The incursion of bluetongue virus serotype 8 (BTV8) in North-Western Europe was firstly detected in the Netherlands in August 2006, and has resulted in one of the largest recorded BT outbreaks. The threat of (re)emergence of a BTV serotype needs a quick response to supply effective vaccines. There is a long record of development and application of inactivated and live-attenuated or modified-live BT vaccines, which both have advantages and disadvantages 
. Further, experimental vaccines have been developed by different approaches, reviewed in Noad and Roy 
. Recently, reverse genetics was developed for BTV1 
, BTV1 and BTV8 
and BTV1, BTV6 and BTV8 
which can be used to further explore the knowledge of BTV and improve current BT vaccines.
Vaccine-related BTV6/net08 has appeared to be avirulent in the field and by experimental infection of different ruminant species 
. Further, BTV6 regenerated by reverse genetics rgBTV6 (in this study named BTVac-6) was indeed avirulent 
. Therefore, we selected this avirulent BTV strain as genetic backbone for vaccine development and changed the serotype by exchange of Seg-2 and Seg-6 for these of BTV1 and BTV8. Seg-2 and Seg-6 encode for the outer shell proteins VP2 and VP5. In particular VP2 induces a protective humoral neutralising immune response, which is highly specific for the respective serotype (see references in: 
). By this method, BT vaccine for another serotype can be made rapidly by exchange of two segments from circulating or (re)emerging BTV serotypes.
The two outer capsid proteins VP2 and VP5 are responsible for virus entry into the host cells. In mammalian cells BTV entry proceeds via virus attachment to the cell, followed by endocytosis and release of a transcriptionally active core particle into the cytoplasm 
. The structural features of VP2 (propeller-like spike) and VP5 (globular) of the outer capsid correlate with their biological roles in virus entry into the cells 
. The most exposed BTV protein VP2 is the highly variable protein among BTV serotypes and is the determinant of the serotype. Antibodies raised against VP2 neutralise virus infectivity supporting the fact that VP2 is the cellular receptor binding protein of the virus 
. The globular outer capsid protein VP5 is likely to be the membrane penetration protein. VP5 protein shares certain secondary structural features with the fusion proteins of enveloped viruses, indicating that it may play a role in virus penetration activity 
. The outer shell proteins VP2 and VP5 of BTV6 were exchanged with that of BTV1 or BTV8 and therefore this might influence some of the functions mentioned above like infectivity and/or virulence.
In this study two ‘serotyped’ BTVs were generated by completing the backbone of BTV6 (eight segments) with Seg-2 and Seg-6 from BTV1 or BTV8 resulting in BTVac-1 and BTVac-8, respectively. In line with this, rgBTV6 
was re-named BTVac-6 in this study. Rescued vaccine viruses (BTVac-x; x presents the serotype) were characterized and compared.
Still, initial virus titres were comparable (within 0.5 log10 TCID50/ml) at 24 hpi for the generated BTVac-x viruses. The maximum titre of BTVac-1 was even slightly higher (0.2–1 log10 TCID50/ml) at all following time points (48, 72 and 96 hpi), whereas the original BTVac-6 was the lowest at each of the following time point (). Apparently, these outer shell proteins fit well on the BTV6 core particle. Since the difference between these BTVac-x viruses is limited to Seg-2 and Seg-6, it can be concluded that the observed small growth advantage for the two serotyped viruses is caused by VP2 and/or VP5. It is unknown whether this small difference in virus replication in vitro also reflects a difference in virus replication in vivo.
Unfortunately, a non-intended lower dose of BTVac-6 was used for vaccination of sheep compared with respect to the ‘serotyped’ vaccine viruses BTVac-1 and BTVac-8. Although the sheep of the BTVac-6 group were slightly delayed in developing fever and PCR-positivity, all sheep seroconverted by ELISA between 7–9 dpi () which was comparable to the other vaccinated groups. This indicated that the lower dose of 1 ml of 101.4 TCID50/ml BTVac-6 is sufficient to vaccinate animals and resulted in comparable seroconversion with respect to vaccination with 1 ml of 105 TCID50/ml of BTVac-1 or BTVac-8. As a consequence of the lower titre of the virus stock, the one-third amount of BTVac-6 in the CombiVac group was also lower and was much less than for the other two BTVac-x vaccine viruses. Apparently, the amount of BTVac-6 in this combination was limited, since BTVac-6 could not be detected in all vaccinated sheep (). Further, two out of four sheep did not raise significant nAb titres specific for serotype 6, and the two other sheep showed only very low nAb titres for serotype 6 (). From these data, however, it is not clear whether this is caused by the limited amount of BTVac-6, negative interference by the excess of other BTVac-x viruses, or by the slightly slower replication rate of BTVac-6 as observed in vitro.
Besides fever, only very mild clinical signs () were observed in vaccinated sheep, which was also seen in experimental infections of BTV6/net08 and rgBTV6 (here named BTVac-6) 
. It can therefore be concluded that VP2 and VP5, of which these originated from virulent BTV8/net07 in BTVac-8, are not associated with virulence. Sheep in the CombiVac group showed similar clinical signs but for a longer period of time. Likely, sheep react stronger after vaccination with immunogenic different viruses and/or needs more time to recover from vaccination. For future research, this observation should have to be taken into account in the light of combining BTVac-x vaccine viruses.
Sheep of the control group showed several days with fever after challenge with BTV8/net07, whereas vaccinated sheep did not. The control group had an average CRI of 8, whereas average CRI's of the vaccinated groups were <2. This indicated that one single vaccination clearly reduce clinical disease in sheep. Sheep in the CombiVac group seemed to be a little less protected (), but it is unclear whether the measured CRI's after challenge are caused by challenge virus, and thus are less protected by CombiVac, or are the result of prolonged clinical reaction by CombiVac vaccination, or are the sum of both.
No challenge virus was detected in vaccinated sheep, indicating that a single vaccination completely blocks replication of challenge virus. No seroconversion or booster of the humoral neutralising immune response specific for serotype 8 was measured for vaccinated sheep after challenge. This confirms the absence of replication of the virulent challenge virus BTV8/net07.
Prior to challenge, vaccinated animals developed significant nAb titres against the respective serotype and very low nAb titres or none at all for the other serotypes. Still all vaccinated sheep, irrespective of the used vaccine virus, were protected from challenge with BTV8/net07, thus including the groups vaccinated with BTVac-1 and BTVac-6. We suggest that cross-protection as observed in this study is due to a nonspecific cell-mediated immune response. Cross-protective immune response to BTV has been described 
. This cross-protective response involves VP2 and NS1 as major ovine CTL immunogens of which NS1 is cross-protective and VP2-specific CTL responses are not 
A longer time period, e.g. four weeks or more, after vaccination with live-attenuated vaccine will show serotype specific protection. Anti-BTV CTL's showing serotype cross-reactivity have been demonstrated to peak between 7 and 21 days after infection 
and even extends to 66 days after multiple immunization 
. Still, the detected serotype specific nAb titre after vaccination with BTVac-x viruses is very promising in the light of long lasting protection for the respective serotypes, and can be expected to be very similar as for live-attenuated vaccines.
The selected vaccine virus background BTVac-x is safe, by which x represents the serotype of the outer shell proteins, even if completed with outer shell proteins of virulent BTV8/net07 (). Apparently, no virulence markers are located on VP2 or VP5 of BTV8/net07. Small differences in nAbs titres could be caused by the lower dose (101.4 TCID50/ml) for BTVac-6 than for the other vaccinated groups (105 TCID50/ml). Further, for combination vaccines consisting of BTVac-x viruses, the amount of each BTVac-x have to be comparable, since negative interference between different BTVAc-x viruses could be expected resulting in a less pronounced viremia and a lower level of nAbs titres ( and ).
The quick generation of BTVac-x vaccine viruses by use of the genetic backbone of vaccine-related BTV6/net08 can also be used to generate safe BTVac-x vaccine virus for other serotypes. Serotyping of rgBTV6 (BTVac-6) presented here for serotypes 1 and 8 is promising for serotyping for other BTV serotypes. Indeed, ‘serotyping’ can be extended for other serotypes but is not unlimited. We were able to ‘serotype’ for TOV, the proposed 25th
, and a few others but not for all BTV serotypes 
. In conclusion, these results show the strategy to develop faster BT vaccines for desired BTV serotypes. However, issues regarding compatibility of Seg-2 and Seg-6 from other serotypes need to be addressed, in particular with respect to protein-protein interactions between these outer shell proteins and the core of BTV6/net08.
Since both live-attenuated and inactivated vaccines currently have a history of safety issues, improvement of safety is an important issue. Recently, protection against BTV8 by replication-defective BTV1 with VP2 of BTV8 was shown 
. These data indicate that VP2 of the respective serotype is sufficient to induce protection, although nonhomologous challenge was not included in this study. Thus, serotype-independent protection could be involved as observed for BTVac-x vaccine viruses in this study. BTVac-x vaccine viruses can be easily generated and are effective in the induction of serotype-specific nAbs as soon as 21 dpi after a single vaccination. Most likely, these tested BTVac-x vaccine viruses will be protective for their respective serotype, like it has been observed for traditionally generated live-attenuated BT vaccines.
Development of DIVA vaccines (DIVA: Differentiating Infected from Vaccinated Animals) is of significant importance to control Bluetongue. BTVac-x vaccines induce a complete immune response against the respective serotypes. Consequently, animals vaccinated with BTVac-x vaccines cannot be distinguished by serological testing from animals infected by the respective BTV serotype. In 2008, BTV1 and BTV6 were detected by genotyping on Seg-10 in the infected and vaccinated ruminant population for BTV8 
. This method is irrespective of the serotype, and combines the high-throughput panBTV PCR assay 
and sequencing of amplicons between the PCR-primer positions. Here, we have improved this method for routine use to differentiate between virulent BTV8 and BTVac-x vaccines. Note that all BTVac-x vaccines have the same genetic background of BTV6, and that field BT-viruses will be detected/identified by the high genetic variation in Seg-10. Thus, BTVac-x vaccines in combination with genotyping on Seg-10 are DIVA-vaccines to detect infectious animals in vaccinated populations.
The system of tailor-made vaccines by exchange of outer shell proteins for those of other serotypes in a defined avirulent genetic backbone offers more advantages, like fully defined genomes, and similar growth characteristics in vivo and in vitro. Moreover, these ‘serotyped’ BTVac-x vaccine viruses share eight out of ten genome segments, and consequently the chance on new undesired (virulent) reassortants after mixing these, e.g. for multi-serotype vaccines, is negligible. In addition, the proteins VP2 and VP5 that differ between BTVac-x vaccine viruses do not harbor virulence markers for the tested BTV serotypes. Further, due to the same replication machinery, negative interference between BTVac-x vaccine viruses after vaccination is reduced to a minimum, although equal amounts of each BTVac-x virus seems to be important as shown in this study. Finally, by the similar growth characteristics in vitro of BTVac-x viruses, similar costs for virus production of each BTVac-x virus could be expected. Noteworthy, these BTVac-x viruses can be used as live-attenuated BT vaccine or, in order to address safety issues, as inactivated BT vaccine. Research is in progress to start ‘serotyping’ of BTV6/net08 for other serotypes in order to develop virus stocks of more BTVac-x viruses for vaccine production.