title: Development of a novel protocol for generating flavivirus reporter particles.
abstract: Infection with West Nile virus (WNV), a mosquito-borne flavivirus, is a growing public and animal health concern worldwide. Prevention, diagnosis and treatment strategies for the infection are urgently required. Recently, viral reverse genetic systems have been developed and applied to clinical WNV virology. We developed a protocol for generating reporter virus particles (RVPs) of WNV with the aim of overcoming two major problems associated with conventional protocols, the difficulty in generating RVPs due to the specific skills required for handling RNAs, and the potential for environmental contamination by antibiotic-resistant genes encoded within the genome RNA of the RVPs. By using the proposed protocol, cells were established in which the RVP genome RNA is replicated constitutively and does not encode any antibiotic-resistant genes, and used as the cell supply for RVP genome RNA. Generation of the WNV RVPs requires only the simple transfection of the expression vectors for the viral structural proteins into the cells. Therefore, no RNA handling is required in this protocol. The WNV RVP yield obtained using this protocol was similar that obtained using the conventional protocol. According to these results, the newly developed protocol appears to be a good alternative for the generation of WNV RVPs, particularly for clinical applications.
authors.affiliation: Laboratory of Environmental Hygiene, Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto-City 603-8555, Japan.
authors.forename: Igor Velado
authors.initials: IV
authors.lastname: Fernández
authors.affiliation: Laboratory of Environmental Hygiene, Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto-City 603-8555, Japan.
authors.forename: Natsumi
authors.initials: N
authors.lastname: Okamoto
authors.affiliation: Laboratory of Environmental Hygiene, Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto-City 603-8555, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Ito
authors.affiliation: Laboratory of Environmental Hygiene, Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto-City 603-8555, Japan; Laboratory of Bacteriology, Department of Animal Medical Sciences, Faculty of Life Science, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto-City 603-8555, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Fukuda
authors.affiliation: Laboratory of Bacteriology, Department of Animal Medical Sciences, Faculty of Life Science, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto-City 603-8555, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Someya
authors.affiliation: Laboratory of Virology, Department of Animal Medical Sciences, Faculty of Life Science, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto-City 603-8555, Japan.
authors.forename: Yosii
authors.initials: Y
authors.lastname: Nishino
authors.affiliation: Laboratory of Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan; Laboratory of Experimental Animal Science, Faculty of Veterinary Medicine, Kitasato University, School of Veterinary Medicine and Animal Science, 35-1 Higashi 23 Bancho, Towada, Aomori 034-8626, Japan.
authors.forename: Nobuya
authors.initials: N
authors.lastname: Sasaki
authors.affiliation: Laboratory of Environmental Hygiene, Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto-City 603-8555, Japan. Electronic address: amaeda@cc.kyoto-su.ac.jp.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Maeda
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D014773:Virology / Q000379:methods*; D014902:West Nile virus / Q000235:genetics / Q000502:physiology*
chemical_list: D008164:Luminescent Proteins
keywords: Protocol; Reporter virus particles; West Nile virus