Comparison of Flavivirus Universal Primer Pairs and Development of a Rapid, Highly Sensitive Heminested Reverse Transcription-PCR Assay for Detection of Flaviviruses Targeted to a Conserved Region of the NS5 Gene Sequences

Abstract

Arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. Because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by PCR would be a useful first-line diagnostic method. The six published Flavivirus genus primer pairs localized in the NS1, NS3, NS5, and 3 NC regions were evaluated in terms of specificity and sensitivity with flaviviruses (including the main viruses pathogenic for humans) at a titer of 10 5 50% tissue culture infectious doses (TCID 50 s) ml ؊1 with a common identification step by agarose gel electrophoresis. Only one NS5 primer pair allowed the detection of all tested flaviviruses with the sensitivity limit of 10 5 TCID 50 s ml ؊1 . Using a heminested PCR with new primers designed in the same region after an alignment of 30 different flaviviruses, the sensitivity of reverse transcription-PCR was improved and allowed the detection of about 200 infectious doses ml ؊1 with all of the tick-and mosquito-borne flaviviruses tested. It was confirmed that the sequenced amplified products in the NS5 region allowed predictability of flavivirus species by dendrogram, including the New York 99 West Nile strain. This technique was successfully performed with a cerebrospinal fluid sample from a patient hospitalized with West Nile virus encephalitis.

Development of Multiplex Real-Time Reverse Transcriptase PCR Assays for Detecting Eight Medically Important Flaviviruses in Mosquitoes

“…The RdRp domain at the carboxy-terminal one-third of NS5 is the most conserved region in the Flavivirus genomes (16,23). The flavivirus consensus amplification primers (amplimers) and virus-specific probe sequences for WNV, JEV, SLEV, and YFV were located within this region ( Fig.…”

“…The situation is further complicated by the detection of WNV activity in Mexico and Central America (3). Therefore, the development of a real-time, multiplex reverse transcriptase (RT) PCR platform for the simultaneous detection of viral RNA genomes in the Americas is crucial for supporting mosquito surveillance efforts and clinical diagnosis.The RNA-dependent RNA polymerase (RdRp) domain, located at the carboxy terminus of nonstructural protein 5 (NS5), is the most conserved coding region in the Flavivirus genomes (15,16,23). The consensus sequence primers (primers FU1 and CFD2; YFV genomic positions, nucleotide [nt] 8997 and nt 9233, respectively) have been used in our previous study for genetic characterization of this domain for all registered flaviviruses (16).…”

“…The RNA-dependent RNA polymerase (RdRp) domain, located at the carboxy terminus of nonstructural protein 5 (NS5), is the most conserved coding region in the Flavivirus genomes (15,16,23). The consensus sequence primers (primers FU1 and CFD2; YFV genomic positions, nucleotide [nt] 8997 and nt 9233, respectively) have been used in our previous study for genetic characterization of this domain for all registered flaviviruses (16).…”

Putative novel lineage of West Nile virus in Uranotaenia unguiculata mosquito, Hungary

“…Samples were tested by nested reverse transcription-PCR assays. These included a screening panel using both universal and virus specific primers targeting a variety of mosquito-borne flaviviruses and orthobunyaviruses supposed to be endemic in the study area [6][7][8].…”

Notification of the first isolation of Cacipacore virus in a human in the State of Rondônia, Brazil

“…Traditionally, the diagnosis of Flavivirus infection has been done by virus isolation or serological testing. To overcome such problems, reverse transcription-polymerase chain reaction (RT-PCR) methods using universal primers have been described for certain flaviviruses [3][4][5][6][7] .…”