Abstract
BackgroundThe genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses.MethodsA Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively.ResultsTwo degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays.ConclusionThe assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.
Highlights
The genus Flavivirus includes several pathogenic agents that cause severe illness in humans
Degenerate primers were selected based on these published sequences, but modified in order to increase the spectrum of flaviviruses included
Neither the set of individual primers nor the set of probes improved the detection limit nor the range of viruses detected and we continued the evaluation with the degenerate set of primers combined with one of the three Locked-Nucleic Acid (LNA)-probes
Summary
The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Reemergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. The genus Flavivirus of the family Flaviviridae consists of more than 70 virus species including many arthropodborne viruses It contains highly pathogenic agents such as the name-giving member yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and dengue virus (DENV), which can cause encephalitis or hemorrhagic. Yellow Fever is endemic in 45 countries throughout Africa and Latin America, with approximately 200,000 cases of human infections worldwide, and 30,000 mortalities [5]. According to ECDC [11], human cases of TEBV infections have increased in the past 30 years dramatically posing a danger to public health in European countries
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