We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.

Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals suffering from respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.

Cytotoxic CD4+CD28− T cells form a rare subset in human peripheral blood. The presence of CD4+CD28− cells has been associated with chronic viral infections, but how these particular cells are generated is unknown. In this study, we show that in primary CMV infections, CD4+CD28− T cells emerge just after cessation of the viral load, indicating that infection with CMV triggers the formation of CD4+CD28− T cells. In line with this, we found these cells only in CMV-infected persons. CD4+CD28− cells had an Ag-primed phenotype and expressed the cytolytic molecules granzyme B and perforin. Importantly, CD4+CD28− cells were to a large extent CMV-specific because proliferation was only induced by CMV-Ag, but not by recall Ags such as purified protein derivative or tetanus toxoid. CD4+CD28− cells only produced IFN-γ after stimulation with CMV-Ag, whereas CD4+CD28+ cells also produced IFN-γ in response to varicella-zoster virus and purified protein derivative. Thus, CD4+CD28− T cells emerge as a consequence of CMV infection.

HIV-1-infected patients co-infected with HBV or HCV were at considerably higher risk of developing LEE when HAART was initiated compared with patients without co-infection, but it is usually not necessary to modify antiretroviral therapy.