The selected primer pairs are listed in Table 1. Primers used for the Sibutramine HCl detection of rhinoviruses have been previously described and were found to be superior to any other assay. At present, the most sensitive and specific methods for detection, typing and identification of viruses are based on molecular biology techniques. Numerous molecular assays have been described, though in silico analysis reveals that the majority of these methods are based on the sequence of only a single viral isolate, and that their performance in laboratory settings is limited. In the current study, a broad and sensitive panel of assays based on a two-step nested PCR was developed for the detection of a majority of human respiratory viruses. Selected primer sets, designed based on complete GenBank information, are complementary to the Magnoflorine-iodide sequences of archival and contemporary viral strains. Such an approach limits the influence of intra�Cspecies sequence variability and facilitates the detection of contemporary and newly emerging strains. The developed assays may thus be considered not only as detection techniques but also as a basic tool for virus discovery. The main disadvantage of developed tests is that detection is limited to viruses that belong to specific viral families/subfamilies, and they cannot be used for all viruses. Furthermore, the diversity of viral species in combination with their low load in clinical samples may result in false-negatives. This particular feature makes these assays inferior to sequence-independent methods, which can detect unknown pathogens. Then again, assays based on degenerated primers offer far higher sensitivity than those based on sequence-independent detection techniques. The detection of unknown viral pathogens in respiratory clinical material is difficult using sequence-independent virus discovery methods, as the low viral load and high background signal from cellular nucleic acids frequently hinders detection. Until now, sequence independent virus discovery techniques were mostly used for virus culture supernatants, or for the discovery of previously unknown DNA viruses. Thus far, no study has identified a novel human respiratory RNA virus using sequence independent amplification techniques. Even though several improvements have been proposed, the sensitivity of these methods is inferior to that of nested or real-time PCR. To test whether it was possible to type the infectious agents in the remaining samples, molecular assays were developed and coupled with ex vivo cell culture techniques based on fully differentiated human airway epithelium.