Prospects of Next-Generation Vaccines for Bluetongue

Bluetongue (BT) is a haemorrhagic disease of wild and domestic ruminants with a huge economic worldwide impact on livestock. The disease is caused by BT-virus transmitted by Culicoides biting midges and disease control without vaccination is hardly possible. Vaccination is the most feasible and cost-effective way to minimize economic losses. Marketed BT vaccines are successfully used in different parts of the world. Inactivated BT vaccines are efficacious and safe but relatively expensive, whereas live-attenuated vaccines are efficacious and cheap but are unsafe because of under-attenuation, onward spread, reversion to virulence, and reassortment events. Both manufactured BT vaccines do not enable differentiating infected from vaccinated animals (DIVA) and protection is limited to the respective serotype. The ideal BT vaccine is a licensed, affordable, completely safe DIVA vaccine, that induces quick, lifelong, broad protection in all susceptible ruminant species. Promising vaccine candidates show improvement for one or more of these main vaccine standards. BTV protein vaccines and viral vector vaccines have DIVA potential depending on the selected BTV antigens, but are less effective and likely more costly per protected animal than current vaccines. Several vaccine platforms based on replicating BTV are applied for many serotypes by exchange of serotype dominant outer shell proteins. These platforms based on one BTV backbone result in attenuation or abortive virus replication and prevent disease by and spread of vaccine virus as well as reversion to virulence. These replicating BT vaccines induce humoral and T-cell mediated immune responses to all viral proteins except to one, which could enable DIVA tests. Most of these replicating vaccines can be produced similarly as currently marketed BT vaccines. All replicating vaccine platforms developed by reverse genetics are classified as genetic modified organisms. This implies extensive and expensive safety trails in target ruminant species, and acceptance by the community could be hindered. Nonetheless, several experimental BT vaccines show very promising improvements and could compete with marketed vaccines regarding their vaccine profile, but none of these next generation BT vaccines have been licensed yet

A cross-sectional study to determine the seroprevalence of bluetongue virus serotype 8 in sheep and goats in 2006 and 2007 in the Netherlands

Background: In August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe. In the course of 2007 it became evident that BTV8 had survived the winter in North-West Europe, re-emerged and spread exponentially. Recently, the European Union decided to start vaccination against BTV8. In order to improve the understanding of the epidemiological situation, it was necessary to execute a cross-sectional serological study at the end of the BT vector season. Cattle were the target species for cross-sectional serological studies in Europe at the end of 2006 and 2007. However, there was no information on the BTV8- seroprevalence in sheep and goats. Results: On the basis of our cross-sectional study, the estimated seroprevalence of BTV8-exposed locations in the Netherlands in 2006 was 0% for goats (95% confidence interval: 0 ¿ 5.6%) and 7.0% for sheep (95% confidence interval: 3.5 ¿ 12.9%). The estimated seroprevalence of BTV-8 exposed locations in 2007 was 47% for goats (95% confidence interval: 36 ¿ 58%) and 70% for sheep (95% confidence interval: 63 ¿ 76%). There was a wide range in within-location seroprevalence in locations with goats and sheep (1 ¿ 100%). A gradient in seroprevalence was seen, with the highest level of seroprevalence in the southern Netherlands, the area where the epidemic started in 2006, and a decreasing seroprevalence when going in a northern direction. Conclusion: There is a much higher estimated seroprevalence of locations with goats exposed to BTV8 than can be inferred from the rather low number of reported clinical outbreaks in goats. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep. The wide range in within-location seroprevalence observed means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between flocks. This should be taken into account when vaccinating animals

Integrated polarization filter for 1550 nm based on a narrow waveguide section

The polarization filter that is presented in this publication is based on a narrow deepetched waveguide. The measured suppression of the TE mode is 20 dB. The achievedbandwidth is larger than 130nm (limited by the setup). The filter width for an operatingrange around 1550 nm is 0.5 μ

Bluetongue virus nonstructural protein NS3/NS3a is not essential for virus replication

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

Bluetongue Viruses Based on Modified-Live Vaccine Serotype 6 with Exchanged Outer Shell Proteins Confer Full Protection in Sheep against Virulent BTV8

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

RNA Elements in Open Reading Frames of the Bluetongue Virus Genome Are Essential for Virus Replication

Members of the Reoviridae family are non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. Bluetongue virus is the prototype orbivirus (family Reoviridae, genus Orbivirus), causing disease in ruminants, and is spread by Culicoides biting midges. Obviously, several steps in the Reoviridae family replication cycle require virus specific as well as segment specific recognition by viral proteins, but detailed processes in these interactions are still barely understood. Recently, we have shown that expression of NS3 and NS3a proteins encoded by genome segment 10 of bluetongue virus is not essential for virus replication. This gave us the unique opportunity to investigate the role of RNA sequences in the segment 10 open reading frame in virus replication, independent of its protein products. Reverse genetics was used to generate virus mutants with deletions in the open reading frame of segment 10. Although virus with a deletion between both start codons was not viable, deletions throughout the rest of the open reading frame led to the rescue of replicating virus. However, all bluetongue virus deletion mutants without functional protein expression of segment 10 contained inserts of RNA sequences originating from several viral genome segments. Subsequent studies showed that these RNA inserts act as RNA elements, needed for rescue and replication of virus. Functionality of the inserts is orientation-dependent but is independent from the position in segment 10. This study clearly shows that RNA in the open reading frame of Reoviridae members does not only encode proteins, but is also essential for virus replicatio

Significance of the oligosaccharides of the porcine reproductive and respiratory syndrome virus glycoproteins GP2a and GP5 for infectious virus production

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains four glycoproteins, GP2a, GP3, GP4 and GP5, the functions of which are still largely unresolved. In this study, the significance of the N-glycosylation of the GP2a and GP5 proteins of PRRSV strain LV was investigated. Both glycoproteins contain two predicted N-glycosylation sites that are highly conserved between North American-type and European-type PRRSV. Using site-directed mutagenesis, single and double mutant full-length PRRSV cDNA clones were generated. After analysing the expression of the mutant proteins and the actual use of the four putative glycosylation sites in the wild-type proteins, the production of mutant virus particles and their infectivities were investigated. The results showed that the N-linked glycans normally present on the GP2a protein are not essential for particle formation, as is the oligosaccharide attached to N53 of the GP5 protein. In contrast, the oligosaccharide linked to N46 of the GP5 protein is strongly required for virus particle production. The specific infectivities of the mutant viruses were investigated by comparing their infectivity-per-particle ratios with that of wild-type virus. The results showed that the lack of either one or both of the N-linked oligosaccharides on GP2a or of the oligosaccharide attached to N53 of GP5 did not significantly affect the infectivities of the viruses. In contrast, the two recombinant viruses lacking the oligosaccharide bound to N46 exhibited a significantly reduced specific infectivity compared with the wild-type virus. The implications of the differential requirements of the modifications of GP2a and GP5 for PRRSV assembly and infectivity are discusse

Rescue of recent virulent and avirulent field strains of Bluetongue virus by reverse genetics

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