Microscopic investigation of AcMNPV infection in the Trichoplusia ni midgut

[Display omitted] •AcMNPV recombinants, Ac-ie1GFP and Ac-ie1GFP+VP39-mCherry fusion, developed for midgut infection study.•At 24hpi single infected midgut cells expressing GFP detected.•At 48hpi cell-to-cell spread of AcMNPV infection detected in midgut.•AcMNPV GP64 is required for cell-to-cell spread infection in midgut. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species for the genus Alphabaculovirus in the family Baculoviridae. In nature, AcMNPV infection begins with ingestion of viral occlusion bodies (OBs) from which occlusion-derived viruses (ODV) are released to infect midgut cells. This study explored the early stages of Trichoplusia ni midgut infection using recombinant viruses expressing green fluorescent protein (GFP) and/or a VP39-mCherry fusion protein under the control of early and late promoters, respectively. Using a recombinant ie1:GFP virus, the anterior midgut region was identified as the predominant site for primary infection. Infection of midguts using the GFP-VP39mCherry-dual labelled recombinant virus revealed that active viral replication and cell-to-cell spread was required for the formation of infection foci and the subsequent spread to uninfected midgut cells and tracheoblasts. The spread of the infection from primary infected cells to secondary cells within the midgut was shown to be dependent upon the membrane fusion protein GP64.