Assessment of a Novel Real-Time Pan-Flavivirus RT-Polymerase Chain Reaction

Abstract

Outbreaks of West Nile virus (WNV) have occurred intermittently in regions around the Mediterranean coast, and the virus may have become established in Northern Italy and Romania, with reported intermittent outbreaks in Spain, Hungary, and France. WNV has also spread rapidly throughout the Americas since its introduction into New York in 1999. This capacity to emerge in new geographical locations and to spread rapidly together with the current increase in incidence of other flaviviruses such as tick-borne encephalitis virus, dengue virus, and Usutu virus has prompted us to design a novel pan-flavivirus RT-polymerase chain reaction for the purpose of surveillance for a range of flaviviruses. The assay utilizes degenerate primers targeting the flavivirus NS5 gene (RNA-dependent RNA polymerase) and detects a range of flaviviruses, including WNV. A small panel of WNV bird samples obtained from the United States has been shown to be detected using this assay. The amplicon generated is of sufficient size to provide sequence data to confirm the identity of the virus detected and undertake limited phylogenetic analysis. Testing using this assay has shown its ability to detect a range of tick-borne flaviviruses, particularly louping ill virus that is endemic in areas of the United Kingdom. The assay has been used to survey 160 bird samples and 1000 mosquito samples from the United Kingdom and found no evidence for WNV.

Recent progress in West Nile virus diagnosis and vaccination

“…Johnson et al designed a pan-flavivirus RT-PCR utilizing degenerate primers targeting the NS5 gene to allow the detection of a range of flaviviruses including WNV. This SYBR Green based RT-PCR was able to detect WNV however the sensitivity was much lower compared to WNV-specific TaqMan RT-PCR assays [76]. SYBR Green has been shown to inhibit the PCR reaction to some extent and melt curve analysis is complicated by dye redistribution during melting.…”

Usutuvirus

“…In der Literatur sind verschiedene Verfahren zum Nachweis von USUVSequenzen beschrieben; unter anderem werden für die Entwicklung des USUVspezifischen Genomnachweises in der Regel Sequenzen aus dem NS5-Genombereich verwendet [23,26]. Von Interesse ist auch die Entwicklung von Pan-Flavivirus-NATs, die es ermöglichen sollen, eine Vielzahl von Flaviviren in klinischen Materialien zu erkennen [10,62,63,64] …”

“…Es ist jedoch zu bedenken, dass bisher keine ausreichenden Informationen zur Dauer der USUV-Virämie beim Menschen vorliegen und es zudem nicht gesichert ist, dass USUV durch Blutprodukte übertragen werden kann. Inwieweit beim Blutspenderscreening auch Pan-Flavivirus-NAT-Systeme eingesetzt werden können, die mit vergleichbarer Spezifität und Sensitivität Genome verschiedener Flaviviren (etwa WNV und USUV) in Blut-und Plasmaspenden erkennen, sollte weiter untersucht werden [64].…”

Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

“…Alternative assays for the diagnosis or detection of flavivirus infection, such as flow cytometry and microarrays, have been reported; however, these assays are not used routinely because of their high costs and complexity (8) (9) (10) . Thus, the principal molecular method for the diagnosis and detection of Flavivirus species is reverse transcription polymerase chain reaction (RT-PCR) developed for the main specific virus or multiplex causing human flavivirus diseases (11) (12) (13) . Here, we describe the evaluation and optimization of a broad specificity SYBR Green I real-time RT-PCR method for the universal detection of flaviviruses.…”