Efficient blue conversion from a 1064 nm microchip laser in long photonic crystal fiber tapers for fluorescence microscopy.

Using a low-cost microchip laser and a long photonic crystal fiber taper, we report a supercontinuum source with a very efficient visible conversion, especially in the blue region (around 420 nm). About 30 % of the total average output power is located in the 350-600 nm band, which is of primary importance in a number of biophotonics applications such as flow cytometry or fluorescence imaging microscopy for instance. We successfully demonstrate the use of this visible-enhanced source for a three-color imaging of HeLa cells in wide-field microscopy.

WELYSSA: a simple tool using mouse monoclonal antibodies for the detection of lyssavirus nucleocapsid in rabies suspected specimens.

A Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assay (ELISA) named WELYSSA was developed for the diagnosis of rabies suspected specimens using prototype viruses from the different genotypes of lyssavirus and from various geographic origins and phylogenetic lineages. It included a panel of 1,660 specimens received for rabies diagnostic testing, and was found to be highly specific (99.9%) and sensitive (97.0%) when compared to other recommended rabies diagnostic methods.

Multicenter comparative study of a new ELISA, PLATELIA RABIES II, for the detection and titration of anti-rabies glycoprotein antibodies and comparison with the rapid fluorescent focus inhibition test (RFFIT) on human samples from vaccinated and non-vaccinated people.

The envelope glycoprotein G of rabies virus induces the production of neutralising antibodies, which are important in protection against rabies. Therefore, titration of anti-envelope glycoprotein antibodies is a good indicator of the degree of immunity in people during anti-rabies treatment or after vaccination. According to the World Health Organization (WHO) guidelines, a booster vaccine dose should be given if the rabies antibody titre falls below 0.5 IU/ml. Titration of anti-rabies antibodies is also useful for plasma centers in the preparation and standardization of human anti-rabies gamma-globulins for therapeutic use and to a lesser extent for the diagnosis of rabies in human sera and cerebrospinal fluid (CSF). This paper presents a new enzyme-linked immunosorbent assay (ELISA), PLATELIA RABIES II, developed for rabies envelope glycoprotein antibody detection or titration and its comparison to the current reference method (RFFIT). The data collected during validation of the test in a multicenter study are analysed to give a sound overall knowledge of the capabilities of the PLATELIA RABIES II, for instance specificity, linearity, accuracy, precision, detection limit and quantitation limit. To this aim, human serum samples from a total of 1348 vaccinated or non-vaccinated people were tested in parallel using the new ELISA and the RFFIT for the presence of anti-rabies antibodies. Data generated indicate a linear relationship across the range of titration between the two methods. The sensitivity reaches 98.6% and the specificity 99.4%. This study indicates that this new ELISA test is as sensitive and specific as the current standardized reference method. The method is simple, safe, rapid and can be considered as a useful alternative to the neutralisation test.

Intravitam diagnosis of human rabies by PCR using saliva and cerebrospinal fluid.

An optimized reverse transcription (RT)-PCR protocol for the intravitam detection of rabies virus genomic RNA was tested with clinical samples obtained from 28 patients suspected of having rabies, 9 of whom were confirmed to have had rabies by postmortem examination. RT-PCR using saliva combined with an immunofluorescence assay performed with skin biopsy samples allowed detection of rabies in the nine patients.

Dynamics of rabies virus quasispecies during serial passages in heterologous hosts.

To understand the mutations and genetic rearrangements that allow rabies virus infections of new hosts and adaptation in nature, the quasispecies structure of the nucleoprotein and glycoprotein genes as well as two noncoding sequences of a rabies virus genome were determined. Gene sequences were obtained from the brain and from the salivary glands of the original host, a naturally infected European fox, and after serial passages in mice, dogs, cats and cell culture. A relative genetic stasis of the consensus sequences confirmed previous results about the stability of rabies virus. At the quasispecies level, the mutation frequency varies, in the following order: glycoprotein region (21.9 x 10(-4) mutations per bp), noncoding sequence nucleoprotein-phosphoprotein region (7.2-7.9 x 10(-4) mutations per bp) and nucleoprotein gene region (2.9-3.7 x 10(-4) mutations per bp). These frequencies varied according to the number, type of heterologous passages and the genomic region considered. The shape of the quasispecies structure was dramatically modified by passages in mice, in which the mutation frequencies increased by 12-31 x 10(-4) mutations per bp, depending on the region considered. Non-synonymous mutations were preponderant particularly in the glycoprotein gene, stressing the importance of positive selection in the maintenance and fixation of substitutions. Two mechanisms of genomic evolution of the rabies virus quasispecies, while adapting to environmental changes, have been identified: a limited accumulation of mutations with no replacement of the original master sequence and a less frequent but rapid selective overgrowth of favoured variants.

Presence of European bat lyssavirus RNas in apparently healthy Rousettus aegyptiacus bats.

Apparently healthy Rousettus aegyptiacus bats were randomly chosen from a Dutch colony naturally infected with European bat lyssavirus subgenotype 1a (EBL1a). These bats were euthanised three months after the first evidence of an EBL1a infection in the colony. EBL1a genomic and antigenomic RNAs of the nucleoprotein gene were detected by nested reverse transcriptase PCR in 75% of the examined Rousettus aegyptiacus bats. The EBL1a RNAs of the nucleoprotein gene were detected mainly in brain tissues, but also in other organs. EBL1a messenger RNAs of the nucleoprotein gene and the glycoprotein gene were detected in brain tissues. The standard fluorescent antibody test revealed the presence of lyssavirus antigens in brain tissues from 7 (17.5%) Rousettus aegyptiacus bats. Furthermore, EBL1a could not be detected by virus isolation on murine neuroblastoma cells or by intracerebral inoculation of suckling mice. Neutralising antibodies directed against EBL1 were detected in 11% of the examined bats. This study shows that at least 85% of the apparently healthy Rousettus aegyptiacus bats must have been infected with EBL1a, and that these bats may survive from an EBL1a infection. Furthermore, the study supports the possibility of a long-term maintenance of EBL1a genome in Rousettus aegyptiacus bats.