Recovery of pathogenic measles virus from cloned cDNA.
Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700-705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773-5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.
Takeda
M
M
Department of Viral Diseases and Vaccine Control, National Institute of Infectious Diseases, Musashi-murayama, Tokyo 208-0011, Japan.
Takeuchi
K
K
Miyajima
N
N
Kobune
F
F
Ami
Y
Y
Nagata
N
N
Suzaki
Y
Y
Nagai
Y
Y
Tashiro
M
M
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008252:Macaca fascicularis; D008459:Measles virus / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity
D018076:DNA, Complementary; D019590:Nucleocapsid Proteins; C055859:P protein, Sendai virus; D010750:Phosphoproteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases