Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings.

Introduction: Zika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, low-cost detection methods are considered highly desirable. Materials and Methods: Here we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene. Results: We show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/µL in under 30 min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5 min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5 min in running buffer. Discussion: Contrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency.

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis.

All flaviviruses, including Zika virus, produce noncoding subgenomic flaviviral RNA (sfRNA), which plays an important role in viral pathogenesis. However, the exact mechanism of how sfRNA enables viral evasion of antiviral response is not well defined. Here, we show that sfRNA is required for transplacental virus dissemination in pregnant mice and subsequent fetal brain infection. We also show that sfRNA promotes apoptosis of neural progenitor cells in human brain organoids, leading to their disintegration. In infected human placental cells, sfRNA inhibits multiple antiviral pathways and promotes apoptosis, with signal transducer and activator of transcription 1 (STAT1) identified as a key shared factor. We further show that the production of sfRNA leads to reduced phosphorylation and nuclear translocation of STAT1 via a mechanism that involves sfRNA binding to and stabilizing viral protein NS5. Our results suggest the cooperation between viral noncoding RNA and a viral protein as a novel strategy for counteracting antiviral responses.