The samples included in the study were diarrhea samples submitted to the Diagnostic Unit, Department of Virology, Erasmus Medical Center, Rotterdam, Netherlands, for diagnosis of gastrointestinal infections in 2007. Out of 1,025 samples screened by standard diagnostic assays for rota-, adeno-, astro-, noro-, entero-, and parechovirus, 165 stool samples were consistently found to be negative for these viruses. Of these 165 samples, 84 were available for additional studies. The percentage of patients with diarrhea of unknown etiology per month was determined, as was the percentage of patients per birth date. Data were compared using Student's t test, and differences were considered significant at a P of <0.05. In addition, 17 diarrhea samples (submitted to the Diagnostic Unit, Department of Virology, Erasmus Medical Center, Rotterdam, Netherlands, for diagnosis of gastrointestinal infections in 2007) in which a virus was previously detected were also available.
The 84 diarrhea samples were collected for the present study, made anonymous, annotated with VS numbers, and stored at −80°C until analyzed by random amplification of partially purified viral RNA, performed as described previously with modifications (1
). A total of 13 diarrhea samples were suspended in phosphate-buffered saline (PBS) until their weight was 10% of the volume and centrifuged at 14,000 rpm for 5 min. Supernatants were filtered through 0.45-μm spin filters (Ultrafree-MC; Millipore), after which Omnicleave endonuclease (Epicentre Biotechnologies) and magnesium chloride (Applied Biosystems) were added to final concentrations of 2 U/μl and 5 mM, respectively, to degrade DNA and RNA. Viral nucleic acids are generally not degraded by nucleases, as they are protected by stable protein capsids and sometimes also by a lipid envelope. Samples were incubated for 1 h at 37°C. RNA was extracted from half of the samples using the Nucleospin RNA XS kit (Machery-Nagel), according to the instructions of the manufacturer. First-strand synthesis was performed by mixing RNA with 1 μM primer FR26RV-N (5′-GCCGGAGCTCTGCAGATATCNNNNNN-3′) and each deoxynucleoside triphosphate (dNTP) at 0.5 mM, which mixture was incubated at 95°C for 5 min and chilled on ice. First-strand buffer (5×; Invitrogen), dithiothreitol (DTT) (Invitrogen), RNasin (Promega), and Superscript III reverse transcriptase (Invitrogen) were added to final concentrations of 1×, 5 mM, 2 U/μl, and 10 U/μl, respectively. The reaction mixture was incubated for 5 min at 25°C and 45 min at 50°C. After a denaturation step at 94°C for 3 min and chilling on ice, 2.5 units of 3′→5′ Exo−
Klenow DNA polymerase (New England Biolabs) was added, and the reaction mixture was incubated at 37°C for 1 h, followed by an enzyme inactivation step at 75°C for 10 min for second-strand synthesis. The reaction mix was used as a template in a 50-μl PCR mixture containing 1× AmpliTaq Gold buffer, 2.5 mM MgCl2
, each dNTP at 0.2 mM, 0.8 μM primer FR20RV (5′-GCCGGAGCTCTGCAGATATC-3′), and 2.5 U AmpliTaq Gold (Applied Biosystems). After 10 min at 94°C, 40 cycles of amplification (94°C for 60 s, 65°C for 60 s, 72°C for 2 min) and 1 cycle of elongation (72°C for 10 min) were performed. The PCR products were separated on an agarose gel, and fragments between 200 and 400 bp, 400 and 800 bp, and 800 and 1,500 bp were excised and purified using the Invisorb Spin DNA extraction kit (Invitek GmbH) according to the manufacturer's instructions. Products were cloned into pCR4-TOPO and introduced into chemically competent Escherichia coli
TOP-10 according to the manufacturer's instructions (Invitrogen).