The Development of New Tools to Investigate Alphavirus Replication Kinetics

Members of the alphavirus genus pose a serious or potential threat to public health in many areas of the world. Nearly all alphaviruses are maintained in nature by transmission cycles that involve alternating replication in a susceptible vertebrate and invertebrate host. The maintenance of this transmission cycle depends on the establishment of a life-long persistent infection in the invertebrate vector host. Although alphavirus replication has been extensively studied in vertebrate models, the strand-specific replication kinetics of alphaviruses during persistent infections of the invertebrate host have not been reported. We investigated the strand-specific replication of different alphavirus genotypes in invertebrate cells.

By comparing different detection strategies and chemistries, we identified an optimal ssqPCR assay design for strand-specific quantification of viral RNAs in infected cells and tissues. We found that primer sets incorporating the use of a non-target tag sequence were able to avoid real-time PCR detection of amplicons that were falsely-primed during reverse-transcription. We also determined that DNA hydrolysis probes increased the sensitivity of ssqPCR assays when compared to a double-stranded DNA-specific dye, SYBR Green.

Using this information, we determined the replication kinetics of two different genotypes of o'nyong nyong virus (ONNV) and chikungunya virus (CHIKV) in infected mosquito cells. We found that (-) strand viral RNAs persisted in invertebrate cells for up to 21 days after infection. We also found that significantly less (-) strand RNA was present in cells infected with opal variants of both ONNV and CHIKV than sense variants at several time points post infection, suggesting that the opal codon has a functional role in (-) strand RNA regulation. We also report the development of an ONNV replicon expression system.

In total, the tools we developed for this report will facilitate future replication studies in the mosquito that may shed light on questions regarding the regulatory role of the opal codon and the persistence of (-) strand RNAs during long-term infections. The strand-specific replication kinetics of ONNV and CHIKV genotypes reported here will serve as a foundation for such investigations.