individuals. To contribute to existing knowledge of the human mycobiome, we investigated the gut mycobiome of the Human Microbiome Project (HMP) cohort by sequencing the Internal Transcribed Spacer 2 (ITS2) region as well as the 18S rRNA gene. Results Three hundred seventeen HMP stool samples were analyzed by ITS2 sequencing. Fecal fungal diversity was significantly lower in comparison to bacterial diversity. Yeast dominated the samples, comprising eight of the top 15 most abundant genera. Specifically, fungal communities were characterized by a high prevalence of Saccharomyces, Malassezia, and Candida, with S. cerevisiae, M. restricta, and C. albicans operational taxonomic units (OTUs) present in 96.8, 88.3, and 80.8% of samples, respectively. There was a high degree of inter- and intra-volunteer variability in fungal communities. However, S. cerevisiae, M. restricta, and C. albicans OTUs were found in 92.2, 78.3, and 63.6% of volunteers, respectively, in all samples donated over an approximately 1-year period. Metagenomic and 18S rRNA gene sequencing data agreed with ITS2 results; however, ITS2 sequencing provided greater resolution of the relatively low abundance mycobiome constituents. Conclusions Compared to bacterial communities, the human gut mycobiome is low in diversity and dominated by yeast including Saccharomyces, Malassezia, and Candida. Both inter- and intra-volunteer variability in the HMP cohort were high, revealing that unlike bacterial communities, an individual’s mycobiome is no more similar to itself over time than to another person’s. Nonetheless, several fungal species persisted across a majority of samples, evidence that a core gut mycobiome may exist. ITS2 sequencing data provided greater resolution of the mycobiome membership compared to metagenomic and 18S rRNA gene sequencing data, suggesting that it is a more sensitive method for studying the mycobiome of stool samples.

The relationship of the gastrointestinal microbiome to health and disease is of major research interest, including the effects of the gut microbiota on age related conditions. Here we report on the outcome of a project to collect stool samples on a large number of community dwelling elderly men using the OMNIgene-GUT stool/feces collection kit (OMR-200, DNA Genotek, Ottawa, Canada). Among 1328 men who were eligible for stool collection, 982 (74%) agreed to participate and 951 submitted samples. The collection process was reported to be acceptable, almost all samples obtained were adequate, the process of sample handling by mail was uniformly successful. The DNA obtained provided excellent results in microbiome analyses, yielding an abundance of species and a diversity of taxa as would be predicted. Our results suggest that population studies of older participants involving remote stool sample collection are feasible. These approaches would allow large scale research projects of the association of the gut microbiota with important clinical outcomes. Keywords: Microbiome, Aging, Men, Gastrointestinal, Genotyping, Stool

Abstract Objective Emerging evidence demonstrated that the structure of fecal microbiome is associated with the likelihood of bronchiolitis in infants. However, no study has examined functional profiles of fecal microbiome in infants with bronchiolitis. In this context, we conducted a case–control study. As a part of multicenter prospective study, we collected stool samples from 40 infants hospitalized with bronchiolitis (cases). We concurrently enrolled 115 age-matched healthy controls. Results First, by applying 16S rRNA gene sequencing to these 155 fecal samples, we identified the taxonomic profiles of fecal microbiome. Next, based on the taxonomy data, we inferred the functional capabilities of fecal microbiome and tested for differences in the functional capabilities between cases and controls. Overall, the median age was 3 months and 45% were female. Among 274 metabolic pathways surveyed, there were significant differences between bronchiolitis cases and healthy controls for 37 pathways, including lipid metabolic pathways (false discovery rate [FDR]

The oral microbiome has the potential to provide an important symbiotic function in human blood pressure physiology by contributing to the generation of nitric oxide (NO), an essential cardiovascular signaling molecule. NO is produced by the human body via conversion of arginine to NO by endogenous nitric oxide synthase (eNOS) but eNOS activity varies by subject. Oral microbial communities are proposed to supplement host NO production by reducing dietary nitrate to nitrite via bacterial nitrate reductases. Unreduced dietary nitrate is delivered to the oral cavity in saliva, a physiological process termed the enterosalivary circulation of nitrate. Previous studies demonstrated that disruption of enterosalivary circulation via use of oral antiseptics resulted in increases in systolic blood pressure. These previous studies did not include detailed information on the oral health of enrolled subjects. Using 16S rRNA gene sequencing and analysis, we determined whether introduction of chlorhexidine antiseptic mouthwash for 1 week was associated with changes in tongue bacterial communities and resting systolic blood pressure in healthy normotensive individuals with documented oral hygiene behaviors and free of oral disease. Tongue cleaning frequency was a predictor of chlorhexidine-induced changes in systolic blood pressure and tongue microbiome composition. Twice-daily chlorhexidine usage was associated with a significant increase in systolic blood pressure after 1 week of use and recovery from use resulted in an enrichment in nitrate-reducing bacteria on the tongue. Individuals with relatively high levels of bacterial nitrite reductases had lower resting systolic blood pressure. These results further support the concept of a symbiotic oral microbiome contributing to human health via the enterosalivary nitrate-nitrite-NO pathway. These data suggest that management of the tongue microbiome by regular cleaning together with adequate dietary intake of nitrate provide an opportunity for the improvement of resting systolic blood pressure.

Crohn’s disease (CD) is a chronic disorder of the gastrointestinal tract characterized by inflammation and intestinal epithelial injury. Loss of function mutations in the intracellular bacterial sensor NOD2 are major risk factors for the development of CD. In the absence of robust bacterial recognition by NOD2 an inflammatory cascade is initiated through alternative PRRs leading to CD. In the present study, MCC950, a specific small molecule inhibitor of NLR pyrin domain-containing protein 3 (NLRP3), abrogated dextran sodium sulfate (DSS)-induced intestinal inflammation in Nod2 −/− mice. NLRP3 inflammasome formation was observed at a higher rate in NOD2-deficient small intestinal lamina propria cells after insult by DSS. NLRP3 complex formation led to an increase in IL-1β secretion in both the small intestine and colon of Nod2 ko mice. This increase in IL-1β secretion in the intestine was attenuated by MCC950 leading to decreased disease severity in Nod2 ko mice. Our work suggests that NLRP3 inflammasome activation may be a key driver of intestinal inflammation in the absence of functional NOD2. NLRP3 pathway inhibition can prevent intestinal inflammation in the absence of robust NOD2 signaling.

ABSTRACT A wide diversity of fungi have been detected in the human gastrointestinal (GI) tract with the potential to provide or influence important functions. However, many of the fungi most commonly detected in stool samples are also present in food or the oral cavity. Therefore, to recognize which gut fungi are likely to have a sustained influence on human health, there is a need to separate transient members of the GI tract from true colonizers. To identify colonizing fungi, the eukaryotic rRNA operon’s second internal transcribed spacer (ITS2) was sequenced from the stool, saliva, and food of healthy adults following consumption of different controlled diets. Unlike most bacterial 16S rRNA genes, the only fungal ITS2 operational taxonomic units (OTUs) detected in stool DNA across multiple diets were also present in saliva and/or food. Additional analyses, including culture-based approaches and sequencing of the 18S rRNA gene, ITS2 cDNA, and DNA extracted using alternative methods, failed to detect additional fungi. Two abundant fungi, Saccharomyces cerevisiae and Candida albicans, were examined further in healthy volunteers. Saccharomyces became undetectable in stool when a S. cerevisiae-free diet was consumed, and the levels of C. albicans in stool were dramatically reduced by more frequent cleaning of teeth. Extremely low fungal abundance, the inability of fungi to grow under conditions mimicking the distal gut, and evidence from analysis of other public datasets further support the hypothesis that fungi do not routinely colonize the GI tracts of healthy adults. IMPORTANCE We sought to identify the fungi that colonize healthy GI tracts and that have a sustained influence on the diverse functions of the gut microbiome. Instead, we found that all fungi in the stool of healthy volunteers could be explained by their presence in oral and dietary sources and that our results, together with those from other analyses, support the model that there is little or no gastrointestinal colonization by fungi. This may be due to Westernization, primate evolution, fungal ecology, and/or the strong defenses of a healthy immune system. Importantly, fungal colonization of the GI tract may often be indicative of disease. As fungi can cause serious infections in immunocompromised individuals and are found at increased abundance in multiple disorders of the GI tract, understanding normal fungal colonization is essential for proper treatment and prevention of fungal pathogenesis.

Background The use of electronic cigarettes (ECs) has increased drastically over the past five years, primarily as an alternative to smoking tobacco cigarettes. However, the adverse effects of acute and long-term use of ECs on the microbiota have not been explored. In this pilot study, we sought to determine if ECs or tobacco smoking alter the oral and gut microbiota in comparison to non-smoking controls. Methods We examined a human cohort consisting of 30 individuals: 10 EC users, 10 tobacco smokers, and 10 controls. We collected cross-sectional fecal, buccal swabs, and saliva samples from each participant. All samples underwent V4 16S rRNA gene sequencing. Results Tobacco smoking had a significant effect on the bacterial profiles in all sample types when compared to controls, and in feces and buccal swabs when compared to EC users. The most significant associations were found in the gut, with an increased relative abundance of Prevotella (P = 0.006) and decreased Bacteroides (P = 0.036) in tobacco smokers. The Shannon diversity was also significantly reduced (P = 0.009) in fecal samples collected from tobacco smokers compared to controls. No significant difference was found in the alpha diversity, beta-diversity or taxonomic relative abundances between EC users and controls. Discussion From a microbial ecology perspective, the current pilot data demonstrate that the use of ECs may represent a safer alternative compared to tobacco smoking. However, validation in larger cohorts and greater understanding of the short and long-term impact of EC use on microbiota composition and function is warranted.

ABSTRACT The vector-borne disease leishmaniasis, caused by Leishmania species protozoa, is transmitted to humans by phlebotomine sand flies. Development of Leishmania to infective metacyclic promastigotes in the insect gut, a process termed metacyclogenesis, is an essential prerequisite for transmission. Based on the hypothesis that vector gut microbiota influence the development of virulent parasites, we sequenced midgut microbiomes in the sand fly Lutzomyia longipalpis with or without Leishmania infantum infection. Sucrose-fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in microbial richness that eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Acetobacteraceae spp. became dominant and numbers of Pseudomonadaceae spp. diminished coordinately as the parasite underwent metacyclogenesis and parasite numbers increased. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and metacyclogenesis. Together, these data suggest that the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation to its infective state prior to disease transmission. IMPORTANCE Leishmania infantum, a parasitic protozoan causing fatal visceral leishmaniasis, is transmitted to humans through the bite of the sand fly Lutzomyia longipalpis. Development of the parasite to its virulent metacyclic state occurs in the sand fly gut. In this study, the microbiota within the Lu. longipalpis midgut was delineated by 16S ribosomal DNA (rDNA) sequencing, revealing a highly diverse community composition that lost diversity as parasites developed to their metacyclic state and increased in abundance in infected flies. Perturbing sand fly gut microbiota with an antibiotic cocktail, which alone had no effect on either the parasite or the fly, arrested both the development of virulent parasites and parasite expansion. These findings indicate the importance of bacterial commensals within the insect vector for the development of virulent pathogens, and raise the possibility that impairing the microbial composition within the vector might represent a novel approach to control of vector-borne diseases.

BackgroundA diverse and abundant gut microbiome can improve cancer patients' treatment response; however, the effect of pelvic chemoradiotherapy (CRT) on gut diversity and composition is unclear. The purpose of this prospective study was to identify changes in the diversity and composition of the gut microbiome during and after pelvic CRT.Materials and methodsRectal swabs from 58 women with cervical, vaginal, or vulvar cancer from two institutions were prospectively analyzed before CRT (baseline), during CRT (weeks 1, 3, and 5), and at first follow-up (week 12) using 16Sv4 rRNA gene sequencing of the V4 hypervariable region of the bacterial 16S rRNA marker gene. 42 of these patients received antibiotics during the study period. Observed operational taxonomic units (OTUs; representative of richness) and Shannon, Simpson, Inverse Simpson, and Fisher diversity indices were used to characterize alpha (within-sample) diversity. Changes over time were assessed using a paired t-test, repeated measures ANOVA, and linear mixed modeling. Compositional changes in specific bacteria over time were evaluated using linear discriminant analysis effect size.ResultsGut microbiome richness and diversity levels continually decreased throughout CRT (mean Shannon diversity index, 2.52 vs. 2.91; all P ConclusionAfter CRT, the diversity of the gut microbiomes in this population tended to return to baseline levels by the 12 week follow-up period, but structure and composition remained significantly altered. These changes should be considered when designing studies to analyze the gut microbiome in patients who receive pelvic CRT for gynecologic cancers.

Viral taxonomic characterization from metagenomic data suffers from high background noise and signal crosstalk. Here, the authors develop VirMAP, a novel pipeline for analyses of metagenomic data that classifies viral reconstructions independent of genome coverage or read overlap.

BackgroundUnderstanding the role of the gut microbiome is pivotal for the future development of therapies for the prevention and management of autoimmune conditions such as type 1 diabetes when sampling during early life may be particularly important. The current standard methods for collecting gut microbiome samples for research is to extract fresh samples or freeze samples immediately after collection. This is often impractical however for population-based studies. The aim of this study was to determine the optimal method for the stabilization of stool bacterial DNA obtained from nappies and transported by post in ambient conditions to the research centre for a national birth cohort study.MethodsFour methods to collect samples were compared to immediate freezing of samples: 1) collecting faeces onto a swab which was immediately frozen, 2) using a commercially available kit with stabilisation solution (OMNIgene•GUT kit) at ambient temperature, 3) collecting onto a swab and 4) collecting into a sterile plain tube. Samples 3) and 4) were returned to the laboratory by post at ambient temperatures. A Bland Altman analysis was used to assess the agreement between the different methods and the frozen standard.ResultsStool samples were collected by parents. For samples transported in ambient conditions, the limits of agreement showed that the OMNIgene•GUT kit had the narrowest 95% limits of agreement with the frozen standard as measured by the number of operational taxonomic units and the Shannon diversity index.ConclusionsAll methods assessed for preserving samples collected from nappies at a distance and delivered by post for gut microbiome analysis showed variation / disagreement from the frozen standard. Overall, the OMNIgene•GUT kit preserved the samples with minimal changes compared to other methods and was practical for parents to use.

Non-steroidal anti-inflammatory drugs (NSAIDs) are routinely used in both veterinary and human medicine. Gastrointestinal injury is a frequent adverse event associated with NSAID use and evidence suggests that NSAIDs induce gastrointestinal microbial imbalance (i.e., dysbiosis) in both animals and people. It is unknown, however, whether cyclooxygenase (COX)-2-selective NSAIDs induce dysbiosis, or if this phenomenon occurs in horses administered any class of NSAIDs. Therefore, our objectives were to determine whether the composition and diversity of the fecal microbiota of adult horses were altered by NSAID use, and whether these effects differed between non-selective and COX-2-selective NSAIDs. Twenty-five adult horses were randomly assigned to 1 of 3 groups: control (n = 5); phenylbutazone (n = 10); or, firocoxib (n = 10). Treatments were administered for 10 days. Fecal samples were collected every 5 days for 25 days. DNA was extracted from feces and the 16S rRNA gene amplified and sequenced to determine the composition of the microbiota and the inferred metagenome. While the fecal microbiota profile of the control group remained stable over time, the phenylbutazone and firocoxib groups had decreased diversity, and alteration of their microbiota profiles was most pronounced at day 10. Similarly, there were clear alterations of the inferred metagenome at day 10 compared to all other days, indicating that use of both non-selective and selective COX inhibitors resulted in temporary alterations of the fecal microbiota and inferred metagenome. Dysbiosis associated with NSAID administration is clinically relevant because dysbiosis has been associated with several important diseases of horses including abdominal pain (colic), colitis, enteric infections, and laminitis.

The oral microbiome has been linked to a number of chronic inflammatory conditions, including obesity, diabetes, periodontitis, and cancers of the stomach and liver. These conditions disproportionately affect Mexican American women, yet few studies have examined the oral microbiota in this at-risk group. We characterized the 16S rDNA oral microbiome in 369 non-smoking women enrolled in the MD Anderson Mano a Mano Mexican American Cohort Study. Lower bacterial diversity, a potential indicator of oral health, was associated with increased age and length of US residency among recent immigrants. Grouping women by overarching bacterial community type (e.g., "Streptococcus," "Fusobacterium," and "Prevotella" clusters), we observed differences across a number of acculturation-related variables, including nativity, age at immigration, time in the US, country of longest residence, and a multi-dimensional acculturation scale. Participants in the cluster typified by higher abundance of Streptococcus spp. exhibited the lowest bacterial diversity and appeared the most acculturated as compared to women in the "Prevotella" group. Computationally-predicted functional analysis suggested the Streptococcus-dominated bacterial community had greater potential for carbohydrate metabolism while biosynthesis of essential amino acids and nitrogen metabolism prevailed among the Prevotella-high group. Findings suggest immigration and adaption to life in the US, a well-established mediator of disease risk, is associated with differences in oral microbial profiles in Mexican American women. These results warrant further investigation into the joint and modifying effects of acculturation and oral bacteria on the health of Mexican American women and other immigrant populations. The oral microbiome presents an easily accessible biomarker of disease risk, spanning biological, behavioral, and environmental factors.

BACKGROUND:Fecal microbiota transplantation (FMT) via colonoscopy or enema has become a commonly used treatment of recurrent C. difficile infection (CDI). AIMS:To compare the safety and preliminary efficacy of orally administered lyophilized microbiota product compared with frozen product by enema. METHODS:In a single center, adults with ≥ 3 episodes of recurrent CDI were randomized to receive encapsulated lyophilized fecal microbiota from 100-200 g of donor feces (n = 31) or frozen FMT from 100 g of donor feces (n = 34) by enema. Safety during the three months post FMT was the primary study objective. Prevention of CDI recurrence during the 60 days after FMT was a secondary objective. Fecal microbiome changes were examined in first 39 subjects studied. RESULTS:Adverse experiences were commonly seen in equal frequency in both groups and did not appear to relate to the route of delivery of FMT. CDI recurrence was prevented in 26 of 31 (84%) subjects randomized to capsules and in 30 of 34 (88%) receiving FMT by enema (p = 0.76). Both products normalized fecal microbiota diversity while the lyophilized orally administered product was less effective in repleting Bacteroidia and Verrucomicrobia classes compared to frozen product via enema. CONCLUSIONS:The route of delivery, oral or rectal, did not influence adverse experiences in FMT. In preliminary evaluation, both routes appeared to show equivalent efficacy, although the dose may need to be higher for lyophilized product. Spore-forming bacteria appear to be the most important engrafting organisms in FMT by the oral route using lyophilized product. TRIAL REGISTRATION:ClinicalTrials.gov NCT02449174.

ObjectivesTo characterize the microbial environment of workers in academic mouse research facilities using endotoxin, 16S qPCR, and 16S amplicon sequencing. To determine whether the work microbiome contributes to the human microbiome of workers.MethodsWe performed area air sampling from the animal rooms, dirty, middle, and setup cage wash locations in four academic mouse research facilities. 10 workers in the dirty cage wash area underwent personal air sampling as well as repeated collection of nasal, oral, and skin samples before and after the work shift. Environmental samples underwent measurement of endotoxin, mouse allergen, bacteria copy number via 16S qPCR, and microbial identification via 16S rDNA sequencing. 16S rDNA sequencing was also performed on human samples before and after the work shift. SourceTracker was used to identify the contribution of the work microbiome to the human microbiome.ResultsMedian endotoxin levels ranged from undetectable to 1.0 EU/m3. Significant differences in mouse allergen levels, bacterial copy number, microbial richness, and microbial community structure were identified between animal, dirty, middle, and setup cage wash locations. Endotoxin levels had only a moderate correlation with microbial composition. Location within a facility was a stronger predictor of microbial community composition (R2 = 0.41, p = 0.002) than facility. The contribution of the work microbiome to the pre-shift human microbiome of workers was estimated to be 0.1 ± 0.1% for the oral microbiome; 3.1 ± 1.9% for the nasal microbiome; and 3.0 ± 1.5% for the skin microbiome.ConclusionsThe microbial environment of academic animal care facilities varies significantly by location rather than facility. Endotoxin is not a proxy for assessment of environmental microbial exposures using 16S qPCR or 16S rDNA sequencing. The work microbiome contributes to the composition of the nasal and skin microbiome of workers; the clinical implications of this observation should be further studied.

Neonatal sepsis in preterm infants is often due to organisms that colonize the skin including Staphylococcus spp. and Candida spp. Development and maturation of the skin microbiome in the neonatal period, especially in preterm infants, may be critical in preventing colonization with pathogens and subsequent progression to neonatal sepsis. Development of the skin microbiome in preterm infants or its determinants in the first 4 weeks of life has not been evaluated.We evaluated the skin microbiome from three body sites, antecubital fossa, forehead and gluteal region, in a prospective cohort of 15 preterm (birth weight < 1500 g and < 32 weeks of gestation) and 15 term neonates. The microbiome community membership and relative abundance were evaluated by amplification and sequencing the bacterial V3-V5 region of the16S rRNA gene on the 454 GS FLX platform. We used linear mixed effects models to analyze longitudinal data.The structure and composition of the skin microbiome did not differ between the three sampling sites for term and preterm infants in the neonatal period. However, skin bacterial richness was positively associated with gestational age in the first four weeks of life. Intravenous antibiotics negatively impacted the bacterial diversity of the skin but we did not see differences with respect to feeding or mode of delivery.Gestational age, which influences the maturity of skin structure and function, is associated with the development of the preterm cutaneous microbiome. Understanding the maturation of a healthy skin microbiome, prevention of pathogen colonization and its role in the development of immunity will be pivotal in the development of novel interventions to prevent infections in critically ill preterm infants.

Disease can be conceptualized as the result of interactions between infecting microbe and holobiont, the combination of a host and its microbial communities. It is likely that genomic variation in the host, infecting microbe, and commensal microbiota are key determinants of infectious disease clinical outcomes. However, until recently, simultaneous, multiomic investigation of infecting microbe and holobiont components has rarely been explored. Herein, we characterized the infecting microbe, host, micro- and mycobiomes leading up to infection onset in a leukemia patient that developed invasive mucormycosis. We discovered that the patient was infected with a strain of the recently described Mucor velutinosus species which we determined was hypervirulent in a Drosophila challenge model and has a predisposition for skin dissemination. After completing the infecting M. velutinosus genome and genomes from four other Mucor species, comparative pathogenomics was performed and assisted in identifying 66 M. velutinosus-specific putatively secreted proteins, including multiple novel secreted aspartyl proteinases which may contribute to the unique clinical presentation of skin dissemination. Whole exome sequencing of the patient revealed multiple non-synonymous polymorphisms in genes critical to control of fungal proliferation, such as TLR6 and PTX3. Moreover, the patient had a non-synonymous polymorphism in the NOD2 gene and a missense mutation in FUT2, which have been linked to microbial dysbiosis and microbiome diversity maintenance during physiologic stress, respectively. In concert with host genetic polymorphism data, the micro- and mycobiome analyses revealed that the infection developed amid a dysbiotic microbiome with low α-diversity, dominated by staphylococci. Additionally, longitudinal mycobiome data showed that M. velutinosus DNA was detectable in oral samples preceding disease onset. Our genome-level study of the host-infecting microbe-commensal triad extends the concept of personalized genomic medicine to the holobiont-infecting microbe interface thereby offering novel opportunities for using synergistic genetic methods to increase understanding of infectious diseases pathogenesis and clinical outcomes.

Effects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×10⁷ plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.