Development and validation of RT-LAMP for detecting yellow fever virus in non-human primates samples from Brazil

Abstract

Monitoring yellow fever in non-human primates (NHPs) is an early warning system for sylvatic yellow fever outbreaks, aiding in preventing human cases. However, current diagnostic tests for this disease, primarily relying on RT-qPCR, are complex and costly. Therefore, there is a critical need for simpler and more cost-effective methods to detect yellow fever virus (YFV) infection in NHPs, enabling early identification of viral circulation. In this study, an RT-LAMP assay for detecting YFV in NHP samples was developed and validated. Two sets of RT-LAMP primers targeting the YFV NS5 and E genes were designed and tested together with a third primer set to the NS1 locus using NHP tissue samples from Southern Brazil. The results were visualized by colorimetry and compared to the RT-qPCR test. Standardization and validation of the RT-LAMP assay demonstrated 100% sensitivity and specificity compared to RT-qPCR, with a detection limit of 12 PFU/mL. Additionally, the cross-reactivity test with other flaviviruses confirmed a specificity of 100%. Our newly developed RT-LAMP diagnostic test for YFV in NHP samples will significantly contribute to yellow fever monitoring efforts, providing a simpler and more accessible method for viral early detection. This advancement holds promise for enhancing surveillance and ultimately preventing the spread of yellow fever.

Keywords: Diagnosis; RT-LAMP; Yellow fever; Yellow fever virus.

Fig. 1

Collection sites for non-human primate (NHP) tissues in municipalities from the Southern region of Santa Catarina State, Brazil. The left side features a map of Brazil, while the right side provides a magnified view of the red box from the Brazil map, indicating the specific sample collection sites (red triangles). The x and y axes on the right map represent longitude and latitude, respectively. The number of collections from each municipality is shown in parentheses. The maps were generated using the sf, maps, and mapdata packages^– from R Software, version 4.3.1.

Fig. 2

YFV detection in NHP tissue samples using RT-LAMP with three primers set (NS5, E, and NS1). Amplification products were visually inspected, with pink indicating negative and yellow indicating positive results. 1–50: positive results, 51–60: negative results, NC: negative control using nuclease-free water instead of RNA, PC: positive control with YFV RNA (vaccine strain 17D). Each code in Table 1 (sample identification 1–60) corresponds to the tests presented in this figure.

Fig. 3

RT-LAMP specificity test assay for YFV detection. The RNA of other flaviviruses circulating in Brazil were tested, including Dengue (DENV-1: DV1 BR90; DENV-2: ICC 265; DENV-3: DV3 BR98; DENV-4: DVT 360) and Zika viruses (ZIKV: BR 2015/15,261). R1–R10: ten independent replicates containing Flavivirus RNA, PC: positive control containing YFV RNA (vaccine strain 17D), NC: negative control using nuclease-free water instead of RNA. Amplification products were visually inspected, with pink indicating negative and yellow indicating positive results.

Fig. 4

Sensitivity of YFV RT-LAMP assay. (a) Assay colorimetric results of five replicates (1–5) and a negative control with water (6) tested with a serial dilution of YFV RNA (10⁶–10⁻³ PFU/mL). (b) Limit of detection of 10¹ PFU/mL established through the probit regression analysis and considering only yellow results as positive. (c) Limit of detection of 10⁻¹ PFU/mL established through the probit regression analysis and considering yellow and orange results as positive.