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.

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.

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.

Safety and efficacy of inactivated African horse sickness (AHS) vaccine formulated with different adjuvants.

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae causing African Horse Sickness (AHS) in equids with a mortality of about 95% in naïve horses. AHS causes serious losses in developing countries where horses play a central role in draft power and transportation. There are nine AHSV serotypes inducing no or low cross-neutralizing antibodies. AHSV is spread by biting Culicoides midges. AHS is endemic in sub-Saharan Africa, and a serious threat outside Africa, since Culicoides species in moderate climate conditions are spreading the closely related bluetongue virus. AHS outbreaks will be devastating for the equestrian industry in developed countries. Live-attenuated vaccines (LAVs) are licensed, marketed and in use in Africa. Their application is controversial with regard to safety issues. LAVs are not allowed in AHS-free countries. We here studied inactivated AHSV with different adjuvants in guinea pigs and horses. Subcutaneous and intramuscular vaccination were studied in horses. Local reactions were observed after prime and boost vaccination. In general, neutralizing antibodies (nAbs) titres were very low after prime vaccination, whereas boost vaccination resulted in high nAb titres for some adjuvants. Vaccinated horses were selected based on local reactions and nAb titres to study efficacy. Unfortunately, not all vaccinated horses survived virulent AHSV infection. Further, most survivors temporarily developed clinical signs and viremia. Further, the current prototype inactivated AHS vaccine is not suitable as emergency vaccine, because onset of protection is slow and requires boost vaccinations. On the other hand, inactivated AHS vaccine is completely safe with respect to virus spread, and incorporation of the DIVA principle based on NS3/NS3a serology and exploring a vaccine production platform for other serotypes is feasible. A superior adjuvant increasing the protective response without causing local reactions will be required to develop payable and acceptable inactivated AHS vaccines.

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.

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.

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.

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.