Combination of ELISA screening and seroneutralisation tests to expedite Zika virus seroprevalence studies

In order to compare VNT and PRNT, we used a panel of sera from blood donors of different origins: 90 from Martinique Island and 10 from Guadeloupe Island (all collected after the 2015–2016 outbreak due to ZIKV Asian genotype); 7 from Cameroon (where low ZIKV seropositivity [13] is presumably linked with the circulation of ZIKV African genotype); 35 from Metropolitan France (where no ZIKV or DENV circulation has been reported). For the performance testing of ELISA+VNT combination strategy, a total of 592 serum specimens collected from Martinique Island (collected before and after the 2015–2016 ZIKV outbreak) were included. A total of 13 serum specimens collected from laboratory staff at least one year after vaccination against several flaviviruses, including Yellow Fever, Japanese Encephalitis and Tick-Borne Encephalitis virus, were included to test the cross reactivity of CPE-based VNT. The consent of the each participant was taken prior to any laboratory testing.

NS1 protein-based Zika IgG ELISA was performed for all samples according to the manufacturer’s instructions. Semiquantitative ratios were calculated and specimens with a ratio value < 0.8 were considered as IgG negative; those with a ratio ≥ 1.1 were considered as positive and those with a ratio ≥ 0.8 to < 1.1 were recorded as equivocal according to the Euroimmun recommendations.

VNT was carried out with H/PF/2013 strain of the ZIKV in 96-well plates containing confluent Vero cells (ATCC CCL-81). H/PF/2013 strain of the ZIKV was selected as this strain gives clear CPE on Vero cells. We used 6 dilutions of each heat inactivated serum (1:10 to 1:320 –corresponding to final testing dilutions 1:20 to 1:640, see below), allowing testing 16 samples or controls per plate. All the serum dilutions and virus additions were done with the automated system (Eppendorf EpMotion 5075). In each VNT, a strong, assured positive control serum from French National Reference Centre for Arboviruses was used (PRNT90 titre: 2560). Sera were mixed vol/vol with 100 TCID50/reaction of ZIKV and incubated 1 h at 37 °C. Then the serum+virus mixture was transferred onto the confluent cell monolayer in duplicate. Two possible readout strategies were evaluated: CPE-based VNT and PCR-based VNT. CPE-based VNT relied on CPE observation directly with light microscopy at day 5 pi. After the 5 days incubation period, gross cytopathic effect can be observed on Vero cells with H/PF/2013 strain of ZIKV. As seen in the Additional File 1, after the incubation period, distinguishing the presence/absence of viral cytophatic effect caused by H/PF/2013 strain of ZIKV is straightforward and easily readable under light microscope. Dilutions of serum associated with CPE were considered as negative, while the absence of CPE indicated a complete neutralisation of ZIKV inoculum (positive result). Consequently, Virus Neutralisation Titre referred to VNT100 and is described as the highest dilution of serum that neutralized virus growth. PCR-based VNT relied on RNA extraction from culture supernatants at day 3 pi and subsequent ZIKV qRT-PCR detection. For the qRT-PCR, ThermoFisher Scientific Express One-Step Superscript qRT-PCR kit with primers 5’-GRGCYCGGCCAATCAG-3′ & 5’-AARGACGGGAGRTCCATTGTG-3′ and the probe FAM-CGCCACCAAGATGA-MGB were used [14]. After the qRT-PCR, the viral load in each serum dilution and the positive control were assessed. As for PCR-based VNT, the presence/absence of viral replication was recorded. All sample dilutions associated with a viral load superior to that of our positive control sample were considered as negative while a viral load inferior or equal to that of our positive control sample was the hallmark of a positive dilution. Consequently, both PCR- and CPE-based VNT were based on the presence or complete absence of virus growth (VNT100). For both tests, serum specimens with a titre ≥40 were considered positive according to the recommendations of the French National Reference Centre for Arboviruses [10].

PRNT was adapted from previously described protocols [15, 16]. It was carried out with H/PF/2013 strain (60 PFU/reaction corresponding to ~ 86 TCID/reaction) in 6 well plates containing confluent Vero cell monolayer and 6 dilutions of each serum (1:5 to 1:160 –corresponding to final testing dilutions 1:10 to 1:320, see below). After one-hour incubation at 37 °C, the vol/vol serum+virus mixture was transferred onto confluent Vero cells monolayer in 6 well plates. Following incubation, inoculums were removed and cell monolayers were immediately covered with carboxymethylcellulose (CMC)/DMEM overlay. Plaques were counted at day 5 pi. Serum specimens with 90% (PRNT90) or 50% (PRNT50) reduction of the number of plaques at titres ≥10 were recorded as positive according to the recommendation of Centers for Disease Control and Prevention [17, 18]. (Additional file 2 shows image of plaque of PRNT at day 5 pi).

In order to estimate the analytical specificity and sensitivity of the VNT for ZIKV, VNT results of the panel were compared with the plaque reduction neutralization test (PRNT) which is considered to be the “gold standard technique”.

The performance of the combined [ELISA screening+VNT confirmation] strategy was evaluated in blood donors from Martinique Island. This choice was justified by the fact that (i) we knew that this population had been heavily exposed to the circulation of ZIKV in 2015–2016 [10], (ii) we also knew that seroprevalence of dengue is ~ 90% in this population [12], (iii) we had samples collected before the arrival of ZIKV (9–10 September 2013) and 6 months after the end of the outbreak (25 January-14 February 2017). Accordingly, all samples were tested using both the ELISA (NS1 protein-based Zika IgG EUROIMMUN assay, Lübeck, Germany) and the CPE-based VNT, and results of the [ELISA screening+VNT confirmation] strategy were compared with those obtained using VNT as a first line test.