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Honey bee viruses are one of the most important pathogens that have contributed to the decrease in honey bee colony health. To analyze the infection dynamics of honey bee viruses, quantification of viral gene expression by RT-qPCR is necessary. However, suitable reference genes have not been reported from viral and RNAi studies of honey bee. Here, we evaluated the expression of 11 common reference genes ( ache 2,  rps 18, β -actin ,  tbp ,  tif ,  rpl 32,  gadph ,  ubc , α -tubulin ,  rpl14 , and  rpsa ) from  Apis mellifera  (Am) and  Apis cerana  (Ac) under Israeli acute paralysis virus (IAPV), chronic bee paralysis virus (CBPV), and Chinese sacbrood virus (CSBV) infection as well as dsRNA-PGRP-SA treatment, and we confirmed their validation by evaluating the levels of the  defensin 1  and prophenoloxidase ( ppo ) genes during viral infection. Our results showed that the expression of selected genes varied under different viral infections.  ache 2,  rps 18, β -actin ,  tbp , and  tif  can be used to normalize expression levels in  Apis mellifera  under IAPV infection, while the combination of  actin  and  tif  is suitable for CBPV-infected experiments. The combination of  rpl 14,  tif ,  rpsa ,  ubc , and  ache 2 as well as more reference genes is suitable for CSBV treatment in  Apis cerana .  Rpl 14,  tif ,  rps 18,  ubc , and α -tubulin  were the most stable reference genes under dsRNA treatment in  Apis mellifera . Furthermore, the geNorm and NormFinder algorithms showed that  tif  was the best suitable reference gene for these four treatments. This study screened and validated suitable reference genes for the quantification of viral levels in honey bee, as well as for RNAi experiments.

Screening and Validation of Reference Genes for RT-qPCR Under Different Honey Bee Viral Infections and dsRNA Treatment