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

<p><span>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 <em>in vivo</em> in the larval gut is the lack of infection synchronization compared to cultured cells. Working with bertha armyworm, <em>Mamestra configurata</em>, larvae infected with the baculovirus Mamestra configurata nucleopolyhedrovirus-A<em> </em><span>&nbsp;</span>(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 analyzing baculovirus gene expression <em>in vivo</em> 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.</span></p>