Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes

Abstract

Background: Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology-which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa.

Results: The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes.

Conclusion: We have developed a rapid, sensitive and specific rRT-PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection.

Figure 1

Alignment of the designed primers and probe with Zika virus strain sequences. Dots indicate identity with the consensus sequence on the top of the alignment.

Figure 2

Sensitivity of the rRT-PCR assay. (A) Standard curve for Zika virus RNA transcript dilution series showing the threshold cycles Ct plotted against the log₁₀ of Zika RNA transcripts and (B) Standard curve obtained with 10-fold serial dilutions of Zika virus. Ct values obtained are plotted against the log of the quantity of infectious virus (pfu/ml).