After successful isolation of the novel bunyavirus, we developed an indirect IFA to detect specific antibodies in patient serum specimens, as previously described [4] . In brief, monolayers of virus-infected Vero E6 cells showing CPE and containing novel bunyavirus RNA were harvested, and one volume of infected cells was mixed with 0.5 volumes of non-infected cells. The mixture was centrifuged at 1,000 x g for 10 minutes, after which cells were resuspended in 16PBS, spotted onto 12-well glass slides, and fixed with acetone for 10 minutes. Sera from patients with FTLS (including 285 acute-phase samples and 95 paired sera), patients with respiratory diseases (80 serum samples), and healthy subjects (50 serum samples) were applied to the cells. Samples (diluted 1:20 in PBS) were screened by first spotting 50 mL of each serum sample per well and incubating for 30 minutes at 37uC. After washing for 10 minutes in PBS, 20 mL of FITC-conjugated goat anti-human IgG (Sihuan Sci-Technics Company, Beijing, China) diluted 1:40 in buffer containing Evans blue was added to each well and incubated for 30 minutes. After washing, slides were mounted in glycerin and examined by immunofluorescence microscopy. A titer of 1:20 was considered positive. 