Synchronization of in vivo MacoNPV-A baculovirus infection by analysis of individual Mamestra configurata larval guts

Many studies have examined the gene expression of baculoviruses during host infection by infecting cultured insect host cells; however, only a few have attempted to characterize the interaction between baculoviruses and insect larvae, which more accurately models the virus-insect relationship. The greatest challenge in assessing gene expression profiles in vivo in the larval gut is the lack of infection synchronization compared to cultured cells. Working with bertha armyworm, Mamestra configurata, larvae infected with the baculovirus Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), viral gene expression was measured using droplet-digital PCR, showing that the rate of infection in individual insects varies extremely widely. Subsequent RNA-Seq of individual guts revealed that gene expression patterns were consistent in individuals with similar levels of viral gene expression. Therefore, the best approach for analysing baculovirus gene expression in vivo is to use the expression of an early viral gene as a means to select individuals that are closely matched in infection progress to produce the most synchronized infection cohorts for measuring gene expression profiles. In this way, a profile of gene expression was identified very early in infection that would have been masked by the activity of a few very robustly infected individuals present in sample pools made up of multiple individuals.