Distribution of RYSV within the CNS of N. cincticeps. (A-I) A dissected adult instar N. cincticeps CNS, immunolabeled with α-tubulin antibody (green) and examined using a confocal microscope. The N. cincticeps CNS consists of the brain (in the head), circum-esophagus connectives, subesophageal ganglion, thoracic ganglion, abdominal ganglion, and the nerve cord. Scale bars, 100 μm. (A-II) Transmission electron micrograph showing the transverse abdominal ganglion of adult instar N. cincticeps. Scale bar, 2 μm. (B-I) Virions matured and budded from the nucleus of the perineurium glial cell of abdominal ganglion. Scale bar, 200 nm. (B-II) Magnified image of the boxed area in (B-I). Scale bar, 100 nm. (B-III) RYSV particles budded from the nucleus and dispersed along the fiber networks in the neuropil of the thoracic ganglion. (B-IV) Magnified image of the boxed area in B-III. (C) The process of RYSV viral particles invading the axons of the CNS of N. cincticeps. The early infection stage is illustrated, depicting RYSV particles attached to the axon in (C-I) and RYSV particles enveloped with a membrane invading into axon in (C-II). The trafficking of RYSV viral particles through the center of the CNS axons (C-III,IV), and the RYSV particles attached to the axon membrane and released from the axons into the subperineurium glial cell in (C-V) and (C-VI). Scale bars, 100 nm. (D-I) Model of the transverse CNS in N. cincticeps. The entire ganglion is enclosed by the neural lamella and a perineurium glial cell. The subperineurium cells are located at the periphery, while the axons occupy the central core of the ganglion. Scale bar, 10 μm. (D-II) Model of RYSV viral particle trafficking in an axon of the N. cincticeps CNS. RYSV particles attach to the membrane and invade the axons. The interaction of the structural protein M with the tubulin in the axons results in the rapid delivery of the viral particles throughout the CNS. Scale bars, 200 nm. ag, abdominal ganglion; ax, axon; br, brain; cc, circum-esophagus connectives; cor, cortex; mit, mitochondria; mt, microtubule; n, nerve(s); nc, nerve cord; nl, neural lamella; nu, nucleus; pc, perineurium glial cells; seg, subesophageal ganglion; spc, subperineurial glial cells; tg, thoracic ganglion; Red arrows, EV, enveloped viral particles; Black arrows, NV, non-enveloped viral particles.