Construction and application of chimeric virus-like particles of tick-borne encephalitis virus and mosquito-borne Japanese encephalitis virus

Abstract

We have previously reported a system for packaging tick-borne encephalitis (TBE) virus subgenomic replicon RNAs into single-round infectious virus-like particles (VLPs) by using in trans expression of viral C/prM/E structural proteins. In this study, the trans-packaging system was applied to the generation of chimeric VLPs with mosquito-borne Japanese encephalitis (JE) virus. Although trans-expression of TBE virus C and JE virus prM/E proteins resulted in the secretion of VLPs, the expression of JE virus C/prM/E proteins did not lead to the secretion of VLPs, suggesting that homologous interaction between C and non-structural proteins or the genomic RNA is important for efficient assembly of infectious particles. Neutralization testing showed that the antigenic characteristics of the VLPs were similar to those of the native virus. Furthermore, the infectivities of the TBE virus-and JE virus-enveloped VLPs for the ISE6 tick cell line and C6/36 mosquito cell line were investigated. The VLPs were able to enter only those cells that were derived from the natural vectors for the respective viruses. TBE virus replicon RNA packaged in VLPs produced TBE virus non-structural proteins in tick cells, but could neither replicate nor produce viral proteins in mosquito cells. These findings indicate the importance of specific cellular factors for virus entry and replication during flavivirus infection of arthropods. These results demonstrate that chimeric VLPs are useful tools for the study of viral genome packaging and cellular factors involved in vector specificity, with the additional safety aspect that these chimeric VLPs can be used instead of full-length chimeric viruses. INTRODUCTIONThe genus Flavivirus (family Flaviviridae) contains important human pathogens, including tick-borne encephalitis (TBE) virus, Japanese encephalitis (JE) virus, yellow fever virus, dengue virus and West Nile (WN) virus. Flaviviruses can be divided into three phylogenetic and ecological groups: the tick-borne group, the mosquito-borne group and the no-known-vector group (Gaunt et al., 2001;Gould et al., 2003;Kuno et al., 1998). The extent of transmission of arthropod-borne viruses depends on both ecological and physiological parameters, of which vector competence is the most important factor. Vector competence is determined by extrinsic and intrinsic factors, such as the physiological ability of vector tissue to become infected and to maintain a particular infectious agent (Hardy et al., 1983;Kramer & Ebel, 2003;Nuttall & Labuda, 2003). The involvement of these factors in flavivirus infection is not well understood.The flavivirus genome consists of a positive-polarity, single-stranded RNA of approximately 11 kb, which encodes three structural proteins, i.e. the core (C), premembrane (prM) and envelope (E) proteins, and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5), within a single long open reading frame (Chambers et al., 1990). The 59-and 39-untranslated regions (UTRs) have predicted secondary structures that ...

Establishment of a neutralization test involving reporter gene-expressing virus-like particles of tick-borne encephalitis virus

“…As shown in Fig. 2B, sequential transfection with the Oshima-REP-luc2A replicon and the pcTBECME plasmid resulted in the production of VLPs, and luciferase expression was confirmed in the cells that were infected with VLPs that contained the Oshima-REP-luc2A replicon (luc-VLPs).In previous studies, the envelope glycoproteins of the VLPs exhibited the same antigenicities as those of the authentic virion (Yoshii et al, 2005;Yoshii et al, 2008). To apply the VLPs that express reporter genes to neutralization testing, we examined whether infection with VLPs was neutralized by virus-specific neutralizing antibodies.…”

“…However, since TBEV is classified as a biosafety level 3 or 4 virus, a high-level biocontainment facility is required to handle the live virus required for neutralization testing. Virus-like particles (VLPs) of flavivirus are produced by the complementation of replicon RNA with the viral structural genes expressed in trans (Gehrke et al, 2003;Khromykh et al, 1998;Scholle et al, 2004;Yoshii et al, 2008). The VLPs of flaviviruses are similar to the native virus with respect to their antigenic and functional characteristics.…”

“…Neutralization testing using VLPs can be also applied to serological diagnoses of other flaviviruses though the construction of pseudo-typed VLPs. In a previous study, pseudo-typed VLPs with envelope proteins derived from Japanese encephalitis virus were constructed (Yoshii et al, 2008). The antigenic characteristics of the pseudo-typed VLPs are similar to those of the native virus.…”

“…In previous studies, the envelope glycoproteins of the VLPs exhibited the same antigenicities as those of the authentic virion (Yoshii et al, 2005;Yoshii et al, 2008). To apply the VLPs that express reporter genes to neutralization testing, we examined whether infection with VLPs was neutralized by virus-specific neutralizing antibodies.…”

Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon

“…In addition, the expression of viral structural proteins in cells harbouring replicon RNA has been shown to produce single-round infectious particles (SRIPs), which are infectious, but progeny viruses cannot be spread from the infected cells, as the packaged genome lacks structural protein genes (Gehrke et al, 2003;Jones et al, 2005;Khromykh et al, 1998;Ng et al, 2007;Scholle et al, 2004;Yun et al, 2009). Furthermore, trans-packaging of replicons by the prM-E proteins from heterologous flaviviruses have been reported (Ansarah-Sobrinho et al, 2008;Yoshii et al, 2008). risk of infection.…”