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.

[Brazil, land of choice for arbovirosis?]. / Le Brésil, une terre d'élection pour les arboviroses?

Scores of arboviroses have been indexed in Brazil with variable rates of morbidity and mortality. Emergence of arboviroses and their transmission by hematophageous vectors are closely related to the country's recent history and in particular to its economic, social and ecological development. Most arboviroses are caused by alphaviruses and flaviviruses. Some species of bunyavirus are also found. Brazil is often regarded as an arbovirus sanctuary where dormant or nonpathogenic viruses can find an optimal context to emerge or reinforce their pathogenic potential in the middle term. Currently yellow fever and especially dengue fever are major public health problems.

Characterization of the antibody response elicited by HIV-1 Env glycomutants in rabbits.

HIV-1 N-glycans are known to shield underlying epitopes towards the protective antibody repertoire. We previously described HIV-1 acute infection Env glycomutants designed from 3D-model in which the removal of clustered N-glycans did not disturb the envelope antigenicity, but increased the neutralization sensitivity. The potential of such immunogens to elicit neutralizing responses was estimated after rabbit immunizations with a DNA/protein protocol. Maturation of the Env-specific antibody response was confirmed by a change in avidity and conformational dependence. For one immunogen, the neutralizing response was increased with a higher breadth compared to the Wild-Type. Our data suggest that Env selective deglycosylation based on 3D data may represent a valuable strategy to improve elicitation of neutralizing antibodies.

[HIV1 Env N-glycans and their interactions with the immune system]. / Les N-glycanes de l'enveloppe du VIH1 et leurs interactions avec le système immunitaire.

N-glycans determine both the conformational and functional characteristics of the HIV1 envelope glycoprotein (Env). In addition, the significant glycosylation modulates the recognition of Env by the different immune system effectors. N-glycans confer to Env the capacity to interact with the type C lectins. Thus, MBLs (mannose binding lectins) recognise the gp120 in a specific manner and exert an antiviral action. Conversely, the interaction between gp120 and DC-SIGN at the dendritic cells surface interferes with the induction of the adaptive immune response. N-glycans also modulate the presentation of the T helper epitopes and thus indirectly influence the induction and the maturation of both the Env cytotoxic cellular and humoral response. Moreover, the N-glycans form a shield which limits the accessibility of the proteic backbone to the antibodies. The constant evolving glycan shield enables neutralising response escape. However, in some cases, N-glycans can constitute clusters which represent a target for the antibodies. The understanding of the multiple roles of N-glycans could significantly contribute to the optimisation of an Env immunogen potentially usable in a vaccine preparation.

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.

Molecular cloning and characterization of MSRV-related sequences associated with retrovirus-like particles.

New sequences have been obtained by successive overlapping RT-PCR extensions from the pol region of a retroviral RNA (multiple sclerosis-associated retroviral element, MSRV) amplified in retrovirus-like particles from patients with multiple sclerosis. gag and pol sequences are related to type C oncoviruses, whereas the env sequence is closer to type D. A tryptophan-like (W) tRNA primer-binding site was identified downstream of the RU5 region in the 5'LTR, and the U3R region cloned in the 3'LTR exhibited potent promoter activity. MSRV clones define a novel family of endogenous elements, HERV-W. From our data, HERV-W RNAs are copackaged in extracellular particles which might be produced by replication-competent or transcomplemented HERV-W copies or by an exogenous member of the HERV-W family.

Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family.

The multiple sclerosis-associated retrovirus (MSRV) isolated from plasma of MS patients was found to be phylogenetically and experimentally related to human endogenous retroviruses (HERVs). To characterize the MSRV-related HERV family and to test the hypothesis of a replication-competent HERV, we have investigated the expression of MSRV-related sequences in healthy tissues. The expression of MSRV-related transcripts restricted to the placenta led to the isolation of overlapping cDNA clones from a cDNA library. These cDNAs spanned a 7.6-kb region containing gag, pol, and env genes; RU5 and U3R flanking sequences; a polypurine tract; and a primer binding site (PBS). As this PBS showed similarity to avian retrovirus PBSs used by tRNATrp, this new HERV family was named HERV-W. Several genomic elements were identified, one of them containing a complete HERV-W unit, spanning all cDNA clones. Elements of this multicopy family were not replication competent, as gag and pol open reading frames (ORFs) were interrupted by frameshifts and stop codons. A complete ORF putatively coding for an envelope protein was found both on the HERV-W DNA prototype and within an RU5-env-U3R polyadenylated cDNA clone. Placental expression of 8-, 3.1-, and 1.3-kb transcripts was observed, and a putative splicing strategy was described. The apparently tissue-restricted HERV-W long terminal repeat expression is discussed with respect to physiological and pathological contexts.

Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. The Collaborative Research Group on Multiple Sclerosis.

The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein-Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial pol fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a pol region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.

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 (approximately 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. Collaborative Research Group on MS.

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.