Chimeric viruses between Rocio and West Nile: the role for Rocio prM-E proteins in virulence and inhibition of interferon-α/β signaling

Abstract

Mosquito-transmitted flavivirus Rocio (ROCV) was responsible for an outbreak of encephalitis in the Ribeira Valley, located in the south coast of Sao Paulo State, Brazil, in 1975-1976. ROCV also causes fatal encephalitis in adult mice. Seroprevalence studies in humans, horses and water buffaloes in different regions of Brazil have suggested that ROCV is still circulating in the country, indicating the risk of re-emergence of this virus. West Nile virus (WNV) is also a mosquito-transmitted encephalitic flavivirus, however, WNV strains circulating in Australia have not been associated with outbreaks of disease in humans and exhibit low virulence in adult mice. To identify viral determinants of ROCV virulence, we have generated reciprocal chimeric viruses between ROCV and the Australian strain of WNV by swapping structural prM and E genes. Chimeric WNV containing ROCV prM-E genes replicated more efficiently than WNV or chimeric ROCV containing WNV prM-E genes in mammalian cells, was as virulent as ROCV in adult mice, and inhibited type I IFN signaling as efficiently as ROCV. The results show that ROCV prM and E proteins are major virulence determinants and identify unexpected function of these proteins in inhibition of type I interferon response.

Figure 1. Generation by CPEC and characterization of ROCV.

(a) The strategy for constructing the full-length infectious cDNA of ROCV by CPEC reaction. (b) Plaque morphology of parental and CPEC-generated ROCV (passage 1) in BHK-21 cells. (c) Replication efficiencies of parental and CPEC-generated ROCV in mouse embryonic fibroblasts (MEF). Cells were infected at MOI = 0.1 with parental or CPEC-generated ROCV, culture supernatant were collected at 0, 24, 48 and 72 hours post-infection, and viral titers were determined by plaque assay on BHK-21 cells. The dashed line represents the LOD of the assay.

Figure 2. Construction and characterization of ROCV/WNV chimeric viruses.

(a) Schematic representation of ROCV/WNV_NSW2011 chimeric viruses. (b) Plaque morphology of parental (ROCV and WNV) and chimeric (ROCV/WNV-prME and WNV/ROCV-prME) viruses in BHK-21 cells (upper) and comparison of plaque size areas (lower). Plaque areas (mm²) for 25–40 randomly selected plaques were measured using ImageJ software (National Institutes of Health, USA) and plotted as mean ± SD. (c) Growth kinetics of parental and chimeric viruses in IFN-competent mouse embryonic fibroblasts (WT MEF) and (d) IFNAR^−/− MEF cells infected at MOI = 0.1. Viral titers were determined by plaque assay on BHK-21 cells at the indicated time points. The dashed lines represent the LOD of the assay. Parametric one-way ANOVA test was used to compare within groups. *P-value ≤ 0.05, ****P-value ≤ 0.0001.

Figure 3. pSTAT1 analysis in IRF3^−/− × IRF7^−/− MEF cells infected with parental and chimeric viruses and treated with mouse IFN-β.

(a) IRF3^−/− × IRF7^−/− MEF cells were infected with parental or chimeric viruses at MOI = 0.1, and treated with 10,000 IU of mouse IFN-β at 48 h.p.i. Cells were gated to the single-cell infected population, and cell counts were plotted to fluorescence intensity of pSTAT1. (b) Relative expression of pSTAT1 normalized to ROCV. Data represent the mean of two independent experiments; each performed in duplicate. Parametric one-way ANOVA test was used to compare within groups. *P-value ≤ 0.05.

Figure 4. Survival of 6-week-old C57BL6 female mice after infection with parental and chimeric viruses.

Six-week-old C57BL6 mice in groups of 10 were infected with 2.6 × 10⁴ pfu of the indicated viruses via the intraperitoneal route and monitored for signs of encephalitis for 21 days post-infection.