Detection of bornavirus-reactive antibodies and BoDV-1 RNA only in encephalitis patients from virus endemic areas: a comparative serological and molecular sensitivity, specificity, predictive value, and disease duration correlation study.

PURPOSE: Human Borna disease virus (BoDV-1) encephalitis is an emerging disease in Germany. This study investigates the spectrum of human BoDV-1 infection, characterizes anti-BoDV-1-antibodies and kinetics, and compares laboratory test performances. METHODS: Three hundred four encephalitis cases, 308 nation-wide neuropsychiatric conditions, 127 well-defined psychiatric cases from Borna disease-endemic areas, and 20 persons with contact to BoDV-1 encephalitis patients or animals were tested for BoDV-1 infections by serology and PCR. RESULTS: BoDV-1 infections were only found in encephalitis patients with residence in, or recent travel to, virus-endemic areas. Antibodies were detected as early as 12 days after symptom onset. Serum antibody levels correlated with disease duration. Serology was ordered after 50% of the disease duration had elapsed, reflecting low awareness. BoDV-1-antibodies were of IgG1 subclass, and the epitope on BoDV-1 antigens was determined. Specificity of the indirect immunofluorescence antibody test (IFAT) and lineblot (LB) from serum and cerebrospinal fluid (CSF), as well as PCR testing from CSF, was 100%. Sensitivity, depending on first or all samples, reached 75-86% in serum and 92-94% in CSF for the IFAT, and 33-57% in serum and 18-24% in CSF for the LB. Sensitivity for PCR in CSF was 25-67%. Positive predictive values were 100% each, while negative predictive values were 99% (IFAT), 91-97% (LB), and 90% (PCR). CONCLUSIONS: There is no hint that BoDV-1 causes other diseases than encephalitis in humans. Awareness has to be increased in virus-endemic areas. Tests are robust but lack sensitivity. Detection of IgG1 against specific peptides may facilitate diagnosis. Screening of healthy individuals is likely not beneficial.

Full genome sequence analysis of parechoviruses from Brazil reveals geographical patterns in the evolution of non-structural genes and intratypic recombination in the capsid region.

Due to high genome plasticity, the evolutionary fate and geographical history of picornaviruses is hard to follow. Here, we determined the complete coding sequences of eight human parechoviruses (HPeV) of types 1, 5 and 6 directly from clinical samples from Brazil. The capsid genes of these strains were not remarkably different from European, North American and Japanese HPeV. Full genome analysis revealed frequent intertypic recombination in the non-structural genome region. In addition, evidence of recombination between viruses of the same type in the capsid-encoding genome region among HPeV1 and HPeV4 was obtained. Bayesian phylogenetic analysis indicated that strains without evidence of recombination with each other in any genome region were separated by no more than 35 years of circulation. Interestingly, in the 3C gene, all Brazilian parechoviruses grouped together regardless of serotype. The most recent common ancestor of these strains dated back 108 years, suggesting long-term endemicity of this particular P3 genome lineage in South America. Our results support the idea that picornavirus replicative genes acquire capsid proteins introduced by new strains. Under certain epidemiological conditions, replicative genes may be maintained in circumscript geographical regions.

Genomic features and evolutionary constraints in Saffold-like cardioviruses.

This study identified the complete genomic sequence of four type 2 and type 3 human Saffold-like cardioviruses (SLCVs) isolated in Germany and Brazil. The secondary structures of the SLCV internal ribosome entry sites (IRESs) were deduced based on RNA base-pairing conservation and co-variation, using an established Theiler's murine encephalomyelitis virus (TMEV) IRES structure as a reference. The SLCV IRES was highly similar to that of TMEV, but motifs critical in TMEV for binding of the polypyrimidine tract-binding protein (PTB) were disrupted. In TMEV, corresponding alterations have been associated with reduced neurovirulence in mice. In the non-structural genome region, there was evidence of multiple intertypic recombination events between different SLCV types. Between viruses of the same type, recombination also occurred in the capsid-encoding genome region. There were apparently no recombination events between mouse TMEV and human SLCV. In another genus of the family Picornaviridae, Enterovirus, natural recombination occurs strictly within species and can serve as an additional criterion for delimiting species. Accordingly, the results of this study suggest that SLCV and TMEV may represent distinct species within the genus Cardiovirus.

Novel human parechovirus from Brazil.

Human parechoviruses (HPeVs) were detected by reverse transcription-PCR in 16.1% of 335 stool samples from children <6 years of age with enteritis in Salvador, Brazil. Whole genome sequencing of 1 sample showed a novel HPeV that has been designated as HPeV8.

A novel diagnostic target in the hepatitis C virus genome.

BACKGROUND: Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (5'-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays. METHODS AND FINDINGS: In this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties. CONCLUSION: This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.

Low-density macroarray for rapid detection and identification of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis which occurs throughout Africa, Eastern Europe, and Asia and results in an approximately 30% fatality rate. A reverse transcription-PCR assay including a competitive internal control was developed on the basis of the most up-to-date genome information. Biotinylated amplification products were hybridized to DNA macroarrays on the surfaces of polymer supports, and hybridization events were visualized by incubation with a streptavidin-horseradish peroxidase conjugate and the formation of a visible substrate precipitate. Optimal assay conditions for the detection of as few as 6.3 genome copies per reaction were established. Eighteen geographically and historically diverse CCHF virus strains representing all clinically relevant isolates were detected. The feasibility of the assay for clinical diagnosis was validated with acute-phase patient samples from South Africa, Iran, and Pakistan. The assay provides a specific, sensitive, and rapid method for CCHF virus detection without requiring sophisticated equipment. It has usefulness for the clinical diagnosis and surveillance of CCHF infections under limited laboratory conditions in developing countries or in field situations.

Detection and prevalence patterns of group I coronaviruses in bats, northern Germany.

We tested 315 bats from 7 different bat species in northern Germany for coronaviruses by reverse transcription-PCR. The overall prevalence was 9.8%. There were 4 lineages of group I coronaviruses in association with 4 different species of verspertilionid bats (Myotis dasycneme, M. daubentonii, Pipistrellus nathusii, P. pygmaeus). The lineages formed a monophyletic clade of bat coronaviruses found in northern Germany. The clade of bat coronaviruses have a sister relationship with a clade of Chinese type I coronaviruses that were also associated with the Myotis genus (M. ricketti). Young age and ongoing lactation, but not sex or existing gravidity, correlated significantly with coronavirus detection. The virus is probably maintained on the population level by amplification and transmission in maternity colonies, rather than being maintained in individual bats.

Circulation of 3 lineages of a novel Saffold cardiovirus in humans.

Cardioviruses cause serious disease, mainly in rodents, including diabetes, myocarditis, encephalomyelitis, and multiple sclerosis-like disseminated encephalomyelitis. Recently, a human virus isolate obtained 25 years ago, termed Saffold virus, was sequenced and classified as a cardiovirus. We conducted systematic molecular screening for Saffold-like viruses in 844 fecal samples from patients with gastroenteritis from Germany and Brazil, across all age groups. Six cardioviruses were identified in patients <6 years of age. Viral loads were 283,305-5,044,412,175 copies/g of stool. Co-infections occurred in 4 of 6 children. No evidence for outbreak-like epidemic patterns was found. Phylogenetic analysis identified 3 distinct genetic lineages. Viral protein 1 amino acids were 67.9%-77.7% identical and had a distance of at least 39.4% from known cardioviruses. Because closely related strains were found on 2 continents, global distribution in humans is suspected. Saffold-like viruses may be the first human cardiovirus species to be identified.

Use of a simulation game for HIV/AIDS education with pre-service teachers.

The article describes the use of a simulation game in HIV/AIDS education with pre-service teachers in Johannesburg, South Africa. The use of a simulation game, as novel experiential pedagogy, was an attempt to raise awareness about HIV and AIDS and to demonstrate that anyone can be at risk of HIV infection. Using a generic qualitative research design, the data were collected over a three-year period by way of video recordings of the simulation game, recordings of large and small group discussions afterwards, and via questionnaires and written reflections by the education students four weeks afterwards. Content analysis and discourse analysis led to the construction of three main themes. First, we found that the novelty factor of the simulation game for raising HIV/AIDS awareness was confirmed both during the game itself and after a period of time had elapsed. Second, in light of many education students' naivety about the intersection of biological, socio-cultural and economic issues at play in the spread of HIV, the game prompted more reflexivity about the disease and helped to broaden the participants' discussions. Lastly, the data revealed the disjuncture between theory and practice in HIV/AIDS education. We propose that in raising awareness of HIV and AIDS, educators should move towards more engaging and challenging pedagogies that address the learning needs of the 'new' generation of university students.

High throughput screening for spores and vegetative forms of pathogenic B. anthracis by an internally controlled real-time PCR assay with automated DNA preparation.

Human infections with Bacillus anthracis have become rare but in cases of intentional release, masses of samples would have to be expected. Current PCR assays for anthrax are appropriate for use in single cases, but they have not been formulated for high throughput screening. This article describes a high throughput real-time PCR for anthrax, including automated sample preparation without the need for pre-culturing of samples. The assay detects single copies of target gene. An internal control monitors the whole assay including sample preparation. The limit of detection in blood was 1,066 (95%CI, 741-1,739) copies/ml, corresponding to 4.4-32.3 organisms/ml. Using spore preparations, 20 colony-forming units (CFU) per sample could be detected reliably (0.8 CFU per PCR). The extraction procedures depleted viable spores from solution by factors of 10,000 (automated procedure) and >100,000 (conventional column procedure). One hundred and ten clinical and environmental specimens were retested, 50 of them sampled during a period of heightened anthrax awareness in 2001. A widely used assay yielded two false positive results (cross-reaction with B. cereus), while the new assay tested all samples negative. The internal control operated stable in all clinical samples. The assay is capable of testing for anthrax in the high throughput mode.

Virus detection and monitoring of viral load in Crimean-Congo hemorrhagic fever virus patients.

We developed a real-time reverse transcription--PCR that detected 1,164 copies/mL of Crimean-Congo hemorrhagic fever virus per milliliter of serum at 95% probability (probit analysis) and was 100% concordant with nested PCR on 63 samples from 31 patients with confirmed infection. Infected patients who died appeared to have higher viral loads; low viral loads correlated with IgG detection.