Frederic Bedin

Chromosomal distribution and coding capacity of the human endogenous retrovirus HERV-W family

Some genomic elements of the multicopy HERV-W endogenous retroviral family have been previously identified in databases. One of them, located on chromosome 7, contains a single complete open reading frame (ORF) putatively encoding an envelope protein. We have experimentally investigated the genomic complexity and coding capacity of the HERV-W family. The human haploid genome contains at least 70, 100, and 30 HERV-W-related gag, pro, and env regions, respectively, widely and heterogeneously dispersed among chromosomes. Using in vitro transcription-translation procedures, three putative HERV-W gag, pro, and env ORFs were detected on chromosomes 3, 6, and 7, respectively, and their sequences analyzed. A 363 amino acid gag ORF containing matrix and carboxy-terminal truncated capsid domains encoded a putative 45-kDa protein. No gag-pro ORF was found, but a pro sequence containing a DTG active site was detected. Finally, the previously described 538 amino acid HERV-W env ORF, located on chromosome 7, was shown to be unique and encoded a putative 80-kDa glycosylated protein. Proteins of molecular mass identical to the one obtained by an in vitro transcription-translation procedure were detected in human placenta, using anti HERV-W Gag- and Env-specific antibodies. The absence of an HERV-W replication-competent provirus versus the existence of HERV-W-related Gag and Env proteins in healthy human placenta is discussed with respect to particle formation, physiology, and pathology.

Development of a pan-retrovirus detection system for multiple sclerosis studies

Introduction ‐ Although recent claims implicating HTLV‐1 in multiple sclerosis (MS) have been refuted, several reports suggest that another, hitherto uncharacterised, retrovirus may be involved. We have developed and applied a novel PCR‐based strategy to explore this possibility. Methods ‐ Degenerate oligonucleotides were used in a semi‐nested format to amplify, from reverse‐transcribed RNA, a region of the pol gene which is well conserved amongst all known retroviruses. Results ‐ The ‘pan‐retrovirus’ detection system was shown to be capable of detecting diverse retroviruses including human lentivirus, human oncovirus, simian D‐type virus and murine oncovirus. The ‘pan‐retrovirus’ technique identified a novel retroviral sequence, designated MSRV‐cpol, in the serum of an MS patient and also in purified virions from MS patient‐derived tissue cultures. Sequence comparisons suggest that in the pol gene MSRV is related (˜75% homology) to the endogenous retroviral element ERV9. Conclusion ‐ These findings lend further support to the concept of retroviral involvement in MS.

Cell cultures and associated retroviruses in multiple sclerosis

Retroviral particles associated with reverse transcriptase (RT) activity in cell‐cultures from MS patients have been reported by different groups. Cell‐cultures have been used for the study and characterization of the corresponding retroviral genome which we have shown is related to ERV9 in the pol region. Previously unpublished details of a study with monocyte cultures are presented together with observations on leptomeningeal and choroid‐plexus cultures. The generation of self‐transformed cultures after inhibition of interferon, followed by the loss of retroviral expression and recurrent apoptosis, is analyzed. Retroviral particles with RT‐activity are produced in monocyte cultures with an apparent correlation with MS disease activity. However, though leptomeningeal and choroid plexus cells from MS can be passaged for a limited period, their evolution in vitro is not compatible with stable retroviral expression. These culture limitations greatly hampered progress on the elucidation of the retroviral genome sequence.

Nonstructural protein NS1 immunodominant epitope detected specifically in dengue virus infected material by a SELDI-TOF/MS based assay

Dengue virus (DV) infection is the most common mosquito‐born viral disease of public health significance. Though most patients only suffer from flu‐like symptoms, a small group of patients experiences more severe forms of the disease. The viral nonstructural protein 1 (NS1), a secreted protein correlating with viremia, is a key element used for dengue diagnosis with potential implications in severe dengue prognosis. Capture‐ELISAs for the early detection of the NS1 protein in the sera during the acute febrile stage are commonly used in routine by diagnostic laboratories. In this study, the detection of NS1 protein in DV‐infected material was assessed by an alternative method combining a single NS1‐directed monoclonal antibody and the SELDI‐TOF/MS technology. According to the epitope mapping, the antibodies used are mainly directed against an immuno‐dominant peptide located on the C‐terminal part of the protein. The NS1 SELDI‐TOF assay is specific, has a sensitivity level close to capture‐ELISAs and is potentially useful for a coupled serotyping/detection assay or for the detection of subtle post‐translational modifications on the protein. J. Med. Virol. 84:490–499, 2012.

Isotope Coded Protein Labeling analysis of plasma specimens from acute severe dengue fever patients

BackgroundDengue fever is the most important arthropod born viral disease of public health significance. Although most patients suffer only from flu-like symptoms, a small group of patient experiences more severe forms of the disease. To contribute to a better understanding of its pathogenesis this study aims to identify proteins differentially expressed in a pool of five viremic plasma from severe dengue patients relative to a pool of five non-severe dengue patients.ResultsThe use of Isotope Coded Protein Labeling (ICPLTM) to analyze plasma depleted of twenty high-abundance proteins allowed for the identification of 51 differentially expressed proteins, which were characterized by mass spectrometry. Using quantitative ELISA, three of these proteins (Leucine-rich glycoprotein 1, Vitamin D binding-protein and Ferritin) were confirmed as having an increased expression in a panel of severe dengue plasma. The proteins identified as overexpressed by ICPLTM in severe dengue plasma involve in clear up action after cell injury, tissue coherence and immune defense.ConclusionThis ICPLTM study evaluating differences between acute severe dengue plasmas and acute non-severe dengue plasmas suggests that the three proteins identified are overexpressed early in the course of the disease. Their possible use as biomarkers for the prognostic of disease severity is discussed.

Differential proteomic analysis of virus-enriched fractions obtained from plasma pools of patients with dengue fever or severe dengue

BackgroundDengue is the most widespread mosquito-borne viral disease of public health concern. In some patients, endothelial cell and platelet dysfunction lead to life-threatening hemorrhagic dengue fever or dengue shock syndrome. Prognostication of disease severity is urgently required to improve patient management. The pathogenesis of severe dengue has not been fully elucidated, and the role of host proteins associated with viral particles has received little exploration.MethodsThe proteomes of virion-enriched fractions purified from plasma pools of patients with dengue fever or severe dengue were compared. Virions were purified by ultracentrifugation combined with a water-insoluble polyelectrolyte-based technique. Following in-gel hydrolysis, peptides were analyzed by nano-liquid chromatography coupled to ion trap mass spectrometry and identified using data libraries.ResultsBoth dengue fever and severe dengue viral-enriched fractions contained identifiable viral envelope proteins and host cellular proteins. Canonical pathway analysis revealed the identified host proteins are mainly involved in the coagulation cascade, complement pathway or acute phase response signaling pathway. Some host proteins were over- or under-represented in plasma from patients with severe dengue compared to patients with dengue fever. ELISAs were used to validate differential expression of a selection of identified host proteins in individual plasma samples of patients with dengue fever compared to patients with severe dengue. Among 22 host proteins tested, two could differentiate between dengue fever and severe dengue in two independent cohorts (olfactomedin-4: area under the curve (AUC), 0.958; and platelet factor-4: AUC, 0.836).ConclusionA novel technique of virion-enrichment from plasma has allowed to identify two host proteins that have prognostic value for classifying patients with acute dengue who are more likely to develop a severe dengue. The impact of these host proteins on pathogenicity and disease outcome are discussed.

Paper-based point-of-care testing for cost-effective diagnosis of acute flavivirus infections†

Flavivirus infections are a serious healthcare concern in tropical and subtropical countries. Although well‐established laboratory tests can provide early diagnosis of acute dengue or Zika infections, access to these tests is limited in developing countries, presenting an urgent need to develop simple, rapid, and robust diagnostic tools. Microfluidic Paper‐based Analytical Devices (μPAD), are typically rapid, cost‐effective, user‐friendly, and they can be used as diagnostic tools for the diagnosis of these infections at Point of Care settings. Early and prompt diagnosis is crucial to improve patient management and reduce the risk of complications. In the present study, we developed and evaluated a wax‐printed paper‐based device for the detection of the dengue and Zika non‐structural NS1 viral protein in blood and plasma. Experiments have been carried out to increase specificity, while maintaining the required sensitivity. As a consequence, the quality of the raw materials and the washing steps were proved to be crucial. The μPAD was able to detect specifically in 6–8 min 10 ng/mL of protein in various sample types. A prototype for the differential detection of dengue and/or Zika NS1 protein was developed. The reading of the results was simplified by using a dedicated application on a smartphone.

Laser-cut paper-based device for the detection of dengue non-structural NS1 protein and specific IgM in human samples

The incidence of flavivirus infections has increased dramatically in recent decades in tropical and sub-tropical areas worldwide, affecting hundreds of millions of people each year. Dengue viruses are typically transmitted by mosquitoes and can cause a wide range of symptoms from flu-like fever to organ impairment and death. Although conventional diagnostic tests can provide early diagnosis of acute dengue infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop affordable, simple, rapid, and robust diagnostic tools that can be used at ‘Point of Care’ settings. Early diagnosis is crucial to improve patient management and reduce the risk of complications. In the present study, a novel laser-cut device made of glass-fiber paper was designed and tested for the detection of the dengue Non Structural 1 (NS1) viral protein and specific IgM in blood and plasma. The device, called PAD, was able to detect around 25 ng/mL of NS1 protein in various sample types in 8 minutes, following a few simple steps. The PAD was also able to detect specific IgM in human plasmas in less than 10 minutes. The PAD appears to have all the potential to assist health workers in early diagnosis of dengue fever or other tropical fevers caused by flaviviruses.