Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family

Impairment of Coronary Arteriolar Endothelium-Dependent Dilation after Multi-Walled Carbon Nanotube Inhalation: A Time-Course Study

Engineered nanomaterials have been developed for widespread applications due to many highly unique and desirable characteristics. The purpose of this study was to assess pulmonary inflammation and subepicardial arteriolar reactivity in response to multi-walled carbon nanotube (MWCNT) inhalation and evaluate the time course of vascular alterations. Rats were exposed to MWCNT aerosols producing pulmonary deposition. Pulmonary inflammation via bronchoalveolar lavage and MWCNT translocation from the lungs to systemic organs was evident 24 h post-inhalation. Coronary arterioles were evaluated 24–168 h post-exposure to determine microvascular response to changes in transmural pressure, endothelium-dependent and -independent reactivity. Myogenic responsiveness, vascular smooth muscle reactivity to nitric oxide, and α-adrenergic responses all remained intact. However, a severe impact on endothelium-dependent dilation was observed within 24 h after MWCNT inhalation, a condition which improved, but did not fully return to control after 168 h. In conclusion, results indicate that MWCNT inhalation not only leads to pulmonary inflammation and cytotoxicity at low lung burdens, but also a low level of particle translocation to systemic organs. MWCNT inhalation also leads to impairments of endothelium-dependent dilation in the coronary microcirculation within 24 h, a condition which does not fully dissipate within 168 h. The innovations within the field of nanotechnology, while exciting and novel, can only reach their full potential if toxicity is first properly assessed

Placental transfer of the polybrominated diphenyl ethers BDE-47, BDE-99 and BDE-209 in a human placenta perfusion system: an experimental study

<p>Abstract</p> <p>Background</p> <p>Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in consumer products. PBDEs may affect thyroid hormone homeostasis, which can result in irreversible damage of cognitive performance, motor skills and altered behaviour. Thus, in utero exposure is of very high concern due to critical windows in fetal development.</p> <p>Methods</p> <p>A human ex vivo placenta perfusion system was used to study the kinetics and extent of the placental transfer of BDE-47, BDE-99 and BDE-209 during four-hour perfusions. The PBDEs were added to the maternal circulation and monitored in the maternal and fetal compartments. In addition, the perfused cotyledon, the surrounding placental tissue as well as pre-perfusion placental tissue and umbilical cord plasma were also analysed. The PBDE analysis included Soxhlet extraction, clean-up by adsorption chromatography and GC-MS analysis.</p> <p>Results and Discussion</p> <p>Placental transfer of BDE-47 was faster and more extensive than for BDE-99. The fetal-maternal ratios (FM-ratio) after four hours of perfusion were 0.47 and 0.25 for BDE-47 and BDE-99, respectively, while the indicative permeability coefficient (IPC) measured after 60 minutes of perfusion was 0.26 h^-1and 0.10 h^-1, respectively. The transport of BDE-209 seemed to be limited. These differences between the congeners may be related to the degree of bromination. Significant accumulation was observed for all congeners in the perfused cotyledon as well as in the surrounding placental tissue.</p> <p>Conclusion</p> <p>The transport of BDE-47 and BDE-99 indicates in utero exposure to these congeners. Although the transport of BDE-209 was limited, however, possible metabolic debromination may lead to products which are both more toxic and transportable. Our study demonstrates fetal exposure to PBDEs, which should be included in risk assessment of PBDE exposure of women of child-bearing age.</p

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

Randomized, Placebo-Controlled Phase 2 Trial of a Lactobacillus crispatus Probiotic Given Intravaginally for Prevention of Recurrent Urinary Tract Infection

In a double-blind placebo-controlled trial of a Lactobacillus crispatus intravaginal suppository probiotic (LACTIN-VÜ, Osel, Inc.) for prevention of recurrent UTI (rUTI) in pre-menopausal women, LACTIN-V was safe, well tolerated, and associated with a reduction in rUTI

Application of Next Generation Sequencing on Norovirus-contaminated oyster samples

The present data set concerne metabarcoding raw reads produced using 4 different PCR targeting polymerase or capside coding region of the genoyupe I and II of norovirus. Test samples of norovirus with serial dilutions in pure water and after a bio-accumulation in oysters. Sequencing was made after VirCapSeq-VERT approach

Application of Next Generation Sequencing on Norovirus‐contaminated oyster samples

Bivalve molluscan shellfish (BMS) contamination with gastroenteritis viruses such as norovirus (NoV) is recognized as a significant public health risk worldwide. These foodborne epidemics are characterized by very low viral concentrations in the implicated foods, and by diverse viruses or multiple NoV strains originating from human sewage, resulting in different strains (co-)infecting the consumers. Next-generation sequencing (NGS) offers promising means to describe the diversity of strains present in BMS or to retrace transmission chains in outbreak settings, but their sensitivity and reproducibility remained to be assessed for this application. In this work, we evaluated the ability of three promising NGS methods to sequence diverse NoV in environmental or food BMS and human stool samples. Using laboratory-prepared samples of known NoV composition, we evaluated the sensitivity, reproducibility, repeatability and selectivity of metabarcoding, capture-based metagenomics and long amplicon sequencing, considering representative NoV strains from genogroup I and II, and the impact of the BMS matrix. The metabarcoding, with separate amplification of polymerase and capsid gene segments followed by Illumina sequencing, was the most sensitive method. It was applied to a selection of 212 BMS samples collected through the European Commission’s NoV baseline survey (BLS), demonstrating a high diversity of NoV sequences found in the BMS which reflect the diversity of NoV strains circulating in the European human population. Besides, a capture-based metagenomics with enrichment of vertebrate viruses was applied on 20 of these BLS samples as well as 20 BMS linked to outbreaks and 10 related human stool samples. In BMS, it yielded NoV sequences compatible with the genomes identified in stool samples, but they were too short to allow definitive confirmation of the infection source. The present report describes NGS methods, including the bioinformatic pipelines, applicable to molecular epidemiology of NoV in BMS, their current limitations and expected outcome