Improved Purification and Quantitative Kinetics Monitoring of Triatoma virus Infection of Rhodnius prolixus

Abstract

Chagas disease control relies on chemical interventions, currently compromised by insecticide resistance. Triatoma virus (TrV) emerges as a biological candidate, requiring standardized methodologies to purify, quantify, and relate viral doses to biological effects in triatomines. We established a comprehensive protocol for the quantitative study of TrV using Rhodnius prolixus as a model, combining physical purification via sucrose gradients and absolute quantification by qPCR targeting the TrVgp1 gene to link viral load with host survival. Primers were validated, and a plasmid standard curve was constructed for absolute quantification. Four isolation methods from Triatoma infestans macerates were compared, including an optimized discontinuous gradient protocol (EGM). High (6 × 106 viral genome copies/µL) and low (2.47 × 105 viral genome copies/µL) doses of EGM-purified TrV were evaluated in oral infections of R. prolixus over 16 days post-inoculation (dpi). The EGM protocol achieved the highest yields, around 109 virus genome copies/µL of purified viral stock. Intestinal viral replication was dose-dependent, exhibiting distinct kinetic phases between low (3–7 dpi) and high (9–12 dpi) inoculum cohorts. Only the high dose significantly reduced host survival. In conclusion, this protocol allows high-yield TrV purification and absolute quantification in R. prolixus, establishing a robust dose–response foundation to assess triatomine susceptibility for integrated biological control programs.