Randomized Placebo-Controlled, Biomarker-Stratified Phase Ib Microbiome Modulation in Melanoma: Impact of Antibiotic Preconditioning on Microbiome and Immunity.

Gut-microbiota modulation shows promise in improving immune-checkpoint blockade (ICB) response; however, precision biomarker-driven, placebo-controlled trials are lacking. We performed a multicenter, randomized placebo-controlled, biomarker-stratified phase I trial in patients with ICB-naïve metastatic melanoma using SER-401, an orally delivered Firmicutesenriched spore formulation. Fecal microbiota signatures were characterized at baseline; patients were stratified by high versus low Ruminococcaceae abundance prior to randomization to the SER-401 arm (oral vancomycin-preconditioning/SER-401 alone/nivolumab + SER-401), versus the placebo arm [placebo antibiotic/placebo microbiome modulation (PMM)/nivolumab + PMM (NCT03817125)]. Analysis of 14 accrued patients demonstrated that treatment with SER-401 + nivolumab was safe, with an overall response rate of 25% in the SER-401 arm and 67% in the placebo arm (though the study was underpowered related to poor accrual during the COVID-19 pandemic). Translational analyses demonstrated that vancomycin preconditioning was associated with the disruption of the gut microbiota and impaired immunity, with incomplete recovery at ICB administration (particularly in patients with high baseline Ruminococcaceae). These results have important implications for future microbiome modulation trials. Significance: This first-of-its-kind, placebo-controlled, randomized biomarker-driven microbiome modulation trial demonstrated that vancomycin + SER-401 and anti-PD-1 are safe in melanoma patients. Although limited by poor accrual during the pandemic, important insights were gained via translational analyses, suggesting that antibiotic preconditioning and interventional drug dosing regimens should be carefully considered when designing such trials.

Impact of a Purified Microbiome Therapeutic on Abundance of Antimicrobial Resistance Genes in Patients With Recurrent Clostridioides difficile Infection.

BACKGROUND: The gastrointestinal microbiota is an important line of defense against colonization with antimicrobial resistant (AR) bacteria. In this post hoc analysis of the phase 3 ECOSPOR III trial, we assessed impact of a microbiota-based oral therapeutic (fecal microbiota spores, live; VOWST Oral Spores [VOS], formerly SER-109]; Seres Therapeutics) compared with placebo, on AR gene (ARG) abundance in patients with recurrent Clostridioides difficile infection (rCDI). METHODS: Adults with rCDI were randomized to receive VOS or placebo orally for 3 days following standard-of-care antibiotics. ARG and taxonomic profiles were generated using whole metagenomic sequencing of stool at baseline and weeks 1, 2, 8, and 24 posttreatment. RESULTS: Baseline (n = 151) and serial posttreatment stool samples collected through 24 weeks (total N = 472) from 182 patients (59.9% female; mean age: 65.5 years) in ECOSPOR III as well as 68 stool samples obtained at a single time point from a healthy cohort were analyzed. Baseline ARG abundance was similar between arms and significantly elevated versus the healthy cohort. By week 1, there was a greater decline in ARG abundance in VOS versus placebo (P = .003) in association with marked decline of Proteobacteria and repletion of spore-forming Firmicutes, as compared with baseline. We observed abundance of Proteobacteria and non-spore-forming Firmicutes were associated with ARG abundance, while spore-forming Firmicutes abundance was negatively associated. CONCLUSIONS: This proof-of-concept analysis suggests that microbiome remodeling with Firmicutes spores may be a potential novel approach to reduce ARG colonization in the gastrointestinal tract.

SER-109: An Oral Investigational Microbiome Therapeutic for Patients with Recurrent Clostridioides difficile Infection (rCDI).

Clostridioides difficile infection (CDI) is classified as an urgent health threat by the Centers for Disease Control and Prevention (CDC), and affects nearly 500,000 Americans annually. Approximately 20−25% of patients with a primary infection experience a recurrence, and the risk of recurrence increases with subsequent episodes to greater than 40%. The leading risk factor for CDI is broad-spectrum antibiotics, which leads to a loss of microbial diversity and impaired colonization resistance. Current FDA-approved CDI treatment strategies target toxin or toxin-producing bacteria, but do not address microbiome disruption, which is key to the pathogenesis of recurrent CDI. Fecal microbiota transplantation (FMT) reduces the risk of recurrent CDI through the restoration of microbial diversity. However, FDA safety alerts describing hospitalizations and deaths related to pathogen transmission have raised safety concerns with the use of unregulated and unstandardized donor-derived products. SER-109 is an investigational oral microbiome therapeutic composed of purified spore-forming Firmicutes. SER-109 was superior to a placebo in reducing CDI recurrence at Week 8 (12% vs. 40%, respectively; p < 0.001) in adults with a history of recurrent CDI with a favorable observed safety profile. Here, we discuss the role of the microbiome in CDI pathogenesis and the clinical development of SER-109, including its rigorous manufacturing process, which mitigates the risk of pathogen transmission. Additionally, we discuss compositional and functional changes in the gastrointestinal microbiome in patients with recurrent CDI following treatment with SER-109 that are critical to a sustained clinical response.

SER-109, an Oral Microbiome Therapy for Recurrent Clostridioides difficile Infection.

BACKGROUND: Current therapies for recurrent Clostridioides difficile infection do not address the disrupted microbiome, which supports C. difficile spore germination into toxin-producing bacteria. SER-109 is an investigational microbiome therapeutic composed of purified Firmicutes spores for the treatment of recurrent C. difficile infection. METHODS: We conducted a phase 3, double-blind, randomized, placebo-controlled trial in which patients who had had three or more episodes of C. difficile infection (inclusive of the qualifying acute episode) received SER-109 or placebo (four capsules daily for 3 days) after standard-of-care antibiotic treatment. The primary efficacy objective was to show superiority of SER-109 as compared with placebo in reducing the risk of C. difficile infection recurrence up to 8 weeks after treatment. Diagnosis by toxin testing was performed at trial entry, and randomization was stratified according to age and antibiotic agent received. Analyses of safety, microbiome engraftment, and metabolites were also performed. RESULTS: Among the 281 patients screened, 182 were enrolled. The percentage of patients with recurrence of C. difficile infection was 12% in the SER-109 group and 40% in the placebo group (relative risk, 0.32; 95% confidence interval [CI], 0.18 to 0.58; P<0.001 for a relative risk of <1.0; P<0.001 for a relative risk of <0.833). SER-109 led to less frequent recurrence than placebo in analyses stratified according to age stratum (relative risk, 0.24 [95% CI, 0.07 to 0.78] for patients <65 years of age and 0.36 [95% CI, 0.18 to 0.72] for those ≥65 years) and antibiotic received (relative risk, 0.41 [95% CI, 0.22 to 0.79] with vancomycin and 0.09 [95% CI, 0.01 to 0.63] with fidaxomicin). Most adverse events were mild to moderate and were gastrointestinal in nature, with similar numbers in the two groups. SER-109 dose species were detected as early as week 1 and were associated with bile-acid profiles that are known to inhibit C. difficile spore germination. CONCLUSIONS: In patients with symptom resolution of C. difficile infection after treatment with standard-of-care antibiotics, oral administration of SER-109 was superior to placebo in reducing the risk of recurrent infection. The observed safety profile of SER-109 was similar to that of placebo. (Funded by Seres Therapeutics; ECOSPOR III ClinicalTrials.gov number, NCT03183128.).

Biofilms harbour Clostridioides difficile, serving as a reservoir for recurrent infection.

C. difficile infection (CDI) is a worldwide healthcare problem with ~30% of cases failing primary therapy, placing a burden on healthcare systems and increasing patient morbidity. We have little understanding of why these therapies fail. Here, we use a clinically validated in vitro gut model to assess the contribution of biofilms towards recurrent disease and to investigate biofilm microbiota-C. difficile interactions. Initial experiments show that C. difficile cells became associated with the colonic biofilm microbiota and are not depleted by vancomycin or faecal microbiota transplant therapies. We observe that transferring biofilm encased C. difficile cells into a C. difficile naïve but CDI susceptible model induces CDI. Members of the biofilm community can impact C. difficile biofilm formation by acting either antagonistically or synergistically. We highlight the importance of biofilms as a reservoir for C. difficile, which can be a cause for recurrent infections.

SER-109, an Investigational Microbiome Drug to Reduce Recurrence After Clostridioides difficile Infection: Lessons Learned From a Phase 2 Trial.

BACKGROUND: Recurrent Clostridioides difficile infection (rCDI) is associated with loss of microbial diversity and microbe-derived secondary bile acids, which inhibit C. difficile germination and growth. SER-109, an investigational microbiome drug of donor-derived, purified spores, reduced recurrence in a dose-ranging, phase (P) 1 study in subjects with multiple rCDIs. METHODS: In a P2 double-blind trial, subjects with clinical resolution on standard-of-care antibiotics were stratified by age (< or ≥65 years) and randomized 2:1 to single-dose SER-109 or placebo. Subjects were diagnosed at study entry by PCR or toxin testing. Safety, C. difficile-positive diarrhea through week 8, SER-109 engraftment, and bile acid changes were assessed. RESULTS: 89 subjects enrolled (67% female; 80.9% diagnosed by PCR). rCDI rates were lower in the SER-109 arm than placebo (44.1% vs 53.3%) but did not meet statistical significance. In a preplanned analysis, rates were reduced among subjects ≥65 years (45.2% vs 80%, respectively; RR, 1.77; 95% CI, 1.11-2.81), while the <65 group showed no benefit. Early engraftment of SER-109 was associated with nonrecurrence (P < .05) and increased secondary bile acid concentrations (P < .0001). Whole-metagenomic sequencing from this study and the P1 study revealed previously unappreciated dose-dependent engraftment kinetics and confirmed an association between early engraftment and nonrecurrence. Engraftment kinetics suggest that P2 dosing was suboptimal. Adverse events were generally mild to moderate in severity. CONCLUSIONS: Early SER-109 engraftment was associated with reduced CDI recurrence and favorable safety was observed. A higher dose of SER-109 and requirements for toxin testing were implemented in the current P3 trial. CLINICAL TRIALS REGISTRATION: NCT02437487, https://clinicaltrials.gov/ct2/show/NCT02437487?term=SER-109&draw= 2&rank=4.

A Phase 1b Safety Study of SER-287, a Spore-Based Microbiome Therapeutic, for Active Mild to Moderate Ulcerative Colitis.

BACKGROUND & AIMS: Firmicutes bacteria produce metabolites that maintain the intestinal barrier and mucosal immunity. Firmicutes are reduced in the intestinal microbiota of patients with ulcerative colitis (UC). In a phase 1b trial of patients with UC, we evaluated the safety and efficacy of SER-287, an oral formulation of Firmicutes spores, and the effects of vancomycin preconditioning on expansion (engraftment) of SER-287 species in the colon. METHODS: We conducted a double-blind trial of SER-287 in 58 adults with active mild-to-moderate UC (modified Mayo scores 4-10, endoscopic subscores ≥1). Participants received 6 days of preconditioning with oral vancomycin (125 mg, 4 times daily) or placebo followed by 8 weeks of oral SER-287 or placebo. Patients were randomly assigned (2:3:3:3) to groups that received placebo followed by either placebo or SER-287 once weekly, or vancomycin followed by SER-287 once weekly, or SER-287 once daily. Clinical end points included safety and clinical remission (modified Mayo score ≤2; endoscopic subscores 0 or 1). Microbiome end points included SER-287 engraftment (dose species detected in stool after but not before SER-287 administration). Engraftment of SER-287 and changes in microbiome composition and associated metabolites were measured by analyses of stool specimens collected at baseline, after preconditioning, and during and 4 weeks after administration of SER-287 or placebo. RESULTS: Proportions of patients with adverse events did not differ significantly among groups. A higher proportion of patients in the vancomycin/SER-287 daily group (40%) achieved clinical remission at week 8 than patients in the placebo/placebo group (0%), placebo/SER-287 weekly group (13.3%), or vancomycin/SER-287 weekly group (17.7%) (P = .024 for vancomycin/SER-287 daily vs placebo/placebo). By day 7, higher numbers of SER-287 dose species were detected in stool samples from all SER-287 groups compared with the placebo group (P < .05), but this difference was not maintained beyond day 7 in the placebo/SER-287 weekly group. In the vancomycin groups, a greater number of dose species were detected in stool collected on day 10 and all subsequent time points through 4 weeks post dosing compared with the placebo group (P < .05). A higher number of SER-287 dose species were detected in stool samples on days 7 and 10 from subjects who received daily vs weekly SER-287 doses (P < .05). Changes in fecal microbiome composition and metabolites were associated with both vancomycin/SER-287 groups. CONCLUSIONS: In this small phase 1b trial of limited duration, the safety and tolerability of SER-287 were similar to placebo. SER-287 after vancomycin was significantly more effective than placebo for induction of remission in patients with active mild to moderate UC. Engraftment of dose species was facilitated by vancomycin preconditioning and daily dosing of SER-287. ClinicalTrials.gov ID NCT02618187.

Fecal microbiota transplantation for the improvement of metabolism in obesity: The FMT-TRIM double-blind placebo-controlled pilot trial.

BACKGROUND: There is intense interest about whether modulating gut microbiota can impact systemic metabolism. We investigated the safety of weekly oral fecal microbiota transplantation (FMT) capsules from healthy lean donors and their ability to alter gut microbiota and improve metabolic outcomes in patients with obesity. METHODS AND FINDINGS: FMT-TRIM was a 12-week double-blind randomized placebo-controlled pilot trial of oral FMT capsules performed at a single US academic medical center. Between August 2016 and April 2018, we randomized 24 adults with obesity and mild-moderate insulin resistance (homeostatic model assessment of insulin resistance [HOMA-IR] between 2.0 and 8.0) to weekly healthy lean donor FMT versus placebo capsules for 6 weeks. The primary outcome, assessed by intention to treat, was change in insulin sensitivity between 0 and 6 weeks as measured by hyperinsulinemic euglycemic clamps. Additional metabolic parameters were evaluated at 0, 6, and 12 weeks, including HbA1c, body weight, body composition by dual-energy X-ray absorptiometry, and resting energy expenditure by indirect calorimetry. Fecal samples were serially collected and evaluated via 16S V4 rRNA sequencing. Our study population was 71% female, with an average baseline BMI of 38.8 ± 6.7 kg/m2 and 41.3 ± 5.1 kg/m2 in the FMT and placebo groups, respectively. There were no statistically significant improvements in insulin sensitivity in the FMT group compared to the placebo group (+5% ± 12% in FMT group versus -3% ± 32% in placebo group, mean difference 9%, 95% CI -5% to 28%, p = 0.16). There were no statistically significant differences between groups for most of the other secondary metabolic outcomes, including HOMA-IR (mean difference 0.2, 95% CI -0.9 to 0.9, p = 0.96) and body composition (lean mass mean difference -0.1 kg, 95% CI -1.9 to 1.6 kg, p = 0.87; fat mass mean difference 1.2 kg, 95% CI -0.6 to 3.0 kg, p = 0.18), over the 12-week study. We observed variable engraftment of donor bacterial groups among FMT recipients, which persisted throughout the 12-week study. There were no significant differences in adverse events (AEs) (10 versus 5, p = 0.09), and no serious AEs related to FMT. Limitations of this pilot study are the small sample size, inclusion of participants with relatively mild insulin resistance, and lack of concurrent dietary intervention. CONCLUSIONS: Weekly administration of FMT capsules in adults with obesity results in gut microbiota engraftment in most recipients for at least 12 weeks. Despite engraftment, we did not observe clinically significant metabolic effects during the study. TRIAL REGISTRATION: ClinicalTrials.gov NCT02530385.

Differences in the Selection Bottleneck between Modes of Sexual Transmission Influence the Genetic Composition of the HIV-1 Founder Virus.

Due to the stringent population bottleneck that occurs during sexual HIV-1 transmission, systemic infection is typically established by a limited number of founder viruses. Elucidation of the precise forces influencing the selection of founder viruses may reveal key vulnerabilities that could aid in the development of a vaccine or other clinical interventions. Here, we utilize deep sequencing data and apply a genetic distance-based method to investigate whether the mode of sexual transmission shapes the nascent founder viral genome. Analysis of 74 acute and early HIV-1 infected subjects revealed that 83% of men who have sex with men (MSM) exhibit a single founder virus, levels similar to those previously observed in heterosexual (HSX) transmission. In a metadata analysis of a total of 354 subjects, including HSX, MSM and injecting drug users (IDU), we also observed no significant differences in the frequency of single founder virus infections between HSX and MSM transmissions. However, comparison of HIV-1 envelope sequences revealed that HSX founder viruses exhibited a greater number of codon sites under positive selection, as well as stronger transmission indices possibly reflective of higher fitness variants. Moreover, specific genetic "signatures" within MSM and HSX founder viruses were identified, with single polymorphisms within gp41 enriched among HSX viruses while more complex patterns, including clustered polymorphisms surrounding the CD4 binding site, were enriched in MSM viruses. While our findings do not support an influence of the mode of sexual transmission on the number of founder viruses, they do demonstrate that there are marked differences in the selection bottleneck that can significantly shape their genetic composition. This study illustrates the complex dynamics of the transmission bottleneck and reveals that distinct genetic bottleneck processes exist dependent upon the mode of HIV-1 transmission.

A Novel Microbiome Therapeutic Increases Gut Microbial Diversity and Prevents Recurrent Clostridium difficile Infection.

BACKGROUND: Patients with recurrent Clostridium difficile infection (CDI) have a ≥60% risk of relapse, as conventional therapies do not address the underlying gastrointestinal dysbiosis. This exploratory study evaluated the safety and efficacy of bacterial spores for preventing recurrent CDI. METHODS: Stool specimens from healthy donors were treated with ethanol to eliminate pathogens. The resulting spores were fractionated and encapsulated for oral delivery as SER-109. Following their response to standard-of-care antibiotics, patients in cohort 1 were treated with SER-109 on 2 consecutive days (geometric mean dose, 1.7 × 10(9) spores), and those in cohort 2 were treated on 1 day (geometric mean dose, 1.1 × 10(8) spores). The primary efficacy end point was absence of C. difficile-positive diarrhea during an 8-week follow-up period. Microbiome alterations were assessed. RESULTS: Thirty patients (median age, 66.5 years; 67% female) were enrolled, and 26 (86.7%) met the primary efficacy end point. Three patients with early, self-limiting C. difficile-positive diarrhea did not require antibiotics and tested negative for C. difficile at 8 weeks; thus, 96.7% (29 of 30) achieved clinical resolution. In parallel, gut microbiota rapidly diversified, with durable engraftment of spores and no outgrowth of non-spore-forming bacteria found after SER-109 treatment. Adverse events included mild diarrhea, abdominal pain, and nausea. CONCLUSIONS: SER-109 successfully prevented CDI and had a favorable safety profile, supporting a novel microbiome-based intervention as a potential therapy for recurrent CDI.

Standardized metadata for human pathogen/vector genomic sequences.

High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium's minimal information (MIxS) and NCBI's BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.

Comparison of illumina and 454 deep sequencing in participants failing raltegravir-based antiretroviral therapy.

BACKGROUND: The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs. METHODS: A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser. RESULTS: Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001). Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454. CONCLUSIONS: In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.

V-Phaser 2: variant inference for viral populations.

BACKGROUND: Massively parallel sequencing offers the possibility of revolutionizing the study of viral populations by providing ultra deep sequencing (tens to hundreds of thousand fold coverage) of complete viral genomes. However, differentiation of true low frequency variants from sequencing errors remains challenging. RESULTS: We developed a software package, V-Phaser 2, for inferring intrahost diversity within viral populations. This program adds three major new methodologies to the state of the art: a technique to efficiently utilize paired end read data for calling phased variants, a new strategy to represent and infer length polymorphisms, and an in line filter for erroneous calls arising from systematic sequencing artifacts. We have also heavily optimized memory and run time performance. This combination of algorithmic and technical advances allows V-Phaser 2 to fully utilize extremely deep paired end sequencing data (such as generated by Illumina sequencers) to accurately infer low frequency intrahost variants in viral populations in reasonable time on a standard desktop computer. V-Phaser 2 was validated and compared to both QuRe and the original V-Phaser on three datasets obtained from two viral populations: a mixture of eight known strains of West Nile Virus (WNV) sequenced on both 454 Titanium and Illumina MiSeq and a mixture of twenty-four known strains of WNV sequenced only on 454 Titanium. V-Phaser 2 outperformed the other two programs in both sensitivity and specificity while using more than five fold less time and memory. CONCLUSIONS: We developed V-Phaser 2, a publicly available software tool (V-Phaser 2 can be accessed via: http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/v-phaser-2 and is freely available for academic use) that enables the efficient analysis of ultra-deep sequencing data produced by common next generation sequencing platforms for viral populations.

Tracking the evolution of dengue virus strains D2S10 and D2S20 by 454 pyrosequencing.

Dengue virus is the most prevalent mosquito-borne virus worldwide. In this study, we used pyrosequencing to analyze the whole viral genome of two mouse-adapted strains, D2S10 and D2S20, that induce a dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)-like lethal disease in mice lacking the type I and/or type II interferon receptors. Previous experiments with D2S10 indicated that N124D and K128E mutations in the envelope protein were responsible for the severe disease induced in mice compared to its parental strain PL046. Here we demonstrate that D2S20 is more virulent than D2S10 and captured the presence of five key amino acid mutations--T70I, N83D, and K122I in envelope (E), and A62T in nonstructural protein 2A (NS2A) and G605V in nonstructural protein 5 (NS5)--that may account for this. These findings set the foundation for further dissection of the viral determinants responsible for dengue disease manifestations in mouse models.

Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification.

RNA viruses are the causative agents for AIDS, influenza, SARS, and other serious health threats. Development of rapid and broadly applicable methods for complete viral genome sequencing is highly desirable to fully understand all aspects of these infectious agents as well as for surveillance of viral pandemic threats and emerging pathogens. However, traditional viral detection methods rely on prior sequence or antigen knowledge. In this study, we describe sequence-independent amplification for samples containing ultra-low amounts of viral RNA coupled with Illumina sequencing and de novo assembly optimized for viral genomes. With 5 million reads, we capture 96 to 100% of the viral protein coding region of HIV, respiratory syncytial and West Nile viral samples from as little as 100 copies of viral RNA. The methods presented here are scalable to large numbers of samples and capable of generating full or near full length viral genomes from clone and clinical samples with low amounts of viral RNA, without prior sequence information and in the presence of substantial host contamination.

Complex dynamic of dengue virus serotypes 2 and 3 in Cambodia following series of climate disasters.

The Dengue National Control Program was established in Cambodia in 2000 and has reported between 10,000 and 40,000 dengue cases per year with a case fatality rate ranging from 0.7 to 1.7. In this study 39 DENV-2 and 57 DENV-3 viruses isolated from patients between 2000 and 2008 were fully sequenced. Five DENV2 and four DENV3 distinct lineages with different dynamics were identified. Each lineage was characterized by the presence of specific mutations with no evidence of recombination. In both DENV-2 and DENV-3 the lineages present prior to 2003 were replaced after that date by unrelated lineages. After 2003, DENV-2 lineages D2-3 and D2-4 cocirculated until 2007 when they were almost completely replaced by a lineage D2-5 which emerged from D2-3 Conversely, all DENV-3 lineages remained, diversified and cocirculated with novel lineages emerging. Years 2006 and 2007 were marked by a high prevalence of DENV-3 and 2007 with a large dengue outbreak and a high proportion of patients with severe disease. Selective sweeps in DENV-1 and DENV-2 were linked to immunological escape to a predominately DENV-3-driven immunological response. The complex dynamic of dengue in Cambodia in the last ten years has been associated with a combination of stochastic climatic events, cocirculation, coevolution, adaptation to different vector populations, and with the human population immunological landscape.

Whole genome pyrosequencing of rare hepatitis C virus genotypes enhances subtype classification and identification of naturally occurring drug resistance variants.

BACKGROUND: Infection with hepatitis C virus (HCV) is a burgeoning worldwide public health problem, with 170 million infected individuals and an estimated 20 million deaths in the coming decades. While 6 main genotypes generally distinguish the global geographic diversity of HCV, a multitude of closely related subtypes within these genotypes are poorly defined and may influence clinical outcome and treatment options. Unfortunately, the paucity of genetic data from many of these subtypes makes time-consuming primer walking the limiting step for sequencing understudied subtypes. METHODS: Here we combined long-range polymerase chain reaction amplification with pyrosequencing for a rapid approach to generate the complete viral coding region of 31 samples representing poorly defined HCV subtypes. RESULTS: Phylogenetic classification based on full genome sequences validated previously identified HCV subtypes, identified a recombinant sequence, and identified a new distinct subtype of genotype 4. Unlike conventional sequencing methods, use of deep sequencing also facilitated characterization of minor drug resistance variants within these uncommon or, in some cases, previously uncharacterized HCV subtypes. CONCLUSIONS: These data aid in the classification of uncommon HCV subtypes while also providing a high-resolution view of viral diversity within infected patients, which may be relevant to the development of therapeutic regimens to minimize drug resistance.

Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis.

Prochlorococcus is the numerically dominant photosynthetic organism throughout much of the world's oceans, yet little is known about the ecology and genetic diversity of populations inhabiting tropical waters. To help close this gap, we examined natural Prochlorococcus communities in the tropical Pacific Ocean using a single-cell whole-genome amplification and sequencing. Analysis of the gene content of just 10 single cells from these waters added 394 new genes to the Prochlorococcus pan-genome--that is, genes never before seen in a Prochlorococcus cell. Analysis of marker genes, including the ribosomal internal transcribed sequence, from dozens of individual cells revealed several representatives from two uncultivated clades of Prochlorococcus previously identified as HNLC1 and HNLC2. While the HNLC clades can dominate Prochlorococcus communities under certain conditions, their overall geographic distribution was highly restricted compared with other clades of Prochlorococcus. In the Atlantic and Pacific oceans, these clades were only found in warm waters with low Fe and high inorganic P levels. Genomic analysis suggests that at least one of these clades thrives in low Fe environments by scavenging organic-bound Fe, a process previously unknown in Prochlorococcus. Furthermore, the capacity to utilize organic-bound Fe appears to have been acquired horizontally and may be exchanged among other clades of Prochlorococcus. Finally, one of the single Prochlorococcus cells sequenced contained a partial genome of what appears to be a prophage integrated into the genome.

Genetic diversity and lineage dynamic of dengue virus serotype 1 (DENV-1) in Cambodia.

In Cambodia, dengue virus (DENV) was first isolated in 1963 and has become endemic with peak epidemic during raining season. Since 2000, the Dengue National Control Program has reported from 10,000 to 40,000 cases per year with fatality rates ranging from 0.7 to 1.7. All four dengue serotypes are found circulating in Cambodia with alternative predominance of serotypes DENV-2 and DENV-3. The DENV-1 represents from 5% to 20% of all circulating viruses, depending upon the year. In this work, 79 clinical strains of DENV-1 were isolated between 2000 and 2009 and their genome fully sequenced. Four distinct lineages with different dynamics were identified. The main evolutionary drive was negative selective pressure but each lineage was characterized by the presence of specific mutations acquired through evolution. Coexistence, extinction and replacement of lineages occurred over the 10-year period. Lineages 1, 2 and 3 were all detected since 2000-2002 and disappeared in 2003, 2004-2005 and 2007, respectively. Lineages 1 and 2 displayed different dynamics. Lineage 1 was very diverse whereas lineage 2 was very homogeneous. Lineage 4 which derived from lineage 3 in 2003 remained the only one at the end of the sampling period in 2008-2009 owing to a selective sweep. The lineages dynamic of DENV-1 viruses and consequences for molecular epidemiology are discussed.