Plaque Reduction neutralization test (PRNT). Two-fold dilutions of test sera were made in 50 ml cell culture medium. Under biohazard level 4 conditions, each of the diluted sera were mixed with 50 ml of NiV diluted to generate ,30 plaque forming units and incubated for 30 min at 37uC. The pre-incubated virusantibody mix was added to Vero cell monolayers grown in 96 well plates and incubated for 30 min at 37uC when the inoculum was removed and replaced 150 ml of cell media. After incubation at 37uC for 24 h, the cells fixed in 100% ice-cold methanol and staining by indirect immunofluorescence assay as follows: The wells in the plate were blocked with BSA/PBS and stained with rabbit sera raised against the G protein of HeV, and goat antirabbit Alexa Fluor 488 conjugate (Invitrogen) diluted 1:1000 in blocking buffer. Viral plaques were visualized and counted, and neutralizing antibody titers were reported based on reduction in plaque count by 50% relative to the untreated control (PRNT 50 ). Antibody levels measured by Immunofluorescence assay (IFA). For IFA, NiV-specific total antibody levels were measured by using NiV G, F and M expressing 293T cells as target antigen. Thirty six hours post-transfection, the cells were harvested, fixed in paraformaldehyde, cytospun (Cytocentrifuge, Thermoscientific) on glass slides to obtain monolayered preparations and then stored at 4uC, and used as antigen within three weeks of preparation. On the day of use, the slides were washed in PBS, permeabilized with Triton-X-100 and blocked with BSA/PBS. After incubation with two fold dilutions of the test sera, the cell monolayers were washed and stained with Alexa fluor 488-conjugated goat anti-mouse antibody according the manufacturer's (Molecular Probes) instructions. Negative and positive controls were run in parallel with each batch.