Novel ELISA-based endpoint dilution protocol to quantify infectious virus titer.
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Negative strand specific RT-qPCR assays to quantify replicative viral RNA.
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Simultaneous application of new methods in individual flies.
Abstract
Quantification of viral replication underlies investigations into host-virus interactions. In Drosophila melanogaster, persistent infections with Drosophila C virus, Drosophila A virus, and Nora virus are commonly observed in nature and in laboratory fly stocks. However, traditional endpoint dilution assays to quantify infectious titers are not compatible with persistently infecting isolates of these viruses that do not cause cytopathic effects in cell culture. Here we present a novel assay based on immunological detection of Drosophila C virus infection that allows quantification of infectious titers for a wider range of Drosophila C virus isolates. We also describe strand specific RT-qPCR assays for quantification of viral negative strand RNA produced during Drosophila C virus, Drosophila A virus, and Nora virus infection. Finally, we demonstrate the utility of these assays for quantification of viral replication during oral infections and persistent infections with each virus.
Graphical abstract
Keywords
virology
Drosophila melanogaster
virus quantification
RT-qPCR
ELISA
Abbreviations
DCV
Drosophila C virus
DAV
Drosophila A virus
TCID50
50% tissue culture infectious dose
CPE
cytopathic effect
ssRT-qPCR
strand specific reverse transcription quantitative polymerase chain reaction