Viral nucleic acids were extracted from respiratory secretions using RNAzol (Biotecx, Houston) as previously described.12,18,24 Primers used for amplification were as fol-lows: complementary DNA (cDNA) synthesis (R1) 5′-ACGGACACCCAAAGTA-3′, upstream primer (R2) 5′-AGCACTTCTGTTTCCC-3′ for picornavirus25; cDNA synthesis (FAM1) 5′-CAGAGACTTGAAGATGTCTTTGC-3′, upstream primer (FAM2) 5′-GCTCTGTCCATGTTATTTG(GA)AT-3′ for influenza virus type A26,27; and cDNA synthesis (CVP3) 5′-IIAAATTGCTIITCTTGTTCTGGC-3′ (I indicates inosine), downstream primer (CVP4) 5′-CCAAAATTCTGATTAGGGCCTCTC-3′ for coronavirus OC43.28 Complementary DNA was synthesized for 1 hour at 43°C in a reaction mix containing 10-mmol/L tromethamine hydrochloride (pH, 8.3), 50-mmol/L potassium chloride, 1.5-mmol/L magnesium chloride, 3.3-µmol/L cDNA synthesis primer, 667-µmol/L deoxynucleoside triphosphates, 20 U of RNase inhibitor (RNasin; Promega Corporation, Madison, Wis), and 5 U of avian myeloblastosis virus reverse transcriptase (Life Sciences, Inc, St Petersburg, Fla). The PCR amplification was performed in a solution containing 10-mmol/L tromethamine hydrochloride (pH, 8.3), 50-mmol/L potassium chloride, 1.5-mmol/L magnesium chloride, 1-µmol/L each of the cDNA synthesis and upstream primers, 200-µmol/L deoxynucleoside triphosphates, and 5 U of Taq polymerase (Perkin-Elmer Corporation, Norwalk, Conn) using a thermal cycler (PTC-100, MJ Research, Inc, Cambridge, Mass). After an initial 4-minute heat denaturation at 94°C, 40 cycles of heat denaturation at 94°C for 1 minute, primer annealing at 49°C (picornavirus), 55°C (influenza virus type A), or 58°C (coronavirus) for 1 minute 30 seconds, and primer extension at 72°C for 1 minute were followed by a final primer extension step at 72°C. Amplified products were 394, 212, and 186 base pairs in length for picornavirus, influenza virus type A, and coronavirus OC43, respectively. Positive results were confirmed by slot-blot hybridization using the following digoxigenin-labeled oligonucleotides27: 5′-TCCTCCGGCCCCTGAATG-3′ (R4) for picornavirus; 5′-TCCTGTCACCTCTGACTAAGGGGATTTTG-3′ (AH2) for influenza A virus; and 5′-AAGCAIAITGCCAAAIAAGTCAGICAGAAAATTTT-3′ (CVPP) for coronavirus OC43.