Development and evaluation of a novel high-throughput image-based fluorescent neutralization test for detection of Zika virus infection

Abstract

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus flavivirus that comprises other important public health viruses, such as dengue (DENV) and yellow fever (YFV). In general, ZIKV infection is a self-limiting disease, however cases of Guillain-Barré syndrome and congenital brain abnormalities in newborn infants have been reported. Diagnosing ZIKV infection remains a challenge, as viral RNA detection is only applicable until a few days after the onset of symptoms. After that, serological tests must be applied, and, as expected, high cross-reactivity between ZIKV and other flavivirus serology is observed. Plaque reduction neutralization test (PRNT) is indicated to confirm positive samples for being more specific, however it is laborious intensive and time consuming, representing a major bottleneck for patient diagnosis. To overcome this limitation, we developed a high-throughput image-based fluorescent neutralization test for ZIKV infection by serological detection. Using 226 human specimens, we showed that the new test presented higher throughput than traditional PRNT, maintaining the correlation between results. Furthermore, when tested with dengue virus samples, it showed 50.53% less cross reactivity than MAC-ELISA. This fluorescent neutralization test could be used for clinical diagnosis confirmation of ZIKV infection, as well as for vaccine clinical trials and seroprevalence studies.

Fig 1. Infection parameters definition.

Virus growth kinetics in C6/36 cell line (A). Zika virus infection in Huh 7.5 cell line with different MOIs (ranging from 0.001 to 1). Dashed line indicates 70% of infection (B). The data presented are the average results of three independent experiments.

Fig 2. Fluorescent neutralization assay setting definition.

Representative image of negative and positive infection controls (A). Assay overview of serial dilution (1/20 to 1/43740) of a negative sample (26885) and two positive samples (LRV/16 1306 and LRV/16 1260) (B). Curve fitting of results and calculation of neutralization titer that inhibit 90% of viral infection (NT₉₀) (C).

Fig 3. Neutralization tests comparison.

Neutralization titers obtained from PRNT or fluorescent neutralization test are compared. A correlation of R = 0.88 was obtained (P = 0.0003).

Fig 4. ZIKV Fluorescent neutralization test validation.

A total of 226 serum samples, among then negative and positive for flavivirus and non-flavivirus infections were examined. The neutralization titers that inhibit 90% of viral infection (NT₉₀) are shown in the graph. Other infections: Leptospirosis, CMV, EBV, Hantavirus, Toxoplasmosis, VDRL. Dashed lines represent the test cut off: negative when NT₉₀ <10, inconclusive when NT₉₀ ≥10 and <20, and positive when NT₉₀ ≥20. Symbols in blue indicate paired samples.

Fig 5. ZIKV neutralization test with paired samples.

Second collections were performed from 3 to 6 months after the first one. Dashed lines represent test cut off: negative when NT₉₀ <10, inconclusive when NT₉₀ ≥10 and <20, and positive when NT₉₀ ≥20.