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Maize fine streak virus (MFSV), an emerging Rhabdovirus sp. in the genus Nucleorhabdovirus, is persistently transmitted by the black-faced leafhopper, Graminella nigrifrons (Forbes). MFSV was transmitted to maize, wheat, oat, rye, barley, foxtail, annual ryegrass, and quackgrass by G. nigrifrons. Parameters affecting efficiency of MFSV acquisition (infection) and transmission (inoculation) to maize were evaluated using single-leafhopper inoculations and enzyme-linked immunosorbent assay. MFSV was detected in ≈20% of leafhoppers that fed on infected plants but <10% of insects transmitted the virus. Nymphs became infected earlier and supported higher viral titers than adults but developmental stage at aquisition did not affect the rate of MFSV transmission. Viral titer and transmission also increased with longer post-first access to diseased periods (PADPs) (the sum of the intervals from the beginning of the acquisition access period to the end of the inoculation access period). Length of the acquisition access period was more important for virus accumulation in adults, whereas length of the interval between acquisition access and inoculation access was more important in nymphs. A threshold viral titer was needed for transmission but no transmission occurred, irrespective of titer, with a PADP of <4 weeks. MFSV was first detected by immunofluorescence confocal laser scanning microscopy at 2-week PADPs in midgut cells, hemocytes, and neural tissues; 3-week PADPs in tracheal cells; and 4-week PADPs in salivary glands, coinciding with the time of transmission to plants.