Abstract Thousands of endoparasitoid wasp species in the families Braconidae and Ichneumonidae harbor “domesticated endogenous viruses” (DEVs) in their genomes. This study focuses on ichneumonid DEVs, named ichnoviruses (IVs), which derive from an unknown virus and produce virions in ovary calyx cells during the pupal and adult stages of female wasps. Females inject IV virions into host insects when laying eggs. Virions infect cells which express IV genes with functions required for wasp progeny development. IVs have a dispersed genome consisting of two genetic components: proviral segment loci that serve as templates for circular dsDNAs that are packaged into capsids, and genes from an ancestral virus controlling virion production. Because of the lack of homology with known viral genes, the molecular control mechanisms of IV genome are largely uncharacterized. We generated a chromosome-scale genome assembly for Hyposoter didymator and identified a total of 67 H. didymator ichnovirus (HdIV) loci distributed across the 12 wasp chromosomes. By analyzing genomic DNA levels, we found that all HdIV loci were locally amplified in calyx cells during the wasp pupal stage, suggesting the implication of viral proteins in DNA replication. We tested a candidate HdIV gene, U16 , encoding a protein with a conserved domain found in primases and which is transcribed in calyx cells during the initial stages of replication. Knockdown of U16 by RNA interference inhibited amplification of all HdIV loci, as well as HdIV gene transcription, circular molecule production and virion morphogenesis in calyx cells. Altogether, our results showed that viral DNA amplification is an early step of IV replication essential for virions production, and demonstrated the implication of the viral gene U16 in this process. Author Summary Parasitoid “domesticated endogenous viruses” (DEVs) provide a fascinating example of eukaryotes acquiring new functions through integration of a virus genome. DEVs consist of multiple loci in the genomes of wasps. Upon activation, these elements collectively orchestrate the production of virions or virus-like particles that are crucial for successful parasitism of host insects. Despite the significance of DEVs for parasitoid biology, the mechanisms regulating key steps in virion morphogenesis are largely unknown. In this study, we focused on the ichneumonid parasitoid Hyposoter didymator , which harbors an ichnovirus consisting of 67 proviral loci. Our findings reveal that all proviral loci are simultaneously amplified in ovary calyx cells of female wasps during the early pupal stage suggesting a hijacking of cellular replication complexes by viral proteins. We tested the implication of such a candidate, U16 , encoding a protein with a weakly conserved primase C-terminal domain. Silencing U16 resulted in inhibited viral DNA amplification and virion production, underscoring the key role of this gene for ichnovirus replication. This study provides evidence that genes involved in viral DNA replication have been conserved during the domestication of viruses in the genomes of ichneumonid wasps.