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.

Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUGMet→GCCAla mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.

Domestic ducks play an important role in the epidemiology of H5N1 avian influenza. Although it is known that vaccines that have a high homology with the challenge virus are able to prevent infection in ducks, little is yet known about the ability of genetically more distant vaccines in preventing infection, disease, and transmission. Here we study the effect of a widely used H5N2 vaccine (A/Chicken/Mexico/232/94/CPA) on the transmission of H5N1 virus (A/Chicken/China/1204/04) in ducks. The quantitative analyses show that despite the low level of homology between the virus and vaccine strain transmission was significantly reduced two weeks after a single or double vaccination. Mortality and disease rates were reduced markedly already one week after a single vaccination.

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate “synthetic” reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replication in vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes.IMPORTANCE African horse sickness virus is transmitted by species of Culicoides biting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replication in vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since the in vitro release of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

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. IN 2008, BTV6 and BTV11 were reported in the Netherlands and Germany, and in Belgium, respectively. 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 BTV serotypes needs a rapid response to supply effective vaccines. Reverse genetics has been developed for BTV1 and more recently also for BTV6. This latter strain, BTV6/net08, is closely related to live-attenuated vaccine for serotype 6 as determined by full genome sequencing. Here, we used this strain as backbone and exchanged segment 2 and 6, respectively Seg-2 (VP2) and Seg-6 (VP5), for those of BTV serotype 1 and 8 using reverse genetics. These so-called ‘serotyped’ vaccine viruses, as mono-serotype and multi-serotype vaccine, were compared for their protective capacity in sheep. In general, all vaccinated animals developed a neutralizing antibody response against their respective serotype. After challenge at three weeks post vaccination with cell-passaged, virulent BTV8/net07 (BTV8/net07/e1/bhkp3) the vaccinated animals showed nearly no clinical reaction. Even more, challenge virus could not be detected, and seroconversion or boostering after challenge was negligible. These data demonstrate that all sheep were protected from a challenge with BTV8/net07, since sheep of the control group showed viremia, seroconversion and clinical signs that are specific for Bluetongue. The high level of cross-protection is discussed.