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.).

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.

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.

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.

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.

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.

Rapid diversification of coevolving marine Synechococcus and a virus.

Marine viruses impose a heavy mortality on their host bacteria, whereas at the same time the degree of viral resistance in marine bacteria appears to be high. Antagonistic coevolution--the reciprocal evolutionary change of interacting species--might reconcile these observations, if it leads to rapid and dynamic levels of viral resistance. Here we demonstrate the potential for extensive antagonistic coevolution between the ecologically important marine cyanobacterium Synechococcus and a lytic virus. In a 6-mo-long replicated chemostat experiment, Synechococcus sp. WH7803 and the virus (RIM8) underwent multiple coevolutionary cycles, leading to the rapid diversification of both host and virus. Over the course of the experiment, we detected between 4 and 13 newly evolved viral phenotypes (differing in host range) and between 4 and 11 newly evolved Synechococcus phenotypes (differing in viral resistance) in each chemostat. Genomic analysis of isolates identified several candidate genes in both the host and virus that might influence their interactions. Notably, none of the viral candidates were tail fiber genes, thought to be the primary determinants of host range in tailed bacteriophages, highlighting the difficulty in generalizing results from bacteriophage infecting γ-Proteobacteria. Finally, we show that pairwise virus-host coevolution may have broader community consequences; coevolution in the chemostat altered the sensitivity of Synechoccocus to a diverse suite of viruses, as well as the virus' ability to infect additional Synechococcus strains. Our results indicate that rapid coevolution may contribute to the generation and maintenance of Synechococcus and virus diversity and thereby influence viral-mediated mortality of these key marine bacteria.

Whole genome deep sequencing of HIV-1 reveals the impact of early minor variants upon immune recognition during acute infection.

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis.

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.

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.

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.

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.

Population genomic sequencing of Coccidioides fungi reveals recent hybridization and transposon control.

We have sequenced the genomes of 18 isolates of the closely related human pathogenic fungi Coccidioides immitis and Coccidioides posadasii to more clearly elucidate population genomic structure, bringing the total number of sequenced genomes for each species to 10. Our data confirm earlier microsatellite-based findings that these species are genetically differentiated, but our population genomics approach reveals that hybridization and genetic introgression have recently occurred between the two species. The directionality of introgression is primarily from C. posadasii to C. immitis, and we find more than 800 genes exhibiting strong evidence of introgression in one or more sequenced isolates. We performed PCR-based sequencing of one region exhibiting introgression in 40 C. immitis isolates to confirm and better define the extent of gene flow between the species. We find more coding sequence than expected by chance in the introgressed regions, suggesting that natural selection may play a role in the observed genetic exchange. We find notable heterogeneity in repetitive sequence composition among the sequenced genomes and present the first detailed genome-wide profile of a repeat-induced point mutation (RIP) process distinctly different from what has been observed in Neurospora. We identify promiscuous HLA-I and HLA-II epitopes in both proteomes and discuss the possible implications of introgression and population genomic data for public health and vaccine candidate prioritization. This study highlights the importance of population genomic data for detecting subtle but potentially important phenomena such as introgression.

Draft genome sequence of four coccolithoviruses: Emiliania huxleyi virus EhV-88, EhV-201, EhV-207, and EhV-208.

The Coccolithoviridae are a group of viruses which infect the marine coccolithophorid microalga Emiliania huxleyi. The Emiliania huxleyi viruses (known as EhVs) described herein have 160- to 180-nm diameter icosahedral structures, have genomes of approximately 400 kbp, and consist of more than 450 predicted coding sequences (CDSs). Here, we describe the genomic features of four newly sequenced coccolithoviruses (EhV-88, EhV-201, EhV-207, and EhV-208) together with their draft genome sequences and their annotations, highlighting the homology and heterogeneity of these genomes to the EhV-86 model reference genome.

Draft genome sequence of the coccolithovirus Emiliania huxleyi virus 202.

Emiliania huxleyi virus 202 (EhV-202) is a member of the Coccolithoviridae, a group of viruses that infect the marine coccolithophorid Emiliania huxleyi. EhV-202 has a 160- to 180-nm-diameter icosahedral structure and a genome of approximately 407 kbp, consisting of 485 coding sequences (CDSs). Here we describe the genomic features of EhV-202, together with a draft genome sequence and its annotation, highlighting the homology and heterogeneity of this genome in comparison with the EhV-86 reference genome.

Genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity.

Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ∼75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection.

High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections.

Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in sequence data in domain III (DIII) of the envelope (E) gene in serial plasma samples (n = 49) taken from 17 patients infected with DENV type 1 (DENV-1), totaling some 8,458 clones. Statistically rigorous approaches were employed to account for artifactual variants resulting from amplification and sequencing, which we suggest have played a major role in previous studies of intrahost genetic variation. Accordingly, nucleotide sequence diversities of viral populations were very low, with conservative estimates of the average levels of genetic diversity ranging from 0 to 0.0013. Despite such sequence conservation, we observed clear evidence for mixed infection, with the presence of multiple phylogenetically distinct lineages present within the same host, while the presence of stop codon mutations in some samples suggests the action of complementation. In contrast to some previous studies we observed no relationship between the extent and pattern of DENV-1 genetic diversity and disease severity, immune status, or level of viremia.

Endemic dengue associated with the co-circulation of multiple viral lineages and localized density-dependent transmission.

Dengue is one of the most important infectious diseases of humans and has spread throughout much of the tropical and subtropical world. Despite this widespread dispersal, the determinants of dengue transmission in endemic populations are not well understood, although essential for virus control. To address this issue we performed a phylogeographic analysis of 751 complete genome sequences of dengue 1 virus (DENV-1) sampled from both rural (Dong Thap) and urban (Ho Chi Minh City) populations in southern Viet Nam during the period 2003-2008. We show that DENV-1 in Viet Nam exhibits strong spatial clustering, with likely importation from Cambodia on multiple occasions. Notably, multiple lineages of DENV-1 co-circulated in Ho Chi Minh City. That these lineages emerged at approximately the same time and dispersed over similar spatial regions suggests that they are of broadly equivalent fitness. We also observed an important relationship between the density of the human host population and the dispersion rate of dengue, such that DENV-1 tends to move from urban to rural populations, and that densely populated regions within Ho Chi Minh City act as major transmission foci. Despite these fluid dynamics, the dispersion rates of DENV-1 are relatively low, particularly in Ho Chi Minh City where the virus moves less than an average of 20 km/year. These low rates suggest a major role for mosquito-mediated dispersal, such that DENV-1 does not need to move great distances to infect a new host when there are abundant susceptibles, and imply that control measures should be directed toward the most densely populated urban environments.