BK polyomavirus with archetypal and rearranged non-coding control regions is present in cerebrospinal fluids from patients with neurological complications.

BK polyomavirus (BKPyV) has recently been postulated as an emerging opportunistic pathogen of the human central nervous system (CNS), but it is not known whether specific strains are associated with the neurotropic character of BKPyV. The presence of BKPyV large T-antigen DNA was examined in 2406 cerebrospinal fluid (CSF) samples from neurological patients with suspected JC polyomavirus infection. Twenty patients had a large T-antigen DNA-positive specimen. The non-coding control region (NCCR) of the BKPyV strains amplified from CSF from these 20 patients, strains circulating in renal and bone marrow transplant recipients and from healthy pregnant women was sequenced. The archetypal conformation was the most prevalent in all groups and 14 of the neurological patients harboured archetypal strains, while the remaining six patients possessed BKPyV with rearranged NCCR similar to previously reported variants from non-neurological patients. Transfection studies in Vero cells revealed that five of six early and four of six late rearranged promoters of these CSF isolates showed significantly higher activity than the corresponding archetypal promoter. From seven of the neurological patients with BKPyV DNA-positive CSF, paired serum samples were available. Five of them were negative for BKPyV DNA, while serum from the remaining two patients harboured BKPyV strains with archetypal NCCR that differed from those present in their CSF. Our results suggest that NCCR rearrangements are not a hallmark for BKPyV neurotropism and the dissemination of a rearranged NCCR from the blood may not be the origin of BKPyV CNS infection.

Development of a one step real-time RT-PCR method for sensitive detection of human astrovirus.

Human astrovirus (HAstV) has been recognized as the second most common cause of diarrhoea among children under 5 years old. To date, the true incidence of HAstV was underestimated when using enzyme immunoabsorbent assays (EIAs) and conventional reverse transcription (RT)-polymerase chain reaction (PCR) methods. The sensitivity of detection of EIA is insufficient and, although RT-PCR is more sensitive than EIA, the time required is a limitation for astrovirus detection. The aim of the study was to develop a real-time RT-PCR method in order to increase the sensitivity, to quantify the viral load and to minimize the time required for HAstV detection. The real-time RT-PCR reported here requires only one rapid step to obtain a high sensitivity (0.0052 infectious units (IU) (0.0026 IU/microl)) in all human astrovirus detected. The real-time RT-PCR detected IUs down to a 10(-6) dilution with an improvement in the detection limit of factor 10(4), whereas the conventional RT-PCR detected down to IUs 10(-2) dilution. This process is able to reduce the time of the assay and avoids the risk of contamination. The method described below has been validated with a panel of 100 clinical samples and the results obtained confirmed the high specificity of the assay; consequently, the application of this assay for molecular diagnosis is feasible as a versatile tool for ascertaining the true implication of HAstV in acute viral gastroenteritis.

Development and validation with clinical samples of internally controlled multiplex real-time PCR for diagnosis of BKV and JCV infection in associated pathologies.

This article describes the development and validation with clinical samples of an internally controlled multiplex quantitative real-time PCR (QRT-PCR) for human polyomaviruses BK (BKV) and JC (JCV). Blood and urine samples from renal transplant recipients with suspected nephropathy, and cerebrospinal fluid (CSF) specimens from AIDS, natalizumab-treated and HIV-negative patients with suspected progressive multifocal leukoencephalopathy, previously checked for BKV and JCV by conventional PCR, were tested by QRT-PCR. All samples positive by conventional PCR were confirmed by QRT-PCR. Four cases of JCV-associated neurological infection, including all those detected in natalizumab-treated patients, and one case of BKV-related neurological infection were only identified by QRT-PCR. BKV was quantified in the CSF of neurological patients for the first time. Analyses of the Quality Control for Molecular Diagnostics 2010 panel were "highly satisfactory" for BKV and "satisfactory" for JCV. The QRT-PCR is specific and reproducible. It improves the sensitivity of conventional PCR for the diagnosis of BKV and JCV infection in various diseases.

Mumps virus diagnosis and genotyping using a novel single RT-PCR.

BACKGROUND: IgM detection is considered as the gold standard for mumps diagnosis. Currently, most cases in developed countries occur in highly vaccinated populations due to secondary vaccine failure. In these patients, pre-existing vaccine-induced antibodies are not able to neutralise the virus, but prevent the typical primary response, so that specific IgM is not always elicited. Consequently, acute infection has to be demonstrated by direct detection of the virus by viral isolation or genomic amplification. RT-PCR allows a diagnosis with the maximum sensitivity to be made and also forms the basis for genotype characterisation by sequencing the SH gene, according to WHO recommendations. However, none of the RT-PCR techniques properly evaluated for the diagnosis of acute mumps infection yields an amplification fragment useful for genotyping, and none of the amplification techniques described for genotyping has proved to be sensitive enough for diagnosis. OBJECTIVES: Development of a RT-PCR for the mumps virus diagnosis and genotyping, properly evaluated in comparison with serological gold-standard technique. STUDY DESIGN: 195 suspected mumps cases and six wild type MuV genotypes were studied. RESULTS: Our method was able to detect 0.001 TCID(50) of mumps virus. Fifty-eight of these showed positive results, of which 54 (93.3%) showed mumps RNA in saliva, while only 20 (34.5%) had mumps IgM in serum. Genotypes G1, G2, H1, H2, D1 and C were identified in positive samples. CONCLUSIONS: The technique described could be a very useful tool for mumps surveillance, management and control.

Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form.

The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus-cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26 Delta C), followed by purification by NTA-Ni(2+) agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies.