Pathogenesis of wild-type- and vaccine-based recombinant peste des petits ruminants virus (PPRV) expressing EGFP in experimentally infected domestic goats.

Peste des petits ruminants virus (PPRV) is a highly contagious morbillivirus related to measles and canine distemper virus, mostly affecting small ruminants. The corresponding PPR disease has a high clinical impact in goats and is characterized by fever, oral and nasal erosions, diarrhoea and pneumonia. In addition, massive infection of lymphoid tissues causes lymphopaenia and immune suppression. This results in increased susceptibility to secondary bacterial infections, explaining the observed high mortality in some outbreaks. We studied the pathogenesis of PPR by experimental inoculation of Dutch domestic goats with a recombinant virulent PPRV strain modified to express EGFP and compared it to an EGFP-expressing vaccine strain of PPRV. After intratracheal inoculation with virulent PPRV, animals developed fever, viraemia and leucopaenia, and shed virus from the respiratory and gastro-intestinal tracts. Macroscopic evaluation of fluorescence at the peak of infection 7 days post-inoculation (dpi) showed prominent PPRV infection of the respiratory tract, lymphoid tissues, gastro-intestinal tract, mucosae and skin. Flow cytometry of PBMCs collected over time demonstrated a cell-associated viraemia mediated by infected lymphocytes. At 14 dpi, pathognomonic zebra stripes were detected in the mucosa of the large intestine. In contrast, vaccine strain-inoculated goats remained largely macroscopically fluorescence negative and did not present clinical signs. A low-level viraemia was detected by flow cytometry, but at necropsy no histological lesions were observed. Animals from both groups seroconverted as early as 7 dpi and sera efficiently neutralized virulent PPRV in vitro. Combined, this work presents a study of the pathogenesis of wild type- and vaccine-based PPRV in its natural host. This study shows the strength of recombinant EGFP-expressing viruses in fluorescence-guided pathogenesis studies.

Pentavalent Disabled Infectious Single Animal (DISA)/DIVA Vaccine Provides Protection in Sheep and Cattle against Different Serotypes of Bluetongue Virus.

Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for "European" serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.

The Bluetongue Disabled Infectious Single Animal (DISA) Vaccine Platform Based on Deletion NS3/NS3a Protein Is Safe and Protective in Cattle and Enables DIVA.

The bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes bluetongue (BT), an OIE-notifiable disease of ruminants. At least 29 BTV serotypes are described as determined by the outer shell proteins VP2 and VP5. Vaccination is the most effective control measure. Inactivated and live-attenuated vaccines (LAVs) are currently available. These vaccines have their specific pros and cons, and both are not DIVA vaccines. The BT Disabled Infectious Single Animal (DISA) vaccine platform is based on LAV without nonessential NS3/NS3a expression and is applicable for many serotypes by the exchange of outer shell proteins. The DISA vaccine is effective and completely safe. Further, transmission of the DISA vaccine by midges is blocked (DISA principle). Finally, the DISA vaccine enables DIVA because of a lack of antibodies against the immunogenic NS3/NS3a protein (DIVA principle). The deletion of 72 amino acids (72aa) in NS3/NS3a is sufficient to block virus propagation in midges. Here, we show that a prototype DISA vaccine based on LAV with the 72aa deletion enables DIVA, is completely safe and induces a long-lasting serotype-specific protection in cattle. In conclusion, the in-frame deletion of 72-aa codons in the BT DISA/DIVA vaccine platform is sufficient to fulfil all the criteria for modern veterinary vaccines.

Vector competence is strongly affected by a small deletion or point mutations in bluetongue virus.

BACKGROUND: Transmission of vector-borne virus by insects is a complex mechanism consisting of many different processes; viremia in the host, uptake, infection and dissemination in the vector, and delivery of virus during blood-feeding leading to infection of the susceptible host. Bluetongue virus (BTV) is the prototype vector-borne orbivirus (family Reoviridae). BTV serotypes 1-24 (typical BTVs) are transmitted by competent biting Culicoides midges and replicate in mammalian (BSR) and midge (KC) cells. Previously, we showed that genome segment 10 (S10) encoding NS3/NS3a protein is required for virus propagation in midges. BTV serotypes 25-27 (atypical BTVs) do not replicate in KC cells. Several distinct BTV26 genome segments cause this so-called 'differential virus replication' in vitro. METHODS: Virus strains were generated using reverse genetics and their growth was examined in vitro. The midge feeding model has been developed to study infection, replication and disseminations of virus in vivo. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV variants and propagation in the midge was examined using PCR testing. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. RESULTS: A 100 nl blood meal containing ±105.3 TCID50/ml of BTV11 which corresponds to ±20 TCID50 infected 50% of fully engorged midges, and is named one Midge Alimentary Infective Dose (MAID50). BTV11 with a small in-frame deletion in S10 infected blood-fed midge midguts but virus release from the midgut into the haemolymph was blocked. BTV11 with S1[VP1] of BTV26 could be adapted to virus growth in KC cells, and contained mutations subdivided into 'corrections' of the chimeric genome constellation and mutations associated with adaptation to KC cells. In particular one amino acid mutation in outer shell protein VP2 overcomes differential virus replication in vitro and in vivo. CONCLUSION: Small changes in NS3/NS3a or in the outer shell protein VP2 strongly affect virus propagation in midges and thus vector competence. Therefore, spread of disease by competent Culicoides midges can strongly differ for very closely related viruses.

Diagnostic DIVA tests accompanying the Disabled Infectious Single Animal (DISA) vaccine platform for African horse sickness.

African Horse Sickness Virus (AHSV) (Orbivirus genus, Reoviridae family) causes high mortality in naïve domestic horses with enormous economic and socio-emotional impact. There are nine AHSV serotypes showing limited cross neutralization. AHSV is transmitted by competent species of Culicoides biting midges. AHS is a serious threat beyond the African continent as endemic Culicoides species in moderate climates transmit the closely related prototype bluetongue virus. There is a desperate need for safe and efficacious vaccines, while DIVA (Differentiating Infected from Vaccinated) vaccines would accelerate control of AHS. Previously, we have shown that highly virulent AHSV with an in-frame deletion of 77 amino acids (aa) in NS3/NS3a is completely safe, does not cause viremia and shows protective capacity. This deletion mutant is a promising DISA (Disabled Infectious Single Animal) vaccine platform, since exchange of serotype specific virus proteins has been shown for all nine serotypes. Here, we show that a prototype NS3 competitive ELISA is DIVA compliant to AHS DISA vaccine platforms. Epitope mapping of NS3/NS3a shows that more research is needed to evaluate this prototype serological DIVA assay regarding sensitivity and specificity, in particular for AHSVs expressing antigenically different NS3/NS3a proteins. Further, an experimental panAHSV PCR test targeting genome segment 10 is developed that detects reference AHSV strains, whereas AHS DISA vaccine platforms were not detected. This DIVA PCR test completely guarantees genetic DIVA based on in silico and in vitro validation, although test validation regarding diagnostic sensitivity and specificity has not been performed yet. In conclusion, the prototype NS3 cELISA and the PCR test described here enable serological and genetic DIVA accompanying AHS DISA vaccine platforms.

African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS Disabled Infectious Single Animal (DISA) vaccine platform.

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. Currently, nine serotypes have been defined showing limited cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horse Sickness (AHS) in equids with a mortality up to 95% in naïve domestic horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are competent vectors of closely related bluetongue virus. AHS outbreaks cause huge economic losses in developing countries. In the developed world, outbreaks will result in losses in the equestrian industry and will have an enormous emotional impact on owners of pet horses. Live-attenuated vaccine viruses (LAVs) have been developed, however, safety of these LAVs are questionable due to residual virulence, reversion to virulence, and risk on virulent variants by reassortment between LAVs or with field AHSV. Research aims vaccines with improved profiles. Reverse genetics has recently being developed for AHSV and has opened endless possibilities including development of AHS vaccine candidates, such as Disabled Infectious Single Animal (DISA) vaccine. Here, virulent AHSV5 was recovered and its high virulence was confirmed by experimental infection of ponies. 'Synthetically derived' virulent AHSV5 with an in-frame deletion of 77 amino acids codons in genome segment 10 encoding NS3/NS3a protein resulted in similar in vitro characteristics as published NS3/NS3a knockout mutants of LAV strain AHSV4LP. In contrast to its highly virulent ancestor virus, this deletion AHSV5 mutant (DISA5) was completely safe for ponies. Two vaccinations with DISA5 as well as two vaccinations with DISA vaccine based on LAV strain AHSV4LP showed protection against lethal homologous AHSV. More research is needed to further improve efficacy, to explore the AHS DISA vaccine platform for all nine serotypes, and to study the vaccine profile in more detail.

One after the other: A novel Bluetongue virus strain related to Toggenburg virus detected in the Piedmont region (North-western Italy), extends the panel of novel atypical BTV strains.

In this rapid communication, a novel atypical bluetongue virus (BTV) strain detected in goats in the Piedmont region (north-western Italy) is described. This strain, BTV-Z ITA2017, is most related in Seg-2/VP-2 (83.8% nt/82.7% aa) to strain TOV of BTV-25. Reactive antisera of goats positive by cELISA for BTV antibodies failed to neutralize a chimeric virus expressing the outermost protein of TOV. Infected animals displayed low levels of RNAemia and absence of clinical signs consistent with bluetongue infection, a scenario described in animals infected with atypical BTV strains.

Bluetongue Disabled Infectious Single Animal (DISA) vaccine: Studies on the optimal route and dose in sheep.

Bluetongue (BT) is a disease of ruminants caused by bluetongue virus (BTV) transmitted by biting midges of the Culicoides genus. Outbreaks have been controlled successfully by vaccination, however, currently available BT vaccines have several shortcomings. Recently, we have developed BT Disabled Infectious Single Animal (DISA) vaccines based on live-attenuated BTV without expression of dispensable non-structural NS3/NS3a protein. DISA vaccines are non-pathogenic replicating vaccines, do not cause viremia, enable DIVA and are highly protective. NS3/NS3a protein is involved in virus release, cytopathogenic effect and suppression of Interferon-I induction, suggesting that the vaccination route can be of importance. A standardized dose of DISA vaccine for serotype 8 has successfully been tested by subcutaneous vaccination. We show that 10 and 100times dilutions of this previously tested dose did not reduce the VP7 humoral response. Further, the vaccination route of DISA vaccine strongly determined the induction of VP7 directed antibodies (Abs). Intravenous vaccination induced high and prolonged humoral response but is not practical in field situations. VP7 seroconversion was stronger by intramuscular vaccination than by subcutaneous vaccination. For both vaccination routes and for two different DISA vaccine backbones, IgM Abs were rapidly induced but declined after 14days post vaccination (dpv), whereas the IgG response was slower. Interestingly, intramuscular vaccination resulted in an initial peak followed by a decline up to 21dpv and then increased again. This second increase is a steady and continuous increase of IgG Abs. These results indicate that intramuscular vaccination is the optimal route. The protective dose of DISA vaccine has not been determined yet, but it is expected to be significantly lower than of currently used BT vaccines. Therefore, in addition to the advantages of improved safety and DIVA compatibility, the novel DISA vaccines will be cost-competitive to commercially available live attenuated and inactivated vaccines for Bluetongue.

Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro.

The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family. IMPORTANCE: Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses.

Experimental infection of small ruminants with bluetongue virus expressing Toggenburg Orbivirus proteins.

Bluetongue virus (BTV) is the prototype orbivirus (Reoviridae family, genus Orbivirus) consisting of more than 24 recognized serotypes or neutralization groups. Recently, new BTV serotypes in goats have been found; serotype 25 (Toggenburg Orbivirusor TOV), serotype 26 (KUW2010/02), and serotype 27 from Corsica, France. KUW2010/02 has been isolated in mammalian cells but is not replicating in Culicoides cells. TOVhas been detected in goats but could not been cultured, although TOV has been successfully passed to naïve animals by experimental infection using viremic blood. Genome segments Seg-2[VP2], Seg-6[VP5], Seg-7[VP7], and Seg-10[NS3/NS3a] expressing the respective TOV proteins were incorporated in BTV using reverse genetics, demonstrating that these TOV proteins are functional in BTV replication. Depending on the incorporated TOV proteins, in vitro replication is, however, decreased compared to the ancestor BTV, in particular by TOV-VP5. Sheep and goats were experimentally infected with BTV expressing both outer capsid proteins VP2 and VP5 of TOV, so-named 'TOV-serotyped BTV'. Viremia was not detected in sheep, and hardly detected in goats after infection with TOV-serotyped BTV. Seroconversion by cELISA, however, was detected, suggesting that TOV-serotyped BTV replicates in small ruminants. One goat was coincidentally pregnant, and the fetus was strong PCR-positive in blood samples and several organs, which conclusively demonstrates that TOV-serotyped BTV replicates in vivo.

Requirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV).

BACKGROUND: Bluetongue virus (BTV) and African horse sickness virus (AHSV) are distinct arthropod borne virus species in the genus Orbivirus (Reoviridae family), causing the notifiable diseases Bluetongue and African horse sickness of ruminants and equids, respectively. Reverse genetics systems for these orbiviruses with their ten-segmented genome of double stranded RNA have been developed. Initially, two subsequent transfections of in vitro synthesized capped run-off RNA transcripts resulted in the recovery of BTV. Reverse genetics has been improved by transfection of expression plasmids followed by transfection of ten RNA transcripts. Recovery of AHSV was further improved by use of expression plasmids containing optimized open reading frames. RESULTS: Plasmids containing full length cDNA of the 10 genome segments for T7 promoter-driven production of full length run-off RNA transcripts and expression plasmids with optimized open reading frames (ORFs) were used. BTV and AHSV were rescued using reverse genetics. The requirement of each expression plasmid and capping of RNA transcripts for reverse genetics were studied and compared for BTV and AHSV. BTV was recovered by transfection of VP1 and NS2 expression plasmids followed by transfection of a set of ten capped RNAs. VP3 expression plasmid was also required if uncapped RNAs were transfected. Recovery of AHSV required transfection of VP1, VP3 and NS2 expression plasmids followed by transfection of capped RNA transcripts. Plasmid-driven expression of VP4, 6 and 7 was also needed when uncapped RNA transcripts were used. Irrespective of capping of RNA transcripts, NS1 expression plasmid was not needed for recovery, although NS1 protein is essential for virus propagation. Improvement of reverse genetics for AHSV was clearly demonstrated by rescue of several mutants and reassortants that were not rescued with previous methods. CONCLUSIONS: A limited number of expression plasmids is required for rescue of BTV or AHSV using reverse genetics, making the system much more versatile and generally applicable. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants, and will greatly benefit the control of these important diseases of livestock and companion animals.

Balance of RNA sequence requirement and NS3/NS3a expression of segment 10 of orbiviruses.

Orbiviruses are insect-transmitted, non-enveloped viruses with a ten-segmented dsRNA genome of which the bluetongue virus (BTV) is the prototype. Viral non-structural protein NS3/NS3a is encoded by genome segment 10 (Seg-10), and is involved in different virus release mechanisms. This protein induces specific release via membrane disruptions and budding in both insect and mammalian cells, but also the cytopathogenic release that is only seen in mammalian cells. NS3/NS3a is not essential for virus replication in vitro with BTV Seg-10 containing RNA elements essential for virus replication, even if protein is not expressed. Recently, new BTV serotypes with distinct NS3/NS3a sequence and cell tropism have been identified. Multiple studies have hinted at the importance of Seg-10 in orbivirus replication, but the exact prerequisites are still unknown. Here, more insight is obtained with regard to the needs for orbivirus Seg-10 and the balance between protein expression and RNA elements. Multiple silent mutations in the BTV NS3a ORF destabilized Seg-10, resulting in deletions and sequences originating from other viral segments being inserted, indicating strong selection at the level of RNA during replication in mammalian cells in vitro. The NS3a ORFs of other orbiviruses were successfully exchanged in BTV1 Seg-10, resulting in viable chimeric viruses. NS3/NS3a proteins in these chimeric viruses were generally functional in mammalian cells, but not in insect cells. NS3/NS3a of the novel BTV serotypes 25 and 26 affected virus release from Culicoides cells, which might be one of the reasons for their distinct cell tropism.

Development of a competitive ELISA for NS3 antibodies as DIVA test accompanying the novel Disabled Infectious Single Animal (DISA) vaccine for Bluetongue.

Recently, we have developed a novel vaccine for Bluetongue named BT Disabled Infectious Single Animal (DISA) vaccine. Due to the lack of non-essential NS3/NS3a protein, BT DISA vaccine is a replicating vaccine, but without the inherent risks of live-attenuated vaccines, such as residual virulence or reversion to virulence by mutations, reassortment with field virus, horizontal spread by vectors and vertical transmission. The immune response induced by BT DISA vaccines is rapidly induced, highly protective and serotype specific which is dependent on the immunodominant and serotype determining VP2 protein. The BT DISA vaccine platform provides the replacement of exclusively VP2 from different serotypes in order to safely formulate multivalent cocktail vaccines. The lack of NS3/NS3a directed antibodies by BT DISA vaccination enables differentiation of infected from vaccinated animals (DIVA principle). A highly conserved immunogenic site corresponding to the late domain was mapped in the N-terminal region of NS3. We here established an NS3-specific competitive ELISA (NS3 cELISA) as serological DIVA test accompanying BT DISA vaccines. To this end, NS3 protein missing putative transmembrane regions was produced in large amounts in bacteria and used as antigen in the NS3 cELISA which was investigated with a variety of sera. The NS3 cELISA displayed a high sensitivity and specificity similar to the commercially available VP7-specific cELISA. Results of previously performed vaccination-challenge trials with BT DISA vaccines clearly demonstrate the DIVA system based on the NS3 cELISA and BT vaccine free of NS3 protein.

VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV.

UNLABELLED: 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.

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA.

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA). BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine. Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses. Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.

Bluetongue virus without NS3/NS3a expression is not virulent and protects against virulent bluetongue virus challenge.

Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially available vaccines and recently developed vaccine candidates have several shortcomings. Therefore, we generated and tested next-generation vaccines for bluetongue based on the backbone of a laboratory-adapted strain of BTV-1, avirulent BTV-6 or virulent BTV-8. All vaccine candidates were serotyped with VP2 of BTV-8 and did not express NS3/NS3a non-structural proteins, due to induced deletions in the NS3/NS3a ORF. Sheep were vaccinated once with one of these vaccine candidates and were challenged with virulent BTV-8 3 weeks after vaccination. The NS3/NS3a knockout mutation caused complete avirulence for all three BTV backbones, including for virulent BTV-8, indicating that safety is associated with the NS3/NS3a knockout phenotype. Viraemia of vaccine virus was not detected using sensitive PCR diagnostics. Apparently, the vaccine viruses replicated only locally, which will minimize spread by the insect vector. In particular, the vaccine based on the BTV-6 backbone protected against disease and prevented viraemia of challenge virus, showing the efficacy of this vaccine candidate. The lack of NS3/NS3a expression potentially enables the differentiation of infected from vaccinated animals, which is important for monitoring virus spread in vaccinated livestock. The disabled infectious single-animal vaccine for bluetongue presented here is very promising and will be the subject of future studies.

Viral replication kinetics and in vitro cytopathogenicity of parental and reassortant strains of bluetongue virus serotype 1, 6 and 8.

Bluetongue virus (BTV), a segmented dsRNA virus, is the causative agent of bluetongue (BT), an economically important viral haemorrhagic disease of ruminants. Bluetongue virus can exchange its genome segments in mammalian or insect cells that have been co-infected with more than one strain of the virus. This process, may potentially give rise to the generation of novel reassortant strains that may differ from parental strains in regards to their phenotypic characteristics. To investigate the potential effects of reassortment on the virus' phenotype, parental as well as reassortant strains of BTV serotype 1, 6, 8, that were derived from attenuated and wild type strains by reverse genetics, were studied in vitro for their virus replication kinetics and cytopathogenicity in mammalian (Vero) cell cultures. The results indicate that genetic reassortment can affect viral replication kinetics, the cytopathogenicity and extent/mechanism of cell death in infected cell cultures. In particular, some reassortants of non-virulent vaccine (BTV-1 and BTV-6) and virulent field origin (BTV-8) demonstrate more pronounced cytopathic effects compared to their parental strains. Some reassortant strains in addition replicated to high titres in vitro despite being composed of genome segments from slow and fast replicating parental strains. The latter result may have implications for the level of viraemia in the mammalian host and subsequent uptake and transmission of reassortant strains (and their genome segments) by Culicoides vectors. Increased rates of CPE induction could further suggest a higher virulence for reassortant strains in vivo. Overall, these findings raise questions in regards to the use of modified-live virus (MLV) vaccines and risk of reassortment in the field. To further address these questions, additional experimental infection studies using insects and/or animal models should be conducted, to determine whether these results have significant implications in vivo.

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 replication.

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 AUG(Met)→GCC(Ala) 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.