Appl Microbiol Biotechnol (2010) 86:1067–1075 1073 Fig. 4 Internalization of scFv3G1. Vero E6 cells infected with HTNV for 10 days were incubated with FITC-labeled scFv3G1 or FITC-labeled MAb3G1 in the absence (a) and in the presence (b) of Texas red-labeled transferrin as an indicator of clathrin-dependent endocytosis at 37°C for 30 min to allow internalization. Nuclei were counterstained with DAPI. Immunofluorescence was observed under confocal microscopy. The colocalization of the scFv3G1 antibody with clathrin-coated endocytic vesicles are indicated by arrows. Scale bar 20 μm scFv3G1 internalization is mediated by a clathrin-dependent endocytotic pathway To further investigate the internalizing pathway of this scFv, Vero E6 cells preinfected with HTNV were incubated with FITC-tagged scFv3G1 and Texas red-tagged transfer- rin, as an indicator of clathrin-dependent endocytosis. As shown in Fig. 4b, merged staining of scFv3G1 and transferrin revealed a colocalization of the recombinant antibody with clathrin-coated endocytic vesicles. Thus, the internalization of scFv3G1 is likely achieved through clathrin-dependent endocytosis. Taken together, these results suggest that the recombinant single-chain antibody, scFv3G1, possesses the HTNV GP-specific binding activity, and subsequent internalization through receptor endocytosis may allow targeted and intracellular delivery of therapeutics against HTNV infection. Discussion Hantaviruses are enveloped, negative-sense RNA viruses, which can cause human infection after inhalation of aerosols (feces, saliva, and urine) from rodent hosts, and