Assessing the health risk of living near composting facilities on lung health, fungal and bacterial disease in cystic fibrosis: a UK CF Registry study

Aim To explore the health risk of living near permitted composting sites (PCSs) on disease severity in children and adults with cystic fibrosis (CF) across the UK. Methods A semi-individual cross-sectional study was used to examine the risk of disease severity in people with CF (pwCF) within and beyond 4 km of PCSs in the UK in 2016. All pwCF registered in the UK CF Registry were eligible for this study. Linear and Poisson regressions, adjusted for age, gender, genotype, BMI, Pseudomonas aeruginosa and deprivation, were used to quantify associations between distance to a PCS and percent predicted forced expiratory volume in one second (ppFEV1), pulmonary exacerbations (#IVdays), and fungal and bacterial infections. Results The mean age of the 9,361 pwCF (3,931 children and 5,430 adults) studied was 20.1 (SD = 14.1) years; 53.3% were male; and 49.2% were homozygous F508del. Over 10% of pwCF (n = 1,015) lived within 4 km of a PCS. We found no statistically significant difference in ppFEV1 and #IVdays/year in children. However, in adults, ppFEV1 was -1.07% lower (95% confidence interval (CI): -2.29%, 0.16%) and #IVdays/year were 1.02 day higher (95%CI: 1.01, 1.04) within 4 km of a PCS. Furthermore, there were statistically significant differences in mean ppFEV1 in CF adults with Aspergillus fumigatus (58.2.% vs 62.0%, p = 0.005) and Candida spp. (56.9% vs 59.9%, p = 0.029) residing within 4 km of a PCS. No associations were identified for allergic bronchopulmonary aspergillosis, P. aeruginosa or Staphylococcus aureus. Conclusions This novel national study provides evidence that adults with CF living near a PCS may experience small reductions in lung function, an increased risk of pulmonary exacerbations, and more frequent fungal infections. If confirmed by studies using refined exposure assessment methods accounting for bioaerosol dispersion, these results could have important implications for the living environment of pwCF

Genetic Relationship between Cocirculating Human Enteroviruses Species C

Recombination events between human enteroviruses (HEV) are known to occur frequently and to participate in the evolution of these viruses. In a previous study, we reported the isolation of a panel of viruses belonging to the Human enterovirus species C (HEV-C) that had been cocirculating in a small geographic area of Madagascar in 2002. This panel included type 2 vaccine-derived polioviruses (PV) that had caused several cases of acute flaccid paralysis in humans. Previous partial sequencing of the genome of these HEV-C isolates revealed considerable genetic diversity, mostly due to recombination. In the work presented herein, we carried out a more detailed characterization of the genomes of viruses from this collection. First, we determined the full VP1 sequence of 41 of these isolates of different types. These sequences were compared with those of HEV-C isolates obtained from other countries or in other contexts. The sequences of the Madagascan isolates of a given type formed specific clusters clearly differentiated from those formed by other strains of the same type isolated elsewhere. Second, we sequenced the entire genome of 10 viruses representing most of the lineages present in this panel. All but one of the genomes appeared to be mosaic assemblies of different genomic fragments generated by intra- and intertypic recombination. The location of the breakpoints suggested potential preferred genomic regions for recombination. Our results also suggest that recombination between type HEV-99 and other HEV-C may be quite rare. This first exhaustive genomic analysis of a panel of non-PV HEV-C cocirculating in a small human population highlights the high frequency of inter and intra-typic genetic recombination, constituting a widespread mechanism of genetic plasticity and continually shifting the HEV-C biodiversity

A Retrospective Overview of Enterovirus Infection Diagnosis and Molecular Epidemiology in the Public Hospitals of Marseille, France (1985–2005)

Human enteroviruses (HEV) are frequent human pathogens and, associated in particular with large outbreaks of aseptic meningitis. Here, we have compiled a database of clinical HEV isolates from the Public Hospitals of Marseille, from 1985 to 2005. Amongst 654 isolates that could be characterized by complete sequencing of the VP1 gene, 98% belonged to species HEV-B; the most frequently isolated serotypes were Echovirus E30, E11, E7, E6 and E4. The high incidence of E30 and the recent emergence of E13 are consistent with reports worldwide and peak HEV isolation occurred mostly in the late spring and summer months. The proportion of echoviruses has decreased across the years, while that of coxsackieviruses has increased. Stool (the most frequent sample type) allowed detection of all identified serotypes. MRC5 (Human lung fibroblasts) cell line was the most conducive cell line for HEV isolation (84.9% of 10 most common serotype isolates, 96.3% in association with BGM (Buffalo green monkey kidney cells)). Previous seroneutralization-based serotype identification demonstrated 55.4% accuracy when compared with molecular VP1 analysis. Our analysis of a large number of clinical strains over 20 years reinforced the validity of VP1 serotyping and showed that comparative p-distance scores can be coupled with phylogenetic analysis to provide non-ambiguous serotype identification. Phylogenetic analysis in the VP1, 2C and 3D regions also provided evidence for recombination events amongst clinical isolates. In particular, it identified isolates with dissimilar VP1 but almost identical nonstructural regions

Molecular Epidemiology and Evolution of Human Enterovirus Serotype 68 in Thailand, 2006–2011

BACKGROUND: Publications worldwide have reported on the re-occurrence of human enterovirus 68 (EV68), a rarely detected pathogen usually causing respiratory illness. However, epidemiological data regarding this virus in particular on the Asian continent has so far been limited. METHODOLOGY/FINDINGS: We investigated the epidemiology and genetic variability of EV68 infection among Thai children with respiratory illnesses from 2006-2011 (n = 1810). Semi-nested PCR using primer sets for amplification of the 5'-untranslated region through VP2 was performed for rhino-enterovirus detection. Altogether, 25 cases were confirmed as EV68 infection indicating a prevalence of 1.4% in the entire study population. Interestingly, the majority of samples were children aged >5 years (64%). Also, co-infection with other viruses was found in 28%, while pandemic H1N1 influenza/2009 virus was the most common co-infection. Of EV68-positive patients, 36% required hospitalizations with the common clinical presentations of fever, cough, dyspnea, and wheezing. The present study has shown that EV68 was extremely rare until 2009 (0.9%). An increasing annual prevalence was found in 2010 (1.6%) with the highest detection frequency in 2011 (4.3%). Based on analysis of the VP1 gene, the evolutionary rate of EV68 was estimated at 4.93 × 10(-3) substitutions/site/year. Major bifurcation of the currently circulating EV68 strains occurred 66 years ago (1945.31 with (1925.95-1960.46)95% HPD). Among the current lineages, 3 clusters of EV68 were categorized based on the different molecular signatures in the BC and DE loops of VP1 combined with high posterior probability values. Each cluster has branched off from their common ancestor at least 36 years ago (1975.78 with (1946.13-1984.97)95% HPD). CONCLUSION: Differences in epidemiological characteristic and seasonal profile of EV68 have been found in this study. Results from Bayesian phylogenetic investigations also revealed that EV68 should be recognized as a genetically diverse virus with a substitution rate identical to that of enterovirus 71 genotype B (4.2 × 10(-3 )s/s/y)

The pathogenic mechanism of the Mycobacterium ulcerans virulence factor, mycolactone, depends on blockade of protein translocation into the ER.

Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer

Discovery and Genomic Characterization of a Novel Ovine Partetravirus and a New Genotype of Bovine Partetravirus

Partetravirus is a recently described group of animal parvoviruses which include the human partetravirus, bovine partetravirus and porcine partetravirus (previously known as human parvovirus 4, bovine hokovirus and porcine hokovirus respectively). In this report, we describe the discovery and genomic characterization of partetraviruses in bovine and ovine samples from China. These partetraviruses were detected by PCR in 1.8% of bovine liver samples, 66.7% of ovine liver samples and 71.4% of ovine spleen samples. One of the bovine partetraviruses detected in the present samples is phylogenetically distinct from previously reported bovine partetraviruses and likely represents a novel genotype. The ovine partetravirus is a novel partetravirus and phylogenetically most related to the bovine partetraviruses. The genome organization is conserved amongst these viruses, including the presence of a putative transmembrane protein encoded by an overlapping reading frame in ORF2. Results from the present study provide further support to the classification of partetraviruses as a separate genus in Parvovirinae

The chemical signatures underlying host plant discrimination by aphids

The diversity of phytophagous insects is largely attributable to speciation involving shifts between host plants. These shifts are mediated by the close interaction between insects and plant metabolites. However, there has been limited progress in understanding the chemical signatures that underlie host preferences. We use the pea aphid (Acyrthosiphon pisum) to address this problem. Host-associated races of pea aphid discriminate between plant species in race-specific ways. We combined metabolomic profiling of multiple plant species with behavioural tests on two A. pisum races, to identify metabolites that explain variation in either acceptance or discrimination. Candidate compounds were identified using tandem mass spectrometry. Our results reveal a small number of compounds that explain a large proportion of variation in the differential acceptability of plants to A. pisum races. Two of these were identified as L-phenylalanine and L-tyrosine but it may be that metabolically-related compounds directly influence insect behaviour. The compounds implicated in differential acceptability were not related to the set correlated with general acceptability of plants to aphids, regardless of host race. Small changes in response to common metabolites may underlie host shifts. This study opens new opportunities for understanding the mechanistic basis of host discrimination and host shifts in insects

SARS-CoV-2 RNA detected in blood products from patients with COVID-19 is not associated with infectious virus [version 2; peer review: 2 approved]

Background: Laboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. SARS-CoV-2 RNA has also been detected in other sample types, but there is limited understanding of the clinical or laboratory significance of its detection in blood. Methods: We undertook a systematic literature review to assimilate the evidence for the frequency of vRNA in blood, and to identify associated clinical characteristics. We performed RT-PCR in serum samples from a UK clinical cohort of acute and convalescent COVID-19 cases (n=212), together with convalescent plasma samples collected by NHS Blood and Transplant (NHSBT) (n=462 additional samples). To determine whether PCR-positive blood samples could pose an infection risk, we attempted virus isolation from a subset of RNA-positive samples. Results: We identified 28 relevant studies, reporting SARS-CoV-2 RNA in 0-76% of blood samples; pooled estimate 10% (95%CI 5-18%). Among serum samples from our clinical cohort, 27/212 (12.7%) had SARS-CoV-2 RNA detected by RT-PCR. RNA detection occurred in samples up to day 20 post symptom onset, and was associated with more severe disease (multivariable odds ratio 7.5). Across all samples collected ≥28 days post symptom onset, 0/494 (0%, 95%CI 0-0.7%) had vRNA detected. Among our PCR-positive samples, cycle threshold (ct) values were high (range 33.5-44.8), suggesting low vRNA copy numbers. PCR-positive sera inoculated into cell culture did not produce any cytopathic effect or yield an increase in detectable SARS-CoV-2 RNA. There was a relationship between RT-PCR negativity and the presence of total SARS-CoV-2 antibody (p=0.02). Conclusions: vRNA was detectable at low viral loads in a minority of serum samples collected in acute infection, but was not associated with infectious SARS-CoV-2 (within the limitations of the assays used). This work helps to inform biosafety precautions for handling blood products from patients with current or previous COVID-19

Protocol for Nearly Full-Length Sequencing of HIV-1 RNA from Plasma

Nearly full-length genome sequencing of HIV-1 using peripheral blood mononuclear cells (PBMC) DNA as a template for PCR is now a relatively routine laboratory procedure. However, this has not been the case when using virion RNA as the template and this has made full genome analysis of circulating viruses difficult. Therefore, a well-developed procedure for sequencing of full-length HIV-1 RNA directly from plasma was needed. Plasma from U.S. donors representing a range of viral loads (VL) was used to develop the assay. RNA was extracted from plasma and reverse-transcribed. Two or three overlapping regions were PCR amplified to cover the entire viral genome and sequenced for verification. The success of the procedure was sensitive to VL but was routinely successful for VL greater than 105 and the rate declined in proportion to the VL. While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads. This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies