Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA.
An infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was constructed by stably inserting genome-length cDNA into the low-copy-number plasmid vector pMC18. Designated pMVE-1-51, the clone consisted of genome-length cDNA of MVE-1-51 under the control of a T7 RNA polymerase promoter. The clone was constructed by using existing components of a cDNA library, in addition to cDNA of the 3' terminus derived by RT-PCR of poly(A)-tailed viral RNA. Upon comparison with other flavivirus sequences, the previously undetermined sequence of the 3' UTR was found to contain elements conserved throughout the genus FLAVIVIRUS: RNA transcribed from pMVE-1-51 and subsequently transfected into BHK-21 cells generated infectious virus. The plaque morphology, replication kinetics and antigenic profile of clone-derived virus (CDV-1-51) was similar to the parental virus in vitro. Furthermore, the virulence properties of CDV-1-51 and MVE-1-51 (LD(50) values and mortality profiles) were found to be identical in vivo in the mouse model. Through site-directed mutagenesis, the infectious clone should serve as a valuable tool for investigating the molecular determinants of virulence in MVE virus.
Hurrelbrink
Robert J
RJ
Department of Microbiology, University of Western Australia, Nedlands, WA 6907, Australia1.
Nestorowicz
Ann
A
Endocrine Division, Lilly Research Laboratories, Indianapolis, IN 46285, USA2.
McMinn
Peter C
PC
Department of Microbiology, University of Western Australia, Nedlands, WA 6907, Australia1.
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018068:Encephalitis Virus, Murray Valley / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity*; D004671:Encephalitis, Arbovirus / Q000473:pathology / Q000821:virology*; D005455:Fluorescent Antibody Technique; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014709:Vero Cells; D010948:Viral Plaque Assay; D014774:Virulence; D014779:Virus Replication
D020413:3' Untranslated Regions; D018076:DNA, Complementary; D012367:RNA, Viral