Abstract
Viruses with segmented RNA genomes must package the correct number of segments for synthesis of infectious virus particles. Recent studies suggest that the members of the Reoviridae family with segmented double-stranded RNA genomes achieve this challenging task by forming RNA networks of segments prior to their recruitment into the assembling capsid albeit direct evidence is still lacking. Here, we investigated the capability of virus recovery by preformed complexes of ten RNA segments of H Virus (EHDV), a Reoviridae member, by transcribing exact T7 cDNA copies of genomic RNA segments in a single in vitro reaction followed by transfection of mammalian cells. The data obtained was further confirmed by RNA complexes generated from Bluetongue virus, another family member. Formation of RNA complexes was demonstrated by sucrose gradient ultracentrifugation, and RNA-RNA interactions inherent to the formation of the RNA complexes were demonstrated by electrophoretic mobility shift assay. Further, we showed that disruption of RNA complex formation inhibits virus recovery, confirming that recruitment of complete RNA networks is essential for packaging and consequently, virus recovery. This efficient reverse genetics system will allow further understanding of evolutionary relationships of Reoviridae members and may also contribute to development of antiviral molecules.
Highlights
Orbiviruses (Reoviridae family) are vectored to particular vertebrate species by arthropods replicating in both insect and mammalian hosts
The in vitro co-transcription products of the Epizootic hemorrhagic disease virus (EHDV)-7 segments showed that ssRNA bands were synthesized in correct sizes similar to the authentic transcripts derived from wild type (WT) viral core (Figure 1B)
At 72 hours post-transfection, a cytopathic effect indistinguishable from that caused by WT Epizootic hemorrhagic disease virus serotype 7 (EHDV-7) virus infection, was detected in ~80% of cells transfected by both RNA products (Figure 1C), demonstrating that infectious virus particles were produced
Summary
Orbiviruses (Reoviridae family) are vectored to particular vertebrate species (e.g., sheep, cattle, horses, deer, etc.) by arthropods (gnats, ticks, or mosquitoes depending on the virus) replicating in both insect and mammalian hosts. The two most common orbiviruses are Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV), each with multiple serotypes. Both BTV and EHDV are transmitted from animal to animal by the same Culicoides vectors (gnats) and are responsible for considerable economic losses to international livestock industries [1, 2] Both viruses have been endemic in many parts of the world and recently emerged in countries and regions that were previously free of these viruses. EHDV and BTV are two distinct serogroups within the orbivirus genus, but are morphologically identical with an icosahedral double-capsid structure and a genome of ten (S1-S10) double-stranded RNA (dsRNA) segments, the majority of which encode only a single protein product. The outer capsid is composed of two major proteins, VP2 and VP5, which surround the inner capsid or viral core formed by two other major proteins, VP7 and VP3 as well www.impactjournals.com/oncotarget as the transcription complex of three minor proteins VP1, VP4 and VP6 that are closely associated with the viral genomic dsRNA segments [2, 4, 5]
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