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

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.