Abstract
Most caliciviruses are refractory to replication in cell culture and only a few members of the family propagate in vitro. Rabbit vesivirus (RaV) is unique due to its ability to grow to high titers in several animal and human cell lines. This outstanding feature makes RaV an ideal candidate for reverse genetics studies, an invaluable tool to understand the molecular basis of virus replication, the biological functions of viral genes and their roles in pathogenesis. The recovery of viruses from a cDNA clone is a prerequisite for reverse genetics studies. In this work, we constructed a RaV infectious cDNA clone using a plasmid expression vector, under the control of bacteriophage T7 RNA-polymerase promoter. The transfection of permissive cells with this plasmid DNA in the presence of T7 RNA-polymerase, provided in trans by a helper recombinant poxvirus, led to de novo synthesis of RNA transcripts that emulated the viral genome. The RaV progeny virus produced the typical virus-induced cytopathic effect after several passages of cell culture supernatants. Similarly, infectious RaV was recovered when the transcription step was performed in vitro, prior to transfection, provided that a 5′-cap structure was added to the 5′ end of synthetic genome-length RNAs. In this work, we report an efficient and consistent RaV rescue system based on a cDNA transcription vector, as a tool to investigate calicivirus biology through reverse genetics.
Highlights
Rabbit vesivirus (RaV) was first isolated in the Veterinary Diagnostic Laboratory at Oregon State University, from feces of rabbits suffering gastrointestinal disorders
To efficiently generate a viable virus progeny, an infectious clone must contain a cDNA of the full virus genomic sequence properly placed under the control of a suitable RNA polymerase promoter to direct the transcription of the genetic construct within the cell or in vitro (Boyer and Haenni, 1994)
In the case of RaV, as for the rest of caliciviruses, the first event occurring after genome uncoating in the cytoplasm, is ORF1 translation
Summary
Rabbit vesivirus (RaV) was first isolated in the Veterinary Diagnostic Laboratory at Oregon State University, from feces of rabbits suffering gastrointestinal disorders. The virus was characterized, and a cDNA copy of the genome was cloned and sequenced. The virus was found to be nonenveloped, isometric and around 30 nm in diameter. The viral genome is 8,295 nucleotides (nt) in size and consists of positive-sense single-stranded RNA with a small protein (VPg) covalently linked to its 5 terminus and a 3 poly-A tail of an average length of 85 nt. The genome comprises 3 open reading frames (ORFs) the most 5 of which encodes a 1,880 amino acids polyprotein that yields the non-structural polypeptides upon self-cleavage. The comparative analyses of domain homology and proteolytic cleavage sites with respect to other caliciviruses and picornaviruses allowed the establishment of the number of mature non-structural proteins, their putative functions, and a hypothetical processing map (Figure 1A). RaV ORF1-encoded non-structural proteins include NS1-2 precursor (unknown function), NS3 (NTPase, helicase), Reverse Genetics System for RaV
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
