Abstract
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.
Highlights
Fever virus, and TBP and PPI were the most stable reference genes[15]
When Spodoptera frugiperda cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the results indicated that ECD was a reliable reference gene for RT-qPCR and was better than 28S as a reference gene for these experiments[16]
In various experimental settings and different tissues, rRNA genes were unsuitable as references gene because their transcription was significantly regulated19. 18S RNA and ACT have been commonly employed as reference genes in Hymenoptera studies[16, 20], a suitable and stable reference gene was significant for the calibration of the qRT-PCR data
Summary
When Spodoptera frugiperda cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the results indicated that ECD was a reliable reference gene for RT-qPCR and was better than 28S as a reference gene for these experiments[16]. PPIA was set as the single, most-optimal internal reference gene for Israeli Acute Paralysis Virus (IAPV) infection experiments in Bombus terrestris[18]. Iap-like[1] and iap-like[2] in HvAV-3h were chosen as the target genes which in order to better verify the stability of the optimal internal gene predicted by the different algorithms and softwares. The results could be used as internal controls in mRNA expression studies in ascovirus-infected S. exigua larvae, fat body cells (IOZCAS-Spex-II-A), and the parasitic wasp M. similis
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