title: Generation of a Single-Cycle Replicable Rift Valley Fever Vaccine.
abstract: Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is an arbovirus that causes severe disease in humans and livestock in sub-Saharan African countries. The virus carries a tripartite, single-stranded, and negative-sense RNA genome, designated as L, M, and S RNAs. RVFV spread can be prevented by the effective vaccination of animals and humans. Although the MP-12 strain of RVFV is a live attenuated vaccine candidate, MP-12 showed neuroinvasiveness and neurovirulence in young mice and immunodeficiency mice. Hence, there is a concern for the use of MP-12 to certain individuals, especially those that are immunocompromised. To improve MP-12 safety, we have generated a single-cycle, replicable MP-12 (scMP-12), which carries L RNA, S RNA encoding green fluorescent protein in place of a viral nonstructural protein NSs, and an M RNA encoding a mutant envelope protein lacking an endoplasmic reticulum retrieval signal and defective for membrane fusion function. The scMP-12 undergoes efficient amplification in the Vero-G cell line, which is a Vero cell line stably expressing viral envelope proteins, while it undergoes single-cycle replication in naïve cells and completely lacks neurovirulence in suckling mice after intracranial inoculation. A single-dose vaccination of mice with scMP-12 confers protective immunity. Thus, scMP-12 represents a new, promising RVF vaccine candidate. Here we describe protocols for scMP-12 generation by using a reverse genetics system, establishment of Vero-G cells, and titration of scMP-12 in Vero-G cells.
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. amurakam@mail.ecc.u-tokyo.ac.jp.
authors.forename: Shin
authors.initials: S
authors.lastname: Murakami
authors.affiliation: Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, TX, 77555-1019, USA.
authors.forename: Kaori
authors.initials: K
authors.lastname: Terasaki
authors.affiliation: Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, TX, 77555-1019, USA.
authors.forename: Shinji
authors.initials: S
authors.lastname: Makino
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D006801:Humans; D059386:Reverse Genetics / Q000379:methods*; D012296:Rift Valley fever virus / Q000276:immunology; D014709:Vero Cells; D014765:Viral Vaccines / Q000235:genetics* / Q000276:immunology
chemical_list: D014765:Viral Vaccines
keywords: Reverse genetics; Rift Valley fever virus; Single-cycle replicable virus; Stable cell establishment; Transfection; Vaccine