Developing Virus-Like Particles (VLPs) and Heterologous VLPs Vaccines for Epizootic Hemorrhagic Disease Virus (EHDV) Serotypes

Epizootic Hemorrhagic Disease Virus (EHDV) is an insect-transmitted pathogen of ruminants, causing periodic and significant losses in wild and captive deer populations and less frequently, a bluetongue-like disease in cattle. The serogroup of EHDV within the Orbivirus genus of the Reoviridae family consists of seven serotypes, in which emerging serotypes pose an increasing risk either regionally or globally, due to the insect vectors. To date, no vaccine against EHDV is commercially available, apart from the live-attenuated vaccine for EHDV-2 (IBAV). In this study, Virus-Like Particles (VLPs) of EHDV-1 and heterologous VLPs of EHDV-2 were generated using baculovirus multigene expression system for the synthesis of the two outer and two inner capsid proteins, essential for the formation of VLPs. The assembly of EHDV-1 recombinant structural proteins into Core-Like particles (CLPs, two proteins) and (VLPs, four proteins) was confirmed by EM analysis. The biological activity of the raised antisera to neutralise EHDV-1 was efficiently confirmed by neutralisation assay at 1:64 dilution. Cross neutralising activities were also detected against EHDV-2 and EHDV-6 serotypes at 1:8 dilution. Results presented in this study validate the potential efficacy of the VLP as a neutralising vaccine and strongly suggest its use as vaccine candidate. Additionally, an alternative approach was also initiated in this research to develop a rational vaccine against EHDV-2 using the reverse genetics system (RG). Towards this, it was first established that in vitro synthesised transcripts from purified EHDV-2 cores could generate infectious virus upon cell transfection. Note that both the generation of core transcripts and recovery of infectious virus of EHDV were not demonstrated previously. Subsequently a complete set of 10 T7 transcripts was synthesised, however, it was not possible to recover any infectious virus, likely to be some unwarranted mutations. Nevertheless, these transcripts will be further investigated for future RG studies

The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides

Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses

The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides

Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses