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Ascoviruses are double-stranded DNA viruses that mainly infect noctuid larvae, and are transmitted by the parasitoid wasp  Microplitis similis  Lyle. Ascovirus-parasitoids wasp-noctuid insects constitute the dissemination system. Selection of suitable reference genes for the dissemination system could play an important role in elucidating the pathogenic molecular mechanisms of ascovirus. Unfortunately, such studies on potential reference genes in the dissemination system of ascoviruses are lacking. In the present study, we evaluated 11 candidate reference genes:  β-actin1  ( ACT1 ),  β-actin2  ( ACT2 ),  elongation factor 1  ( EF1 ),  elongation factor 2  ( EF2 ),  ribosomal protein L10  ( L10 ),  ribosomal protein L17A  ( L17A ),  superoxide dismutase  ( SOD ),  28S ribosome  ( 28S ),  Tubulin  ( TUB ) and  18S ribosome  ( 18S ). The samples were originally from various virus concentrations and points-in-time of experimental treatments using RefFinder and four algorithms. The results showed that  EF1  was the most stable internal gene in  S. exigua  and  M. similis  and that  EF2  was the most stable in the IOZCAS-Spex-II-A cell line, and the stability of reference genes were confirmed via the expression levels of two inhibitor of apoptosis-like ( iap -like) genes from  Heliothis virescens  ascovirus 3 h (HvAV-3h). This study provides a crucial basis for future research that explores the molecular mechanisms of the pathogenesis of ascoviruses.

Selection of reference genes for expression analysis using RT-qPCR in the dissemination system of Heliothis virescens ascovirus 3 h (HvAV-3h)