There is a real and present danger of catastrophic Yellow fever epidemic transmission between humans if the virus were introduced into the urban environment. For this reason, there is a need for a rapid and sensitive technique for detecting the Yellow fever virus with high specificity. Molecular assays based on RT-PCR are now utilized for specific and sensitive detection of arboviruses. qRT-PCR is widely considered an efficient tool for detection of viruses due to its speed and reproducibility . qRT-PCR has also been successfully applied for the study of several arbovirus-vector interaction [7, 11, 15, 16].
The problem associated with false negative test results with probe based assays prompted us to develop a SYBR Green I based qRT-PCR assay. The false negative results are primarily attributed to mutation in the target sequence of the probe [13, 17, 18]. Thus, Whiley and Sloots reported non-detection of a strain of Respiratory Syncytial Virus (RSV) in a TaqMan probe-based assay that resulted from a single nucleotide mismatch , and the same limitation has been observed in a TaqMan-based assay for detection of West Nile virus . More recently, an LNA (locked nucleic acid) probe-based assay failed to detect a Brazilian YFV isolate, also due to a single transversion (G-A) in the target sequence of the probe . Moreover, detailed analysis of YFV genome database at NCBI revealed presence of similar transversions (G-T) in the probe target region (reported by Drosten et al., 2002) of five YFV sequences (GenBank Accession no. DQ235229, AY968065, U52422, U52395, U52392). The probe independent SYBR Green-I based qRT-PCR assay has the potential to detect such mutant strains . YFV being a RNA virus is prone to emergence of novel mutant strains, which warrants for development of sensitive probe independent assays.
The SYBR Green based qRT-PCR that we have developed provides a simple and economic alternative for high throughput screening, with the important advantage that the whole process of RT and qPCR can be completed within 1 hour. The analysis of amplification plot in conjunction with melting curve, as practised in this study has resulted in discriminating non-specific amplification. This is recommended in all SYBR Green based Real-time RT-PCR assay to avoid false positive results, since SYBR Green can also intercalate into non-specific DNA including primer-dimer . The assay was found to be 10-fold more sensitive compared to conventional RT-PCR, which confirms its utility in detecting low viremic samples (we are using this as a model system for general studies of infection in certain mosquito organs). The quantitation of the viral load in samples was carried out using a standard curve obtained by plotting cycle threshold (Ct) values versus known virus concentration. The detection limit of this assay was found to be 1 PFU/mL, (30 RNA copies/reaction; equivalent to 3000 RNA copies/ml), similar to other TaqMan based assays [9–12]. A comparison of real-time RT-PCR with viral plaque assay revealed a significant correlation between YFV genome to infectious particle, which varied from 1000 to 5000:1 . Similar 3-log ratio difference was also reported for DENV-2 . The specificity of the assay was established through melting curve analysis by differential melting temperature (Tm). This aids in differentiating non specific amplifications. The specificity of this assay was also verified by including a battery of closely related flaviviruses (DENV 1 through 4, JEV, WNV, SLEV) and alphavirus (CHIKV). The non reactivity with these related viruses confirm the utility of this assay in YFV endemic areas, where these related viruses are also co-circulating. Further, the analysis of nucleotide sequence of the amplicon in YFV positive samples confirmed the specificity of the assay.
The qRT-PCR assay was subsequently applied to monitor the infection rate of YFV in three Ae. aegypti mosquito colonies during the extrinsic incubation period. Variation in infection rate was clearly discernable among different Aedes colonies and among individual mosquitoes. Variation in infection rate was also noticed at different days of post infection. The fluctuation in infection rate at different days of extrinsic incubation period was also reported for DENV and CHIKV [7, 19, 20] This may be attributed to the differential susceptibility of the different mosquito populations. The fluctuation in infection rate and viremia on different days has been attributed to a range of factors including digestion of blood meal at early stage to decline in nutrient availability in later stage [7, 19].
Its utility for screening of YFV infected mosquito pools was further confirmed from that fact that there was neither inhibition of detection nor with quantification with addition of up to 49 uninfected mosquitoes to one infected mosquito. The detection of a single infected mosquito in a pool size up to 50 makes this a reliable assay for surveillance of YFV.