A rapid and specific real time RT-PCR assay for diagnosis of Toscana virus infection.

BACKGROUND: To scan a virus (TOSV) belongs to the Phlebovirus genus within the Bunyaviridae family. TOSV is an arbovirus transmitted by sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis. OBJECTIVES: Development and assessment of a new sensitive and specific real-time RT-PCR assay for TOSV diagnosis. STUDY DESIGN: TOSV-specific primers and probe targeting the S-segment of the genome were designed, based on recent TOSV sequences available in public databases. Sensitivity was assessed using 10-fold serial dilutions of a RNA transcript and serial dilutions of TOSV strains isolated from infected human beings. Specificity was determined by testing RNA extracts from closely related Phleboviruses. The assay was then used for TOSV infection diagnosis in 971 clinical samples and for TOSV detection in 2000 sandflies. RESULTS: The real-time RT-PCR assay exhibited a sensitivity of under 257 copies per reaction for the RNA transcripts and 0.0056 and 0.014 TCID50 of Italian and Spanish TOSV genotypes per reaction, respectively. No other close Phleboviruses were detected. TOSV was identified in 17 clinical samples and in 3 sandflies. CONCLUSIONS: The assay described is a rapid, robust and reliable real-time RT-PCR test for accurate diagnosis of human TOSV infection as well as for the surveillance of TOSV in vector populations.

The microbial detection array for detection of emerging viruses in clinical samples--a useful panmicrobial diagnostic tool.

Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods.

Dengue surveillance in the French armed forces: a dengue sentinel surveillance system in countries without efficient local epidemiological surveillance.

Surveillance of travel-acquired dengue could improve dengue risk estimation in countries without ability. Surveillance in the French army in 2010 to 2011 highlighted 330 dengue cases, mainly in French West Indies and Guiana: DENV-1 circulated in Guadeloupe, Martinique, French Guiana, New Caledonia, Djibouti; DENV-3 in Mayotte and Djibouti; and DENV-4 in French Guiana.

Toscana virus inhibits the interferon beta response in cell cultures.

Toscana virus (TOSV) is an emerging pathogen in the Mediterranean basin where it causes summertime outbreaks of aseptic meningitis and meningoencephalitis. Many aspects of TOSV biology remain unknown including the possible implication of an amplifying mammalian host besides its vector. The three experiments described here were designed to assess the relationship between TOSV and type-I interferon (IFN) response. The main findings were as follows. First, TOSV growth in Vero cells is sensitive to an antiviral state induced by low-dose addition of exogenous IFN beta (IFN-ß) (10IU/ml). Second, no IFN-ß mRNA or IFN-ß was detectable after infection of HeLa and 293T cells by TOSV. Finally, TOSV inhibits IFN-ß production induced by Sendaï virus, a well known inducer of IFN-ß production. In addition to showing that TOSV can inhibit the IFN-ß response, these findings suggest that anti-IFN capability is maintained by regular contact with that of a mammalian host.

Phenotypic and genotypic characterization of dengue virus isolates differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome.

Dengue viruses (DENV) cause 50-100 million cases of acute febrile disease every year, including 500,000 reported cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Viral factors have been proposed to influence the severity of the disease, but markers of virulence have never been identified on DENV. Three DENV serotype-1 isolates from the 2007 epidemic in Cambodia that are derived from patients experiencing the various clinical forms of dengue were characterized both phenotypically and genetically. Phenotypic characteristics in vitro, based on replication kinetics in different cell lines and apoptosis response, grouped isolates from DF and DHF patients together, whereas the virus isolate from a DSS patient showed unique features: a lower level of replication in mammalian cells and extensive apoptosis in mosquito cells. Genomic comparison of viruses revealed six unique amino acid residues in the membrane, envelope, and in non-structural genes in the virus isolated from the DSS patient.

Quantitative label-free RNA detection using surface-enhanced Raman spectroscopy.

Surface-Enhanced Raman Spectroscopy (SERS) was performed to detect label-free RNA. We defined conditions which make it possible to probe the four bases of RNA, in single strands of polyadenosine (pA), polyuridine (pU), polycytosine (pC) and polyguanosine (pG). We therefore present below a quantitative analysis of mixtures of non-hybridized single strands, based on the deconvolution of the SERS mixture spectrum into the relative contributions of the SERS spectra of each constituent.

Chikungunya virus, southeastern France.

In September 2010, autochthonous transmission of chikungunya virus was recorded in southeastern France, where the Aedes albopictus mosquito vector is present. Sequence analysis of the viral genomes of imported and autochthonous isolates indicated new features for the potential emergence and spread of the virus in Europe.

Cellular receptors, differentiation and endocytosis requirements are key factors for type I IFN response by human epithelial, conventional and plasmacytoid dendritic infected cells by measles virus.

While the antiviral response during measles virus (MeV) infection is documented, the contribution of the hosting cell type to the type I interferon (IFN-alpha/beta) response is still not clearly established. Here, we report that a signature heterogeneity of the IFN-alpha/beta response according to the cell type. The MeV tropism dictated by the expression of appropriate cellular receptor appeared to be crucial for epithelial cells. For conventional DCs (cDCs), the maturation state played a prominent role. In response to both wild type MeV isolates and laboratory/vaccine strains, immature cDCs produced higher levels of IFN-alpha than mature cDCs, despite the reduced expression levels of both CD46 and CD150 receptors by the former ones. While in epithelial cells and cDCs the MeV transcription was required to activate the IFN-alpha/beta response, plasmacytoid DCs (pDCs) rapidly produced large amounts of IFN-alpha mostly independently of the viral infection cycle. This argues for a significant contribution of pDCs in response to MeV infection and/or vaccination.

Real-time reverse-transcription loop-mediated isothermal amplification for rapid detection of rift valley Fever virus.

The development and validation of a one-step, single-tube, real-time accelerated reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the L RNA segment of Rift Valley fever virus (RVFV) are described. The assay was performed at a constant temperature (63 degrees C), with a real-time follow-up using a LightCycler and a double-stranded-DNA-intercalating fluorochrome. The assay is highly sensitive and comparable to real-time RT-PCR, with a detection limit of approximately 10 RNA copies per assay. However, the RT-LAMP assay is much faster than traditional RT-PCR and generates results in <30 min for most diluted samples. The specificity of the primers was established using other, related arboviruses as well as virus-containing and virus-free sera. The RT-LAMP assay reported here is thus a valuable tool for the rapid detection of RVFV in field diagnostic laboratories.

Circulation of Chikungunya virus in Gabon, 2006-2007.

This study reports the first isolation and partial genetic characterization of Chikungunya virus (CHIKV) from patients during a 2006-2007 dengue-like syndrome outbreak in Gabon. The isolated viruses were phylogenetically close to strains isolated in the Democratic Republic of the Congo 7 years ago and to strains isolated more recently in Cameroon. These results indicate a continuing circulation of a genetically stable CHIKV population during 7 years in Central Africa.

Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response.

BACKGROUND: Double stranded RNA (dsRNA) is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA. METHODOLOGY AND PRINCIPAL FINDINGS: During measles virus infection, the IFN-beta gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-beta gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-beta expression, provided that it was delivered into the cytosol as a 5'-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-beta induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5'-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5'-triphosphate RNA end. CONCLUSIONS/SIGNIFICANCE: RIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5'-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader.

Dynamics of viral RNA synthesis during measles virus infection.

We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until approximately 24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is approximately 3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5'-end abortive replication fragment RNAs.

Optimized SYBR green real-time PCR assay to quantify the absolute copy number of measles virus RNAs using gene specific primers.

A sensitive and specific RT-QPCR based on real-time analysis of PCR products stained with SYBR green, was designed and carefully optimised to quantify individual measles virus RNA species. Pairs of specific primers were designed to detect N, P, M, F, H, or L sequences. To detect the genome and/or antigenome, two primers were chosen so as to amplify a 221 nt fragment (L-Tr) encompassing L gene end and trailer. Every gene-specific PCR assay was able to detect = 10 copies/sample, with a dynamic range of 4-5 log10 copies. No significant fluorescent signal was detected from non-infected cell cDNA template. When measles virus microccocal nuclease resistant genomic RNA was reverse transcribed, a 1:1 ratio was observed between single gene amplicons except for L-Tr which displayed a 2.6-fold excess over the other genes. This likely reflects the presence of some shorter abortive genome since the use of a plasmid encoding the entire virus genome resulted in 1:1 ratio for L-Tr segment when compared to others amplicons. Thus, this RT-QPCR assay appears suitable for follow-up studies of viral RNA populations during infection and may also be useful for reliable detection of measles virus in clinical samples.

Cell surface activation of the alternative complement pathway by the fusion protein of measles virus.

Measles virus (MV)-infected cells are activators of the alternative human complement pathway, resulting in high deposition of C3b on the cell surface. Activation was observed independent of whether CD46 was used as a cellular receptor and did not correlate with CD46 down-regulation. The virus itself was an activator of the alternative pathway and was covered by C3b/C3bi, resulting in some loss in infectivity without loss of virus binding to target cells. The cell surface expression of MV fusion (F), but not haemagglutinin, envelope protein resulted in complement activation of the Factor B-dependent alternative pathway in a dose-dependent manner and F-C3b complexes were formed. The underlying activation mechanism was not related to any decrease in cell surface expression of the complement regulators CD46 and CD55. The C3b/C3bi coating of MV-infected cells and virus should ensure enhanced targeting of MV antigens to the immune system, through binding to complement receptors.