Multiple genome segments determine virulence of bluetongue virus serotype 8

Bluetongue virus (BTV) causes bluetongue, a major hemorrhagic disease of ruminants. In order to investigate the molecular determinants of BTV virulence, we used a BTV-8 strain minimally passaged in tissue culture (termed BTV8_L in this study) and a derivative strain passaged extensively in tissue culture (BTV8_H), in in vitro and in vivo studies. BTV8_L was pathogenic in both IFNAR^-/- mice and in sheep, while BTV8H was attenuated in both species. To identify genetic changes which led to BTV8_H attenuation we generated 34 reassortants between BTV8_L and BTV8_H. We found that partial attenuation of BTV8L in IFNAR^-/- mice was achieved by simply replacing genomic segment 2 (Seg2, encoding VP2) or Seg10 (encoding NS3) with the BTV8_H homologous segments. Fully attenuated viruses required at least two genome segments from BTV8H, including Seg2 with either Seg1 (encoding VP1), Seg6 (encoding VP6 and NS4) or Seg10 (encoding NS3). Conversely, full reversion of virulence of BTV8H required at least five genomic segments of BTV8_L. We also demonstrated that BTV8_H acquired an increased affinity for glycosaminoglycan receptors during passaging in cell culture due to mutations in its VP2 protein. Replication of BTV8_H was relatively poor in IFN-competent primary ovine endothelial cells compared to BTV8_L, and this phenotype was determined by several viral genomic segments, including Seg4 and Seg9. This study demonstrated that multiple viral proteins contribute to BTV-8 virulence. VP2 and NS3 are primary determinants of BTV pathogenesis, but VP1, VP5, VP4, VP6 and VP7 also contribute to virulence. IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants and it is listed as a notifiable disease by the World Organisation for Animal Health (OIE). The clinical outcome of BTV infection varies considerably and depends on environmental, host and virus specific factors. Over the years, BTV serotypes/strains with various degrees of virulence (including non-pathogenic strains) have been described in different geographical locations. However, no data are available to correlate the BTV genotype to virulence. This study shows that BTV virulence is determined by different viral genomic segments. The data obtained will help to characterize thoroughly the pathogenesis of bluetongue. The possibility to determine the pathogenicity of virus isolates on the basis of their genome sequence will help to design control strategies that fit the risk posed by new emerging BTV strains.