The Sensitivity of Massively Parallel Sequencing for Detecting Candidate Infectious Agents Associated with Human Tissue

Massively parallel sequencing technology now provides the opportunity to sample the transcriptome of a given tissue comprehensively. Transcripts at only a few copies per cell are readily detectable, allowing the discovery of low abundance viral and bacterial transcripts in human tissue samples. Here we describe an approach for mining large sequence data sets for the presence of microbial sequences. Further, we demonstrate the sensitivity of this approach by sequencing human RNA-seq libraries spiked with decreasing amounts of an RNA-virus. At a modest depth of sequencing, viral transcripts can be detected at frequencies less than 1 in 1,000,000. With current sequencing platforms approaching outputs of one billion reads per run, this is a highly sensitive method for detecting putative infectious agents associated with human tissues

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Genomic characterization of viruses infecting freshwater polar cyanobacteria

There is wide recognition that cyanobacteria are major primary producers in polar freshwater regions. Filamentous cyanobacteria are commonly found in benthic mats and biofilms at the bottom of lakes, ponds and streams, while picocyanobacteria dominate the planktonic communities of many polar lakes. However, no representative viruses infecting this group of organisms have been characterized. This dissertation, which is a culmination of experiments and genomic and metagenomic analyses, presents the first characterization of viruses infecting freshwater polar cyanobacteria and the discovery of previously unknown groups of viruses. First, I isolated and genetically characterized a polar freshwater cyanophage (S-EIV1) that represents a new evolutionary lineage of bacteriophages that are globally widespread and abundant. Second, I described a new group of viruses (Cyanophage A-1 and Cyanophage N-1) infecting freshwater filamentous cyanobacteria that contain a distinct DNA polymerase. Third, during genomic analysis of Cyanophage N-1, I identified a DNA repeat region similar to a Clustered Regularly Interspaced Short Palindromic (CRISPR) array. The CRISPR array had direct repeats with high similarity to those commonly found in filamentous cyanobacteria. I showed that the viral-encoded CRISPR was transcribed and have the potential be viral-mediated transferred to its host. Finally, DNA-stable isotope probing (DNA-SIP) was used to recover and sequence viruses infecting primary producers in a polar cyanobacterial mat. Arctic freshwater systems are some of the most threatened environments because of rapid climate change, and viruses encompass the greatest genetic and biological diversity on Earth. This work presents previously unknown groups of viruses and a newly discovered virus-host system that provide new tools for investigating host-virus interactions and examining arctic viral diversity.Science, Faculty ofEarth, Ocean and Atmospheric Sciences, Department ofGraduat

Complete genome sequence of the cyanophage S-PRM1 isolated from Singapore coastal waters

Here, we report the genomic sequence and comparative analysis of the cyanophage S-PRM1 which was recently isolated from Singapore coastal waters using Synechococcus sp. strain WH7803. The genome of 144,311 bp was sequenced using a combination of Illumina and Oxford Nanopore technologies. The calculated OrthoANI (Average Nucleotide Identity by Orthology) values of S-PRM1 were between 65.24% and 89.39% when compared with previously sequenced cyanomyoviruses with the highest identity with cyanophage S-CAM9. Auxiliary metabolic genes (AMGs) involved in photosynthesis, carbon metabolism, phosphate acquisition, DNA biosynthesis were found in the genome of S-PRM1.NRF (Natl Research Foundation, S’pore)Accepted versio

Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B

Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages.Published versio

temporal and spatial dynamics of Bacteria, Archaea and protists in equatorial coastal waters

International audienceSingapore, an equatorial island in South East Asia, is influenced by a biannual reversal of wind directions which defines two monsoon seasons. We characterized the dynamics of the microbial communities of Singapore coastal waters by collecting monthly samples between February 2017 and July 2018 at four sites located across two straits with different trophic status, and sequencing the V6-V8 region of the small sub-unit ribosomal RNA gene (rRNA gene) of Bacteria, Archaea, and Eukaryota. Johor Strait, which is subjected to wider environmental fluctuations from anthropogenic activities, presented a higher abundance of copiotrophic microbes, including Cellvibrionales and Rhodobacterales. The mesotrophic Singapore Strait, where the seasonal variability is caused by changes in the oceanographic conditions, harboured a higher proportion of typically marine microbe groups such as Synechococcales, Nitrosupumilales, SAR11, SAR86, Marine Group II Archaea and Radiolaria. In addition, we observed seasonal variability of the microbial communities in the Singapore Strait, which was possibly influenced by the alternating monsoon regime, while no seasonal pattern was detected in the Johor Strait

Sequence summary and detection of viral DNA.

<p>*ppm: pairs per million.</p

Circos [16]plot detailing HaRNAV sequence recovery.

<p>The red and blue lines represent reads aligning on the minus and plus strand, respectively. The Heterosigma akashiwo RNA virus has an 8,587 bp ss-RNA linear genome with a single CDS, shown in green on the circos plot. The read depth of coverage is shown in the centre of the plot. The genome is depicted by alternating black-white arcs of 500 bp in size.</p

Complete genome sequence of Lysinibacillus sp. strain SGAir0095, isolated from tropical air samples collected in Singapore

Lysinibacillus sp. strain SGAir0095 was isolated from tropical air samples collected in Singapore, and its complete genome was sequenced with a hybrid strategy using single-molecule real-time sequencing and short reads. The genome consists of one chromosome of 4.14 Mbp and encompasses 3,885 protein-coding genes, 39 rRNAs, and 101 tRNAs.MOE (Min. of Education, S’pore)Published versio

Complete genome sequence of Pseudomonas sp. strain SGAir0191, isolated from tropical air collected in Singapore

Pseudomonas sp. strain SGAir0191 was isolated from an air sample collected in Singapore, and its genome was sequenced using a combination of long and short reads to generate a high-quality genome assembly. The complete genome is approximately 5.07 Mb with 4,370 protein-coding genes, 19 rRNAs, and 73 tRNAs.MOE (Min. of Education, S’pore)Published versio