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Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA
Robert J Hurrelbrink1, Ann Nestorowicz2, Peter C McMinn1
1Department of Microbiology, University of Western Australia, Nedlands, WA 6907, Australia1.
Abstract:
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.
Insights
Researchers created an infectious clone of Murray Valley encephalitis virus (MVEV). This MVEV clone accurately replicates parental virus characteristics, offering a tool for virulence studies.
Area of Science:
- Virology
- Molecular Biology
Background:
- Murray Valley encephalitis virus (MVEV) is a significant flavivirus pathogen.
- Understanding MVEV virulence determinants is crucial for disease control.
Purpose of the Study:
- To construct a functional infectious cDNA clone of the MVEV prototype strain 1-51 (MVE-1-51).
- To characterize the biological properties of the virus derived from the infectious clone.
- To establish a tool for investigating MVEV virulence factors.
Main Methods:
- Genome-length cDNA of MVE-1-51 was inserted into a low-copy-number plasmid vector under a T7 RNA polymerase promoter.
- The 3' untranslated region (UTR) sequence was determined and analyzed for conserved flavivirus elements.
- RNA transcribed from the clone was transfected into BHK-21 cells to generate infectious virus.
- Clone-derived virus (CDV-1-51) was compared to the parental MVE-1-51 in vitro (plaque morphology, replication, antigenicity) and in vivo (virulence, LD50).
Main Results:
- An infectious cDNA clone, pMVE-1-51, was successfully constructed.
- The 3' UTR contained conserved elements found in the Flavivirus genus.
- Transfection of transcribed RNA yielded infectious virus (CDV-1-51).
- CDV-1-51 exhibited similar in vitro characteristics and identical in vivo virulence to the parental MVE-1-51.
- LD50 values and mortality profiles were indistinguishable between CDV-1-51 and MVE-1-51.
Conclusions:
- The infectious cDNA clone of MVEV is a viable tool for MVEV research.
- The clone faithfully replicates the parental virus phenotype.
- This tool will facilitate studies on the molecular basis of MVEV virulence through site-directed mutagenesis.