Since 1998, Bluetongue virus (BTV)-serotypes 1, 2, 4, 9, and 16 have invaded European countries around the Mediterranean Basin. In 2006, a huge BT-outbreak started after incursion of BTV-serotype 8 (BTV8) in North-Western Europe. More recently, BTV6 and BTV11 were reported in North-Western Europe in 2008. These latter strains are closely related to live-attenuated vaccine, whereas BTV8 is virulent and can induce severe disease in ruminants, including cattle. In addition, Toggenburg orbivirus (TOV) was detected in 2008 in Swiss goats, which was recognized as a new serotype of BTV (BTV25). The (re-)emergency of known and unknown BTV-serotypes needs a rapid response to supply effective vaccines, and research to study this phenomenon. Recently, orbivirus research achieved an important breakthrough by the establishment of reverse genetics for BTV1. Here, reverse genetics for two recent BTV strains representing virulent BTV8 and avirulent BTV6 was developed. For this purpose, extensive sequencing of full-genomes was performed, resulting in the consensus sequences of BTV8/net07 and BTV6/net08. The recovery of ‘synthetic BTV’, respectively rgBTV8 and rgBTV6, completely from T7-derived RNA transcripts was confirmed by silent mutations by which these ‘synthetic BTVs’ could be genetically distinguished from wild type BTV, respectively wtBTV6 and wtBTV8. The in vitro and in vivo properties of rgBTV6 or rgBTV8 were comparable to the properties of their parent strains. The asymptomatic or avirulent properties of rgBTV6 and the virulence of rgBTV8 were confirmed by experimental infection of sheep. Reverse genetics of the vaccine-related BTV6 provides a perfect start to develop new generations of BT-vaccines. Reverse genetics of the virulent BTV8 will accelerate research on the special features of BTV8, like transmission by species of Culicoides in a moderate climate, transplacental transmission, and pathogenesis in cattle.

Safe and effective coronavirus disease–19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1–skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and recently emerging variants with substitutions in the Spike protein have led to growing concerns over increased transmissibility and decreased vaccine coverage due to immune evasion. Here, sera from recipients of a single dose of our Ad26.COV2.S COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants of concern. All tested variants demonstrated susceptibility to Ad26.COV2.S-induced serum neutralization albeit mainly reduced as compared to the B.1 strain. Most pronounced reduction was observed for the B.1.351 (Beta; 3.6-fold) and P.1 (Gamma; 3.4-fold) variants that contain similar mutations in the receptor-binding domain (RBD) while only a 1.6-fold reduction was observed for the widely spreading B.1.617.2 (Delta) variant.

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Since 1998, several serotypes of Bluetongue virus (BTV) have invaded several southern European countries. In 2006, the unknown BTV serotype 8 (BTV8/net06) unexpectedly invaded North-West Europe and has resulted in the largest BT-outbreak ever recorded. More recently, in 2008 BTV serotype 6 was reported in the Netherlands and Germany. This virus, BTV6/net08, is closely related to modified-live vaccine virus serotype 6, except for genome segment S10. This genome segment is closer related to that of vaccine virus serotype 2, and therefore BTV6/net08 is considered as a result of reassortment. Research on orbiviruses has been hampered by the lack of a genetic modification method. Recently, reverse genetics has been developed for BTV based on ten in vitro synthesized genomic RNAs. Here, we describe a targeted single-gene modification system for BTV based on the uptake of a single in vitro synthesized viral positive-stranded RNA. cDNAs corresponding to BTV8/net06 genome segments S7 and S10 were obtained by gene synthesis and cloned downstream of the T7 RNA-polymerase promoter and upstream of a unique site for a restriction enzyme at the 3'-terminus for run-off transcription. Monolayers of BSR cells were infected by BTV6/net08, and subsequently transfected with purified in vitro synthesized, capped positive-stranded S7 or S10 RNA from BTV8/net06 origin. "Synthetic" reassortants were rescued by endpoint dilutions, and identified by serotype-specific PCR-assays for segment 2, and serogroup-specific PCRs followed by restriction enzyme analysis or sequencing for S7 and S10 segments. The targeted single-gene modification system can also be used to study functions of viral proteins by uptake of mutated genome segments. This method is also useful to generate mutant orbiviruses for other serogroups of the genus Orbivirus for which reverse genetics has not been developed yet.