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 ...