Abstract
Kunjin virus (KUNV) is an attenuated strain of the severe neurotropic West Nile virus (WNV). The virus has a single-strand positive-sense RNA genome that encodes a polyprotein. Following gene expression, the polyprotein is cleaved into structural proteins for viral packaging and nonstructural proteins for viral replication and expression. Removal of the structural genes generate subgenomic replicons that maintain replication capacity. Co-expression of these replicons with the viral structural genes produces reporter virus-like particles (RVPs) which infect cells in a single round. In this study, we aimed to develop a system to generate multivalent RVPs based on KUNV to elicit an immune response against different viruses. We selected the Ebola virus (EBOV) glycoprotein (GP) and the matrix protein (VP40) genes, as candidates to be delivered by KUNV RVPs. Initially, we enhanced the production of KUNV RVPs by generating a stable cell line expressing the KUNV packaging system comprising capsid, precursor membrane, and envelope. Transfection of the DNA-based KUNV replicon into this cell line resulted in an enhanced RVP production. The replicon was expressed in the stable cell line to produce the RVPs that allowed the delivery of EBOV GP and VP40 genes into other cells. Finally, we immunized BALB/cN mice with RVPs, resulting in seroconversion for EBOV GP, EBOV VP40, WNV nonstructural protein 1, and WNV E protein. Thus, our study shows that KUNV RVPs may function as a WNV vaccine candidate and RVPs can be used as a gene delivery system in the development of future EBOV vaccines.
Highlights
The genus Flavivirus includes important viruses that cause worldwide morbidity and mortality
The replicon is driven by a Cytomegalovirus (CMV) promoter expressing an open reading frame (ORF) of Kunjin virus (KUNV) where most of the structural cassette is replaced with firefly luciferase reporter gene (Luc)
We found that cell clone 9 had high levels of E protein compared to all the other clones (Figure 1B)
Summary
The genus Flavivirus (family Flaviviridae) includes important viruses that cause worldwide morbidity and mortality. The genus includes the mosquito-borne neurotropic West Nile virus (WNV) whose infection may lead to severe meningoencephalitis. Over the last two decades, the virus has caused more than 2000 fatal cases in America [1]. The Kunjin virus (KUNV) is a naturally attenuated. WNV strain, which has caused a limited number of nonfatal cases since 1960 [2,3]. The difference in virulence of these two viruses is due to the high sensitivity of KUNV to the type I interferon response [4]. The KUNV nonstructural protein (NS) 5, cannot inhibit interferon-dependent JAK–STAT signaling as the WNV NS5 [5]
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