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

Abstract

. This assay had a specificity of 100% and various sensitivities of at least 3.5 PFU/ml for YFV, 2.0 PFU/ml for JEV, 10.0 PFU/ml for WNV, and 10.0 PFU/ml for SLEV. Additionally, we have developed an in vitro transcription system to generate RNase-resistant RNA templates for each of these eight viruses. These templates can be incorporated into the assay as RNA copy number controls and/or as external controls for RNA-spiked mosquito pools for quality assurance purposes. Although further study with mosquitoes collected in the field is needed, the incorporation of this assay into mosquito surveillance could be used as an early-warning system for the detection of medically important flaviviruses, particularly when the cocirculation of multiple viruses in the same region is suspected.Flaviviruses are significant causes of disease worldwide and can be classified serologically into several antigenic complexes (12,20). Medically important members of the Japanese encephalitis virus (JEV) serocomplex include JEV, St. Louis encephalitis virus (SLEV), and West Nile virus (WNV). Each of these viruses causes similar disease syndromes in humans, ranging from an asymptomatic or a mild flu-like illness to clinical encephalitis (18). Until 1999, SLEV was the only mosquito-borne flavivirus causing significant human morbidity and mortality in the United States (22). WNV, traditionally found in Europe, Africa, the Middle East, and Asia, emerged in New York City in the late summer of 1999 (1). By 2005 the distribution of WNV had expanded into areas of known recent SLEV activity, such as the states of Florida, Mississippi, Louisiana, Texas, Arizona, Colorado, and California (3). Mosquito surveillance for detection of virus activity during the transmission season is an essential tool for implementation of an effective mosquito control strategy. As WNV and SLEV occupy similar ecological niches in North America, it is critical to develop a cost-effective assay capable of detecting the presence of either one or both viruses simultaneously in the mosquito pools. In addition, dengue virus (DENV) serotypes 1 to 4 (DENV-1 to DENV-4, respectively), SLEV, and yellow fever virus (YFV) are endemic in Latin America (5). 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 nucleotide sequences flanked by these two primers were variable...

Infection of the central nervous system with dengue virus 3 genotype I causing neurological manifestations in Brazil

“…RNA was extracted (Viral RNA QIAamp Extraction Kit; Qiagen, Valencia, USA), followed by reverse transcription using random primers (MMLV; Promega, Madison, USA). The cDNA was used as the template in the quantitative PCR (qPCR) with primers designed to amplify the NS5 region of the viruses from the genus Flavivirus (11) . qPCR was performed with a commercial mix (SYBR® Green …”

“…The primers used for amplifi cation were forward,5´-GTGTCCCAGCCGGCGGTGTCATCAGC-3´, and reverse, 5´-TACAACATGATGGGAAAGCGAGAGAAAAA-3´ ( 11) . PCR failed to detect HSV-1, HSV-2, VZV, CMV, and viruses of the genus Enterovirus.…”

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

“…Serological tests can cross-react between flaviviruses or have false negative results (18) . Compared with conventional RT-PCR, real-time RT-PCR has several advantages such as rapidity, low risk of false positive results, high sensitivity, specificity, and the possibility of viral quantification (12) (19) (20) . In fact, a previously described real-time RT-PCR method (11) was able to detect a broad range of flaviviruses found in Brazil (3) (21) (22) .…”

“…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.…”

Two New Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction Assays with Unique Target Sites for the Specific and Sensitive Detection of Lineages 1 and 2 West Nile Virus Strains

“…Even for The 2 new qRT-PCR assays were eventually tested with other species of genus Flavivirus, such as YFV, JEV, TBEV, and MVEV, in order to verify the specificity of the detection. For the adjustment of comparable RNA amounts for the different species, a SYBR Green real-time RT-PCR based on the pan-Flavi primer set (as previously published 7 ) was used, which targeted the conserved NS5 region of this genus. As shown in Table 4, assay 1 detected MVEV (Ct 5 33.8), and assay 2 detected JEV (Ct 5 28.4).…”