Abstract
ABSTRACTAfrican horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids. The virion particle is composed of seven proteins organized in three concentric layers, an outer layer made of VP2 and VP5, a middle layer made of VP7, and inner layer made of VP3 that encloses a replicase complex of VP1, VP4, and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious candidate vaccines against all AHSV serotypes, taking into account not only immunogenic and safety properties but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established a highly efficient reverse genetics (RG) system for AHSV serotype 1 (AHSV1) and, subsequently, a VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required the exchange of two to five RNA segments to achieve equivalent titers of particles. All reassortant-defective viruses could be amplified and propagated to high titers in cells complemented with VP6 but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor (IFNAR)-knockout mice. Thus, these defective viruses have the potential to be used for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis, and pathogenesis.IMPORTANCE African horse sickness virus is transmitted by biting midges and causes African horse sickness in equids, with mortality reaching up to 95% in naive horses. Therefore, the development of efficient vaccines is extremely important due to major economic losses in the equine industry. Through the establishment of a highly efficient RG system, replication-deficient viruses of all nine AHSV serotypes were generated. These defective viruses achieved high titers in a cell line complemented with VP6 but failed to propagate in wild-type mammalian or insect cells. Importantly, these candidate vaccine strains showed strong protective efficacy against AHSV infection in an IFNAR−/− mouse model.
Highlights
African horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids
The core particle consists of two concentric layers: the surface VP7 layer and the inner VP3 layer that encloses the genome of 10 double-stranded RNA (dsRNA) segments and a replicase complex of three enzymes, VP1, VP4, and VP6 [11]
Instead of the use of deletions reported for VP6-deficient bluetongue virus (BTV) [22], we introduced 11 stop codons (TAA or TGA) throughout the S9 gene (S9multistop), disrupting the open reading frames (ORFs) of VP6 and NS4 but retaining the length of the S9 RNA, minimizing the genetic pressure on defective AHSV serotype 1 (AHSV1) recovery and replication
Summary
African horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids. We sought to develop highly efficacious candidate vaccines against all AHSV serotypes, taking into account immunogenic and safety properties and virus productivity and stability parameters, which are essential criteria for vaccine candidates To achieve this goal, we first established a highly efficient reverse genetics (RG) system for AHSV serotype 1 (AHSV1) and, subsequently, a VP6-defective AHSV1 strain in combination with in trans complementation of VP6. All reassortant-defective viruses could be amplified and propagated to high titers in cells complemented with VP6 but were totally incompetent in any other cells These replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor (IFNAR)-knockout mice. Through the establishment of a highly efficient RG system, replication-deficient viruses of all nine AHSV serotypes were generated These defective viruses achieved high titers in a cell line complemented with VP6 but failed to propagate in wild-type mammalian or insect cells. At the level of individual proteins, only the structure of a top domain of VP7 has been reported for AHSV4 [16]; a molecular understanding of the structure-function relationship of AHSV proteins is not available and structure-based rational vaccine design for AHSV is not currently possible
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