Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.
Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.
Ikegami
Tetsuro
T
Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA.
Won
Sungyong
S
Peters
C J
CJ
Makino
Shinji
S
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005821:Genetic Techniques; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D012296:Rift Valley fever virus / Q000235:genetics* / Q000254:growth & development* / Q000472:pathogenicity; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D018076:DNA, Complementary; C423923:NSs protein, Bunyamwera bunyavirus; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D008156:Luciferases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases