A Baculovirus-Expressed Dicistrovirus That Is Infectious to Aphids

Abstract

Detailed investigation of virus replication is facilitated by the construction of a full-length infectious clone of the viral genome. To date, this has not been achieved for members of the family Dicistroviridae. Here we demonstrate the construction of a baculovirus that expresses a dicistrovirus that is infectious in its natural host. We inserted a full-length cDNA clone of the genomic RNA of the dicistrovirus Rhopalosiphum padi virus (RhPV) into a baculovirus expression vector. Virus particles containing RhPV RNA accumulated in the nuclei of baculovirus-infected Sf21 cells expressing the recombinant RhPV clone. These virus particles were infectious in R. padi, a ubiquitous aphid vector of major cereal viruses. The recombinant virus was transmitted efficiently between aphids, despite the presence of 119 and 210 vector-derived bases that were stably maintained at the 5 and 3 ends, respectively, of the RhPV genome. The maintenance of such a nonviral sequence was surprising considering that most RNA viruses tolerate few nonviral bases beyond their natural termini. The use of a baculovirus to express a small RNA virus opens avenues for investigating replication of dicistroviruses and may allow large-scale production of these viruses for use as biopesticides.

Successful Transcription but not Translation or Assembly ofSolenopsis InvictaVirus 3 in a Baculovirus-Driven Expression System

“…Positive-strand RNA viruses exhibit comparatively higher mutation rates because of the lack of proofreading machinery, template switching, and other factors which could have contributed to our difficulties. However, previous use of this method with Rhopalosiphum padi virus (RhPV) showed that non-virus nucleotide insertions at the 5' and 3' UTR termini had no effect on the ability to produce infectious virus (Pal et al 2007). Whether our deletions had an impact on translation was not determined.…”

“…At present, there is no available means of propagating infectious SINV-3 particles in vitro, which has hampered its evaluation and development as a microbial insecticide. However, Pal et al (2007) successfully produced a similar virus, Rhopalosiphum padi virus (RhPV) (Picornavirales: Dicistroviridae), in quantity with a recombinant baculovirus system capable of expressing the entire genome of the virus in surrogate insect cells. As there is no available cell line for fire ants, and the method had been established for a similar virus, baculovirus-driven expression seemed to be a viable solution to the production of SINV-3.…”

“…In this study, our objective was to utilize a baculovirus expression system as reported by Pal et al (2007) to generate and produce SINV-3. Although unsuccessful at producing assembled virus particles, the SINV-3/bacmid construct did yield full-length transcripts of the SINV-3 genome from infected Sf21 cells.…”

“…Although unsuccessful at producing assembled virus particles, the SINV-3/bacmid construct did yield full-length transcripts of the SINV-3 genome from infected Sf21 cells. Recently, Pal et al (2014) published a corrigendum to their original work (Pal et al 2007) in which they discuss their inability to repeat the expression of RhPV. Indeed, the authors apparently never successfully expressed RhPV with the baculovirus system (Pal et al 2014).…”

“…Recently, Pal et al (2014) published a corrigendum to their original work (Pal et al 2007) in which they discuss their inability to repeat the expression of RhPV. Indeed, the authors apparently never successfully expressed RhPV with the baculovirus system (Pal et al 2014). This new information compelled us to make the scientific community aware of our own failure to manufacture SINV-3 in a surrogate system.…”