Temperature-gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein-nucleic acid interactions. A linear and highly reproducible temperature-gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature-gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand-separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double-stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (-)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch-formation during the renaturation of (+)- and (-)strands from different clones. Examples are given for different viroid clones and HIV-clones from one and the same patient. In another example, clones with point mutations from site-directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the repressor and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.

Human parvovirus 4 has been considered to be transmitted only parenterally. However, after novel genotype 3 of parvovirus 4 was found in 2 patients with no parenteral risks, we tested infants in Ghana. A viremia rate of 8.6% over 2 years indicates that this infection is common in children in Africa.

BackgroundHuman enterovirus (EV) and parechovirus (HPeV) are significant causes of encephalitis and meningitis in children. The aim of this study was to determine the prevalence, type and viral RNA concentration of EV and HPeV in cerebrospinal fluid (CSF) samples in an unselected cohort of patients <18 years admitted to Bonn university hospital from 1998 to 2008.MethodsA total of 327 CSF samples from 327 patients were retrospectively tested by real-time reverse-transcriptase PCR (RT-PCR) for EV and HPeV, and by real-time PCR for cytomegalovirus (CMV), herpes simplex virus 1/2 (HSV), and varizella zoster-virus (VZV). Samples had been submitted for routine virological work-up due to suspected meningitis or encephalitis and had been stored at −20 °C hereafter. Positive samples for EV and HPeV were sequenced within the gene encoding the VP1 region (EV), the VP2 and the VP3/VP1 junction region (HPeV).ResultsThe overall prevalence was 4.3 % (14/327) for EV, 0.6 % (2/327) for HPeV, and 0.3 % (1/327) for HSV and VZV, respectively. CMV was not detected in this cohort. In children less than 3 months of age the prevalence was 7.7 % (2/26) for EV and 7.7 % (2/26) for HPeV, respectively. Frequency of EV detection ranged from 0 to 12 % per year and highest rates were observed from June to September. All typed EV belonged to species B. Both HPeV infections were detected in the fall of 2008 and were typed as HPeV genotype 3. Viral RNA concentrations were highest in patients with HPeV infection, followed by echovirus 30 and other EV. In total, 86 % (12/14) of EV infections presented as aseptic meningitis, whereas both HPeV infections presented as severe sepsis-like illness.ConclusionsEV and HPeV were equally prevalent in children <3 months of age. Beyond the detection of EV and HPeV, the determination of viral RNA concentration and typing of EV and HPeV might prove beneficial for patient management and public health.

Of 44 children born to human immunodeficiency virus (type 1) (HIV)-infected mothers, 11 have become seronegative. After the loss of maternal antibodies all children were analysed for several immunological functions and virological parameters in order to determine their HIV status. All children to date are clinically healthy and have normal immune functions. HIV-1 was detected by p24 antigen in one child, by in situ hybridization in nine children while viral cultures were all negative. These data suggest that the rate of vertical transmission of HIV-1 may be underestimated if seronegative children are considered to be not infected. They also suggest that molecular biological techniques are more sensitive than HIV antigen assay or viral cultures.