The genus Flavivirus of the family Flaviviridae consists of more than 70 virus species including many arthropod-borne 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 fever and pose some of the most challenging emerging diseases in humans .
Flaviviruses comprise three epidemiologically distinct groups; the mosquito-borne group, the tick-borne group and the unknown vector viruses . They are enveloped positive-stranded RNA viruses with a genome of approximately 11 kb. The viral genome encodes a large single polyprotein from which three structural proteins, Capsid (C), Envelope (E) and Membrane (M) and seven non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, are produced .
Flaviviruses pose a threat to public health worldwide, especially in developing countries. In recent years, there has been a dramatic increase in cases of DENV infections globally. The disease is endemic in more than 100 countries in Africa, the Americas, the Eastern Mediterranean, South-east Asia, and the Western Pacific. About 50–100 million cases of DENV infection are reported worldwide every year, resulting in 500,000 cases of hemorrhagic fever requiring hospitalization and 25,000 fatalities . 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 . WNV is one of the most widespread flaviviruses. WNV lineage 1 is endemic in the Americas, India, and Australia. Both lineages 1 and 2 are found in Africa and Europe [6–8]. The recent WNV outbreak reported in August 2010 in Greece was the first large outbreak in humans in Europe since the Romanian outbreak in 1996–1997 . JEV is the leading cause of viral encephalitis in Asia with 30,000–50,000 clinical cases reported annually and 10,000 deaths . TBEV, virus-causing infection of the central nervous system, has spread extensively in some European regions. According to ECDC , human cases of TEBV infections have increased in the past 30 years dramatically posing a danger to public health in European countries.
A number of flaviviruses have repeatedly demonstrated their ability to expand their geographical range. Some current examples are the introduction of Usutu virus and Bagaza virus into central Europe [12, 13], or Zika virus into Yap island and Cambodia . In recent years, also a high number of mosquito-only flaviviruses including Culex and Aedes flaviviruses were discovered throughout the world [15–18]. Apart from the human pathogenic and mosquito-only flaviviruses, several novel flaviviruses were latterly discovered: Nounane in Cote d’Ivoire , T’Ho virus in Mexico , Lammi virus in Finnland , and Duck tembusu virus in China . In contrast to mosquito-only flaviviruses, these novel flaviviruses were shown to be phylogenetically related to human pathogenic flaviviruses.
The clinical diagnosis of flavivirus infections is not unambiguous due to unspecific symptoms varying from mild, febrile illness to viral hemorrhagic fever. Many of these viruses have a common transmission vector and can co-circulate in the same area. All these factors make a precise identification of the pathogen difficult.
Today most diagnostic laboratories use serological assays for flavivirus testing. These tests are commonly based on the enzyme-linked immunosorbent assay (ELISA) and detect virus-specific IgM and IgG antibodies. Antibodies are undetectable prior to 5–7 days after onset of infection which hampers the usefulness of the serological methods. Molecular techniques, in contrast, can be used already in the acute phase and are known to be rapid, highly specific and sensitive.
Since the early 1990s, several group-specific and generic molecular assays for detection of flaviviruses have been developed as reviewed previously [23–25]. A number of attempts to detect several flaviviruses in a single reverse transcription-polymerase chain reaction (RT-PCR) assay have been made . These assays vary in the selected target region, assay format and detection method. The highly conserved NS3 and NS5 genes have been used predominantly for flavivirus testing, mainly as nested, hemi-nested RT-PCR or SYBR green real-time PCR, without using a probe for sequence verification.
The aim of this study was to develop a rapid, sensitive and reliable TaqMan probe-based quantitative RT-PCR (qRT-PCR) assay for simultaneous detection of several flaviviruses, using the conserved NS5 gene region. The introduction of Locked-Nucleic Acid (LNA) bases in the probe increases robustness, specificity and sensitivity of the assay and has been shown to allow quantification of the viral load.