Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA

• Published in: Journal of general virology Vol. 80; no. 12; pp. 3115 - 3125
• Main Authors: Hurrelbrink, Robert J, Nestorowicz, Ann, McMinn, Peter C
• Format: Journal Article
• Language: English
• Published: England Soc General Microbiol 01.12.1999
• Subjects: 3' Untranslated Regions - genetics; Animals; Base Sequence; Cell Line; Chlorocebus aethiops; Cloning, Molecular; DNA, Complementary - genetics; Encephalitis Virus, Murray Valley - genetics; Encephalitis Virus, Murray Valley - immunology; Encephalitis Virus, Murray Valley - pathogenicity; Encephalitis, Arbovirus - pathology; Encephalitis, Arbovirus - virology; Flavivirus; Fluorescent Antibody Technique; Mice; Molecular Sequence Data; Murray valley encephalitis virus; Mutagenesis, Site-Directed; Phage T7; Precipitin Tests; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral - biosynthesis; RNA, Viral - genetics; RNA, Viral - metabolism; Vero Cells; Viral Plaque Assay; Virulence; Virus Replication
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/0022-1317-80-12-3115

Department of Microbiology, University of Western Australia, Nedlands, WA 6907, Australia 1 Endocrine Division, Lilly Research Laboratories, Indianapolis, IN 46285, USA 2 Author for correspondence: Peter McMinn.Fax +61 8 9346 2912. e-mail peter.mcminn{at}health.wa.gov.au 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.