A TaqMan RT-qPCR assay for absolute quantification of citrus leprosis virus C lineage SJP: disclosing the subgenomic/genomic ratio in plant and mite vector, plant organ-specific viral loads, and the kinetics of viral accumulation in plants

Citrus leprosis virus C (CiLV-C) causes citrus leprosis, a re-emergent viral disease affecting citrus production in Latin America. Here, we developed two TaqMan RT-qPCR assays to detect and quantify CiLV-C lineage SJP, prevalent in the Brazilian citrus belt and the world’s main sweet orange production area. Assays targeted sequences within the genes p29 and RdRp. ORF p29 is transcribed in a subgenomic RNA (sgRNA) and codes for the putative capsid protein. ORF RdRp , coding for the replicase, is directly translated from the genomic RNA (gRNA). After assessing the efficiency and sensitivity of the assays, the targets were quantified in (i) symptomatic tissues of field-collected sweet orange ( Citrus sinensis ) samples, in a time course after infection in both (ii) Arabidopsis thaliana and (iii) sweet orange plants, and in (iv) the mite vector Brevipalpus yothersi . Sweet orange fruits support higher quantities of CiLV-C molecules than stems and leaves. Amounts of viral molecules in late lesions of different developmental stages collected in the field remain stable, but CiLV-C quantities progressively increase from the early stages of the infection to the appearance of symptoms in both A. thaliana and C. sinensis. The high sensibility of the assays allowed the quantification of CiLV-C in early infection periods, even during the asymptomatic period of plants, and in scant amounts of B. yothersi individuals. In plants, a higher accumulation of p29 than RdRp was reported (sgRNA/gRNA up to 95), reflecting the transcription of the p29 sgRNA. In mites, p29 quantities were only slightly higher than RdRp (sgRNA/gRNA of 2), adding a new tool to evaluate the putative replication of CiLV-C in its vector, a challenging aspect of the study of mite-virus interplay. The methods developed here contribute to a more accurate analysis of citrus leprosis epidemiology and shed light on unknown features of the virus-vector interaction.