Advanced Yellow Fever Virus Genome Detection in Point-of-Care Facilities and Reference Laboratories

Abstract

Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcriptionquantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.

Low Thymic Activity and Dendritic Cell Numbers Are Associated with the Immune Response to Primary Viral Infection in Elderly Humans

“…Sera were tested for the presence of YFV-17D genomes at days 0, 2, 4, 7, and 10 after vaccination by quantitative real-time PCR (RT-PCR) as described elsewhere (23). A serial dilution of in vitro-generated viral RNA of known concentration was simultaneously run to standardize the measurement.…”

Resurgence of Yellow Fever in Angola, 2015–2016

“…All results of RT-PCRs were negative. A real time RT-PCR for YFV, targeting the 5′ noncoding region ( 9 ), produced positive results for samples from all 3 patients. The samples were then tested by using a pan-flavivirus RT-PCR targeting the flavivirus NS5 gene region using primers FU1 8993F and cFD2 9258R ( 10 ).…”

Detection of antibodies against Icoaraci, Ilhéus, and Saint Louis Encephalitis arboviruses during yellow fever monitoring surveillance in non‐human primates (Alouatta caraya) in southern Brazil

“…We attempted to isolate the viruses by inoculating the samples in cell cultures (C6/36‐clone Aedes albopictus cells) followed by indirect immunofluorescence (IFI) using polyclonal antibodies for the main arbovirus antigenic groups in Brazil. We used real‐time quantitative PCR (RT‐qPCR) when attempting to detect YFV and DENV in captures B, C, and D and Zika and Chikungunya in capture C. These arboviruses were all widely circulating in humans in Brazil during the study period.…”