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West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system.

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.

SUBMITTER: Li W

PROVIDER: S-EPMC5468312 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system.

Li Wei W Ma Le L Guo Li-Ping LP Wang Xiao-Lei XL Zhang Jing-Wei JW Bu Zhi-Gao ZG Hua Rong-Hong RH

Scientific reports 20170612 1

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 ...[more]

PMID: 28607390

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Project description:The genus Flavivirus currently consists of approximately 80 single-strand positive-sense RNA viruses. These replicate in a range of hosts including myriad vertebrate, insect, and tick species. As a consequence of this broad host range, the majority of flaviviruses can be propagated in most vertebrate and insect cell cultures. This ability to infect arthropods and vertebrates usually is essential for maintenance of these viruses in nature. But recently, there has been the discovery of a number of flaviviruses that infect mosquitoes but not vertebrates. It remains largely unknown why certain flaviviruses infect vertebrates and mosquitoes while others infect mosquitoes or vertebrates exclusively.Here, we initiated in vitro host range studies of Rabensburg virus (RABV), an intermediate between the mosquito-specific and horizontally transmitted flaviviruses, to provide information on the factor(s) that underlie the varying host range of flaviviruses. RABV is an intermediate between the mosquito-specific and horizontally transmitted flaviviruses because it does not infect mammalian or avian cell cultures, house sparrows, or chickens, but it does share genetic characteristics with the Japanese Encephalitis serogroup of flaviviruses.In vitro growth kinetic assays revealed the complete abrogation of RABV growth on Vero and E6 cells incubated at temperatures 35°C and higher, but surprisingly RABV infected, replicated efficiently, and displayed overt cytopathic effects (CPE) on Vero and E6 cell cultures incubated below 35°C. In contrast, RABV was fully viable, replicated efficiently, and displayed overt CPE on C6/36 cells incubated at 28°C or 37°C, thus implicating temperature as an important factor limiting the host range of RABV.These data are critical for further study to more fully identify the determinants that mediate the evolution of biological transmission among flaviviruses. It also will be useful for studies that look to provide a comprehensive molecular definition of flavivirus-host cell interactions. And it will provide a cadre of information to design wet lab experiments to investigate the genetic changes that facilitate host switching, which may lead to new vertebrate pathogens or transmission pathways.

Dataset Information

West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system.

Publications

West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system.

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