Abstract
West Nile virus (WNV) is a neurotropic pathogen which causes zoonotic disease in humans. Recently, there have been an increasing number of infected cases and there are no clinically approved vaccines or effective drugs to treat WNV infections in humans. The purpose of this study was to facilitate vaccine and antiviral drug discovery by developing a packaging cell line-restricted WNV infectious replicon particle system. We constructed a DNA-based WNV replicon lacking the C-prM-E coding region and replaced it with a GFP coding sequence. To produce WNV replicon particles, cell lines stably-expressing prM-E and C-prM-E were constructed. When the WNV replicon plasmid was co-transfected with a WNV C-expressing plasmid into the prM-E-expressing cell line or directly transfected the C-prM-E expressing cell line, the replicon particle was able to replicate, form green fluorescence foci, and exhibit cytopathic plaques similar to that induced by the wild type virus. The infectious capacity of the replicon particles was restricted to the packaging cell line as the replicons demonstrated only one round of infection in other permissive cells. Thus, this system provides a safe and convenient reporter WNV manipulating tool which can be used to study WNV viral invasion mechanisms, neutralizing antibodies and antiviral efficacy.
Highlights
West Nile virus (WNV) is a neurotropic flavivirus and the etiologic agent responsible for West Nile encephalitis in humans[1]
Similar packaging systems have been developed for several other flaviviruses, such as WNV replicons[10,11,12], Kunjin virus (KUNV)[13], tick-borne encephalitis virus (TBEV)[14, 15] and dengue virus[16, 17]
All goose and horse sera positive in the ELISA were positive by PRNT (Table 1). These results indicate that the ΔWNV-GFP/BWNV-CME cell system could be used for testing neutralizing antibodies
Summary
West Nile virus (WNV) is a neurotropic flavivirus and the etiologic agent responsible for West Nile encephalitis in humans[1]. As described (11), after serial passages they detected a rapid loss of replicon-encoded reporter gene activity In both of these cell lines, the WNV structural protein genes were not integrated into the cell genome. We successfully established a WNV reporter replicon particles packaging system based genome integrated cell lines simultaneously expressing two (prM-E) or all three (C-prM-E) WNV structural proteins. When the reporter replicon particles infect BWNV-CME cell lines, they can spread and morphologically alter the infected cells by mimicking the wild type virus, in which plaque formation can be readily visualised and enumerated.
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