Improved detection of Zika virus RNA in human and animal specimens by a novel, highly sensitive and specific real-time RT-PCR assay targeting the 5'-untranslated region of Zika virus

Abstract

Background: Highly sensitive and specific laboratory diagnostics are especially important for controlling the rapidly expanding Zika virus (ZIKV) epidemic, as the clinical features of ZIKV infection may be mild or indistinguishable from other arbovirus infections. Non-vector-borne transmissions may occur due to persistent virus shedding in ZIKV-infected patients’ urogenital tract, blood, and/or bodily fluids. Some existing RT-PCR assays utilizing primers/probes designed before the present epidemic emerged might misdiagnose up to 20-80% of ZIKV infected patients because of limited sensitivity and nucleotide mismatches. Methods: We developed and evaluated five novel real-time RT-PCR assays targeting conserved regions in the 5’ untranslated region (5’UTR), envelope (E’), non-structural protein 2A (NS2A), NS5, and 3’-UTR of the ZIKV genome. Results: The ZIKV-5’-UTR assay exhibited the lowest in-vitro limit of detection (5-10 RNA copies/reaction and 3.0 x 10-1 plaque-forming units/ml). Compared to the modified version of a widely adopted RT-PCR assay targeting the ZIKV-E gene, the ZIKV-5’UTR assay showed better sensitivity in human clinical specimens, and representative mouse specimens, including many organs which are known to be involved in human ZIKV infection but difficult to obtain in clinical settings. The ZIKV-5’UTR assay detected ZIKV RNA in all 84/84 (100.0%) ZIKV-E’-positive and an additional 30/296 (10.1%, P<0.01) ZIKV-E’-negative mouse specimens. The higher sensitivity of the ZIKV-5’UTR assay was most significant in kidney and testis/epididymis specimens (P<0.01). No in-vitro or in-vivo cross-reactivity was found between the ZIKV-5’UTR assay and other common flaviviruses/arboviruses. Conclusions: The highly sensitive and specific ZIKV-5’UTR assay may help to improve the laboratory diagnosis of ZIKV infection.