Replication is a key step in successful virus infections. The replication strategies of positive-strand RNA viruses share remarkable similarities: all replicate and express their genomes through negative-strand RNA intermediates that are used as templates for the production of positive-strand progeny RNAs packaged in new virion particles . Therefore, the presence of negative-strand RNA intermediates should serve as a reliable marker for active virus replication in infected hosts.
In an attempt to develop an in vitro RNA replication assay for honey bee viruses, we first evaluated the existing methods, including conventional RT-PCR and Tagged RT-PCR for specificity of strand-specific detection of DWV RNA. The results showed that DWV RNA could be amplified by conventional RT-PCR without any primer for reverse transcription. The reason for nonspecific cDNA synthesis by conventional RT-PCR could be attributed to different events, including false-priming by antigenomic viral RNA or cellular RNAs, as well as self primering due to the secondary structure at the 5'UTR of viral RNA during reverse transcription, as earlier reports suggested [19, 25–27]. Tagged RT-PCR was developed to resolve the problem of PCR amplification of falsely-primed cDNA associated with conventional RT-PCR [28, 29]. However, the finding that cDNAs generated by tag-forward or tag-reverse primers were amplified by subsequent PCR with a single reverse primer or forward primer, respectively, suggested that the residue of tagged-primer from reverse transcription possibly served in subsequent PCR and led to amplification of non-strand-specific products, making necessary a purification step to assure total elimination of remaining primer from RT products or primer concentration reduction in RT reaction . The evidence that conventional RT-PCR and Tagged RT-PCR without purification, failed to discriminate between the two strands of viral RNA in this study, suggests that caution should be taken with regards to the strand specificity of such methods.
In order to circumvent the problems of false priming and contamination of residue primers from RT, it is worthwhile to develop improved procedures for analysis of virus replication. We report here the development of a novel strand-specific RT-PCR coupled with a biotinylated primer for reverse transcription and purification of biotinylated cDNA with magnetic beads. Using biotinylated oligonucleotide primers during the reverse transcription can lead to subsequent synthesis of biotinylated cDNAs, which have a high binding affinity to the streptavidin-coated magnetic beads. The purification step of streptavidin magnetic beads-cDNA complex, ensures the capture of only biotinylated cDNAs. The disappearance of a positive signal for non-strand-specific amplification of magnetic-bead purified biotinylated cDNA in our study suggests that the assay developed is a significant modification of conventional or Tagged RT-PCR. The purification of biotinylated cDNA with magnetic beads is a key step in assuring the strand-specific detection and elimination of the residues of RNA, non-incorporated RT primers as well as enzymes and salts that would interfere with subsequent PCR amplification.
While quantitative detection of DWV infection and tissue tropism of DWV in the host were previously reported [30, 31], there is lack of information on quantification of virus replicative intermediates and differentiation of positive and negative-strand DWV RNA in the different tissues of infected bees, which could provide important insight into the complexity of virus replication strategies leading to disease pathogenesis. To gain a better understanding of active sites of DWV replication in infected bees, we applied the method developed in our study to localize and quantify the positive and negative-strand DWV RNA in different host tissues. Our results showed that replication of DWV was spread throughout the body of bees with symptomatic infection. The detection of replicative intermediates, negative-strand DWV RNA, in the hemolymph, gut, wings, legs, head, thorax, and abdomen indicated that active replication occurred in these sites.
The observation that different tissues showed distinct kinetics of DWV replication, together with the fact that differences in the titers of positive-strand DWV RNAs were not significant among tissues examined, suggested that DWV had a tropism to certain host tissues in the replication. Among seven tissues examined, the most abundant amount of negative-strand DWV RNA was detected in the wings, which suggests that the wings are likely the predominant tissue site of DWV replication. Our earlier studies [15, 31] demonstrated that colony foods could act as a vehicle for transmission of DWV and suggested that the lining of the digestive tract was likely the primary site of the DWV infection via food-borne transmission, with the virus spreading to secondary sites from the digestive tract. The presence of positive-strands, likely from food, and the absence of replication (negative-strands) of DWV RNA only in the gut and abdomen of asymptomatic bees, indicated that these sites are critical for the DWV pathogenesis course, signifying the necessity of an unknown event at these sites, such as a co-infection or a differential genetic background, to generate the permissive environment for a massive replication observed in symptomatic bees, likely responsible for the symptoms observed.
Compared to the titer of positive-strand DWV RNA, the relatively low amount of negative-strand DWV RNA implies that a regulatory mechanism may exist to facilitate the viral replication. These findings would stimulate further investigation to unveil the regulatory mechanisms that honey bees use to control the pattern of replication. Both positive and negative-strand RNAs of DWV were also detected in Varroa mites (data not shown) using the magnetic beads methodology. This finding is in agreement with preliminary works [22, 23] and supports the conclusion that the Varroa mite may be a biological vector of DWV.
In sum, strand-specific detection and quantification of DWV RNA were achieved using qRT-PCR incorporated with biotinylated primers and purification of biotinylated cDNA with magnetic beads. The elucidation of DWV replication profiles in honey bees would have broad implications for future development of therapeutic strategies for viral diseases. The assay developed in this study represents a useful tool to study not only replication of honey bee viruses but also other single-stranded RNA viruses.