Towards establishing a reverse genetics system to recover infectious African horse sickness virus

Abstract

African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, is an arthropod-borne virus that is capable of causing severe disease in horses. Although progress has been made regarding structure-function analyses of individual AHSV proteins, studies into AHSV biology would be greatly enhanced if a reverse genetic system was available whereby individual genome segments could be genetically manipulated. Consequently, the aim of this study was essentially to develop a reverse genetic system for AHSV that would allow recovery of infectious from recombinant sources and/or allow for the targeted introduction of cDNA-derived genome segments into the viral genome. Towards establishing a reverse genetic system for AHSV, it was first determined whether in vitro-transcribed AHSV ssRNA is infectious. The results indicated that infectious virus could be recovered following transfection of permissive cells with purified AHSV-4 core-derived ssRNA. These results therefore suggested that infectious AHSV may be recovered from recombinant sources, provided that the AHSV ssRNA bear authentic 5’- and 3’-terminal sequences and are capped at their 5’ end. Subsequently, two DNA-based and a synthetic mRNA-based reverse genetic approach was evaluated for their ability to recover AHSV-4. The use of an entirely plasmid DNA-based reverse genetic system, in which full-length cDNA copies of the AHSV-4 genome segments are flanked by an upstream T7 RNA polymerase promoter and by a downstream hepatitis delta virus (HDV) ribozyme sequence, failed to recover AHSV-4 in BSR-T7 mammalian cells. Likewise, transfection of the mammalian cells with the T7 transcription cassettes of each cloned AHSV-4 cDNA genome segment did not result in the recovery of infectious AHSV-4. Similar results were obtained when a mixture of in vitro-synthesised and -capped AHSV-4 T7 transcripts, using the T7 transcription cassettes as templates in these reactions, were transfected into BSR cells. The inability to recover infectious AHSV-4 from these recombinant sources may have been due to different technical complexities, including inefficient capping of transcripts that may lead to the activation of antiviral responses and difficulties associated with transfection of cells with a full complement of the ten DNA constructs or ssRNA transcripts. Consequently, it was next investigated whether recombinant AHSV could be generated by targeted replacement of a single genome segment with a cDNA-derived genome segment. Transfection of BSR cell monolayers with a mixture of in vitro-synthesised and -capped AHSV-4 segment 10 T7 transcripts and AHSV-3 core-derived ssRNA yielded reassortant plaques, of which the identity was confirmed by a serogroup discriminating polymerase chain reaction assay and nucleotide sequencing of the genome segment 10 amplicon. The recovery of recombinant AHSV containing a plasmid cDNA-derived genome segment not only represents a valuable milestone toward the development of a reverse genetic system for AHSV, but also is a powerful tool for studies aimed at understanding AHSV biology. This reverse genetic approach is potentially applicable to all genome segments and has the potential to be used as a tool for future investigations into the functions of viral proteins in replicating AHSV, as well as the elucidation of genetic factors involved in viral pathogenesis and virulence.