AUTHOR=Deng Yanchun , Zhao Hongxia , Yang Sa , Zhang Li , Zhang Lina , Hou Chunsheng 

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.01715

DOI=10.3389/fmicb.2020.01715

ISSN=1664-302X

ABSTRACT=<p>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 (<italic>ache</italic>2, <italic>rps</italic>18, β<italic>-actin</italic>, <italic>tbp</italic>, <italic>tif</italic>, <italic>rpl</italic>32, <italic>gadph</italic>, <italic>ubc</italic>, α<italic>-tubulin</italic>, <italic>rpl14</italic>, and <italic>rpsa</italic>) from <italic>Apis mellifera</italic> (Am) and <italic>Apis cerana</italic> (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 <italic>defensin 1</italic> and prophenoloxidase (<italic>ppo</italic>) genes during viral infection. Our results showed that the expression of selected genes varied under different viral infections. <italic>ache</italic>2, <italic>rps</italic>18, β<italic>-actin</italic>, <italic>tbp</italic>, and <italic>tif</italic> can be used to normalize expression levels in <italic>Apis mellifera</italic> under IAPV infection, while the combination of <italic>actin</italic> and <italic>tif</italic> is suitable for CBPV-infected experiments. The combination of <italic>rpl</italic>14, <italic>tif</italic>, <italic>rpsa</italic>, <italic>ubc</italic>, and <italic>ache</italic>2 as well as more reference genes is suitable for CSBV treatment in <italic>Apis cerana</italic>. <italic>Rpl</italic>14, <italic>tif</italic>, <italic>rps</italic>18, <italic>ubc</italic>, and α<italic>-tubulin</italic> were the most stable reference genes under dsRNA treatment in <italic>Apis mellifera</italic>. Furthermore, the geNorm and NormFinder algorithms showed that <italic>tif</italic> 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.</p>