Detection of viral antigens and isolation methods has long been used for the diagnosis of respiratory virus infections. The objective was to determine the ability of HuH7 cells to support the replication of prototype and wild strains of respiratory viruses. The cell culture-adapted strains of influenza viruses A and B, parainfluenza viruses 1-4, respiratory syncytial viruses A and B, both strains of the human metapneumoviruses, numerous rhinoviruses, most of the adenoviruses, coronaviruses 229E and OC43, and a number of enteroviruses (poliovirus type 3, coxsackie virus B1, echovirus type 30) replicate in HuH7. The kinetic study of the replication of influenza A and B viruses showed that there were infected cells in HuH7 and MDCK lines as early as 24 hr post-infection. However, the replication of influenza A and B viruses was more rapid and intense on MDCK cells than on HuH7 cells. During the three winters of 1999-2000, 2000-2001, and 2001-2002, of the 1,226 (23.3%) direct fluorescent assay-positive nasal aspirates from children admitted to hospital, 788 were positive for respiratory syncytial virus, 228 for influenza virus, 133 for parainfluenza virus, and 77 for adenovirus. Of the 4,032 direct fluorescent assay-negative nasal aspirates, 571 virus isolates were identified by using HuH7 cell culture; 272 rhinoviruses, 100 influenza viruses A and B, 85 enteroviruses, 40 adenoviruses, 35 coronaviruses, 31 parainfluenza viruses, and 10 respiratory syncytial viruses. Interestingly, 100/328 (30.5%) influenza viruses A and B, 40/189 (21.1%) adenoviruses, and 31/164 (19%) parainfluenza viruses type 1-3, not detected by direct fluorescent assay, were identified by isolation in HuH7 cell culture.