Andy Haegeman

Rapid Generation of Replication-Deficient Monovalent and Multivalent Vaccines for Bluetongue Virus: Protection against Virulent Virus Challenge in Cattle and Sheep

ABSTRACT Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak.

Classical swine fever: Comparison of oronasal immunisation with CP7E2alf marker and C-strain vaccines in domestic pigs

Effective oronasal vaccination against classical swine fever (CSF) is essential to achieve protection in wild boar. However the currently available live CSF vaccines, e.g. C-strain, do not allow serological differentiation between infected and vaccinated animals (DIVA). A modified live marker vaccine candidate (CP7E2alf) has been recently developed (Reimann et al., 2004). This communication reports the comparison of CP7E2alf and C-strain virus vaccines during 98 days following oronasal immunisation in domestic pigs. C-strain vaccine virus was consistently detected in tonsils of all (n=30) animals from 3 to 77 days post vaccination (dpv) and in blood (n=36) between 3 and 13dpv by CSFV-specific rRT-PCR. CP7E2alf virus RNA was detected in 6 animals slaughtered between 4 and 63dpv by a BVDV-specific rRT-PCR. The chimeric virus was not detected in blood samples. As detected by CSFV E2-specific antibody ELISA and virus neutralisation tests, seroconversion first occurred at 11dpv in the C-strain vaccinated group and between 11 and 15dpv in the CP7E2alf vaccinated group. The serological response was still present at 98dpv. The CP7E2alf serological response remained negative using the CSFV E(rns) ELISA whereas seroconversion occurred in the C-strain vaccinated group. In conclusion, the primary replication site of CP7E2alf vaccine virus was found to be the tonsils as in the C-strain and virulent field strains. Persistence of CP7E2alf in the tonsils was also demonstrated up to 63dpv. Both vaccines showed immunogenicity after oronasal administration in domestic pigs. In contrast to the C-strain, CP7E2alf vaccine allowed the use of DIVA approaches in serological tests. This study confirms CP7E2alf as a promising marker vaccine candidate for oronasal vaccination programmes to control CSF in domestic pigs and wild boar.

Proof of concept for the reduction of classical swine fever infection in pigs by a novel viral polymerase inhibitor

5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a representative of a class of imidazopyridines with potent in vitro antiviral activity against pestiviruses including classical swine fever virus (CSFV). This study analysed whether the lead compound, BPIP, was able to reduce virus replication in infected piglets. The compound, administered in feed, was readily bioavailable and was well tolerated. Eight specific-pathogen-free pigs received a daily dose of 75 mg kg(-1) (mixed in feed) for a period of 15 consecutive days, starting 1 day before infection with the CSFV field isolate Wingene. BPIP-treated pigs developed a short, transient viraemia (one animal remained negative) and leukopenia (three animals did not develop leukopenia). Virus titres at peak viraemia (7 days post-infection) were markedly lower (approximately 1000-fold) than in untreated animals (P=0.00005) and the viral genome load in blood was also significantly lower (P<or=0.001) in drug-treated animals than in untreated animals over the entire experiment. At the end of the experiment (day 33), no infectious virus was detectable in the tonsils of BPIP-treated animals, although low levels of viral RNA were detected. The inability to isolate infectious virus from the tonsils indicates that the risk of a persistent CSFV infection is negligible. Further optimization of the antiviral potency and bioavailability of this lead compound may result in molecules completely suppressing virus replication. A potent antiviral could potentially be used as a primary control measure against virus spread in case of an outbreak, in addition to present countermeasures. This study provides the first proof of concept for the prophylaxis/treatment of CSFV infection in pigs.

The reduction of CSFV transmission to untreated pigs by the pestivirus inhibitor BPIP: A proof of concept

5-[(4-Bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a representative molecule of a novel class of highly active in vitro inhibitors of the replication of Classical swine fever virus (CSFV). We recently demonstrated in a proof of concept study that the molecule has a marked effect on viral replication in CSFV-infected pigs. Here, the effect of antiviral treatment on virus transmission to untreated sentinel pigs was studied. Therefore, BPIP-treated pigs (n=4), intra-muscularly infected with CSFV, were placed into contact with untreated sentinel pigs (n=4). Efficient transmission of CSFV from four untreated seeder pigs to four untreated sentinels was observed. In contrast, only two out of four sentinel animals in contact with BPIP-treated seeder animals developed a short transient infection, of which one was likely the result of sentinel to sentinel transmission. A significant lower viral genome load was measured in tonsils of sentinels in contact with BPIP-treated seeder animals compared to the positive control group (p=0.015). Although no significant difference (p=0.126) in the time of onset of viraemia could be detected between the groups of contact animals, a tendency towards the reduction of virus transmission was observed. Since sentinel animals were left untreated in this exploratory trial, the study can be regarded as a worst case scenario and gives therefore an underestimation of the potential efficacy of the activity of BPIP on virus transmission.

Classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the European network of excellence for epizootic disease diagnosis and control

The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT-PCR protocols and a standardized protocol commonly used by the Friedrich-Loeffler-Institute (FLI) on a panel of well-characterized samples. In general, all participants produced results within the acceptable range. The FLI assay, several in-house assays, and the commercial kits had high analytical sensitivity and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without modifications. In conclusion, the ring trial showed reliability of classical swine fever diagnosis on an international level and helped to optimize CSFV-specific RT-PCR diagnostics.

Estimating internal pelvic sizes using external body measurements in the double-muscled Belgian Blue beef breed

In the double-muscled (DM) Belgian Blue beef (BBB) breed, caesarean section (CS) is being applied systematically as a management tool to prevent dystocia. As a matter of fact, CS is the only possible way of calving in the breed. High birth weight and a relatively small pelvic area are the main causes of dystocia and, in the DM-BBB breed, the reasons for the systematically applied CS. Selection for lower birth weight and larger pelvic sizes might reduce dystocia and routine CS. Few data on inner pelvic sizes of pedigree animals are available. Using external measurements to estimate the inner pelvic sizes might be an option to resolve this problem. In this study, animals of the DM-BBB breed were measured and weighed on farms and in abattoirs. External and internal pelvic sizes increased with live weight and age of the animals. Gender had a significant influence on inner pelvic traits. Increased muscular conformation was associated with decreased inner pelvic dimensions. Models with weight, gender, age, withers height and outer pelvic width (TcTc) can be used to estimate inner pelvic sizes ( R 2 between 0·35 and 0·77). The estimated inner pelvic sizes can then be used to genetically evaluate pelvic traits in the DM-BBB breed. Improving weight, withers height and TcTc width in combination with lowering muscular conformation may help to decrease the high rate of caesarean section in the DM-BBB.

Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines

Eradication of bluetongue virus is possible, as has been shown in several European countries. New serotypes have emerged, however, for which there are no specific commercial vaccines. This study addressed whether heterologous vaccines would help protect against 2 serotypes. Thirty-seven sheep were randomly allocated to 7 groups of 5 or 6 animals. Four groups were vaccinated with commercial vaccines against BTV strains 2, 4, and 9. A fifth positive control group was given a vaccine against BTV-8. The other 2 groups were unvaccinated controls. Sheep were then challenged by subcutaneous injection of either BTV-16 (2 groups) or BTV-8 (5 groups). Taken together, 24/25 sheep from the 4 experimental groups developed detectable antibodies against the vaccinated viruses. Furthermore, sheep that received heterologous vaccines showed significantly reduced viraemia and clinical scores for BTV-16 when compared to unvaccinated controls. Reductions in clinical signs and viraemia among heterologously vaccinated sheep were not as common after challenge with BTV-8. This study shows that heterologous protection can occur, but that it is difficult to predict if partial or complete protection will be achieved following inactivated-BTV vaccination.

Investigation of a Possible Link Between Vaccination and the 2010 Sheep Pox Epizootic in Morocco

Sheep pox is endemic in most parts of Northern Africa and has the potential to cause severe economic problems. Live attenuated vaccines are used in Morocco, and in many other countries, to control the disease. Sheep pox virus (SPPV) re-appeared in 2010 causing a nodular clinical form previously not observed in Morocco. The severe clinical signs observed during the course of this outbreak and initial reports citing similarity in nucleotide sequence between the Moroccan vaccine strain and field isolates warranted a more in depth analysis of this epizootic. In this study, sequence analysis showed that isolates obtained from four provinces of eastern Morocco were identical, demonstrating that a single SPPV strain was responsible for the 2010 epizootic. In addition, the genome fragments sequenced and phylogenetic analyses undertaken as part of this study showed significant differences between field isolates and the Moroccan vaccine strain. New PCR methods were developed to differentiate between wild-type isolates and vaccine strains of SPPV. Using these methods, no trace of wild-type SPPV was found in the vaccine and no evidence was found to suggest that the vaccine strain was causing clinical disease.

Development and validation of three Capripoxvirus real-time PCRs for parallel testing

Capripoxviruses have the potential to cause outbreaks with a severe socio-economic impact. The latter, combined with an altered virus dissemination pattern, warrants its status as an important emerging disease. Disease control or eradication programmes can only be applied successfully if the necessary diagnostic tools are available allowing clear and unequivocal identification of the pathogen. Real-time PCR combines high sensitivity/specificity with a reduced analysis time and is thus a proven useful tool for identification of many pathogens, including Capripoxviruses. In order for a real-time PCR to be used in a diagnostic capacity, the different analytical and diagnostic parameters need to be evaluated to assure data quality. The implementation of parallel testing using multiple real-time PCRs with similar characteristics can improve further Capripoxvirus diagnosis. It was therefore the purpose of this study to develop a triplet real-time PCR panel with similar high sensitivity/specificity and provide sufficient validation data regarding the performance characteristics that the panel can be used in parallel, depending on the purpose and local situation.

Intra-host variation structure of classical swine fever virus NS5B in relation to antiviral therapy

Classical swine fever (CSF) is one of most important diseases of the Suidea with severe social economic consequences in case of outbreaks. Antivirals have been demonstrated, in recent publications, to be an interesting alternative method of fighting the disease. However, classical swine fever virus is an RNA virus which presents a challenge as intra-host variation and the error prone RNA dependent RNA polymerase (RdRp) could lead to the emergence/selection of resistant variants hampering further treatment. Therefore, it was the purpose of this study to investigate the intra-host variation of the RdRp gene, targeted by antivirals, in respect to antiviral treatment. Using the non-unique nucleotide changes, a limited intra-host variation was found in the wild type virus with 2 silent and 2 non-synonymous sites. This number shifted significantly when an antiviral resistant variant was analyzed. In total 22nt changes were found resulting in 14 amino acid changes whereby each genome copy contained at least 2 amino-acid changes in the RdRp. Interestingly, the frequency of the mutations situated in close proximity to a region involved in antiviral resistance in CSFV and bovine viral diarrhea virus (BVDV) was elevated compared to the other mutations. None of the identified mutations in the resistant variant and which could potentially result in antiviral resistance was present in the wild type virus as a non-unique mutation. In view of the spectrum of mutations identified in the resistance associated region and that none of the resistance associated mutations reported for another strain of classical swine fever for the same antiviral were observed in the study, it can be suggested that multiple mutations confer resistance to some degree. Although the followed classical approach allowed the analysis the RdRp as a whole, the contribution of unique mutations to the intra-host variation could not be completely resolved. There was a significant difference in de number of unique mutations found between: 1/wild type virus and the antiviral resistant variant and 2/between both and the number to be expected from the error rate of the RT-PCR process. This indicates that the some of the unique mutations contributed to the intra-host variation and that the antiviral pressure also shifted this pattern. This is important as one of the non-synonymous mutations found in the resistant variant and which was located in the antiviral resistance associated region, was present in the wild type virus as a unique mutation. The findings presented in this study not only show the importance of intra-host variation analysis but also warrants further research certainly in view of the potential inclusion of antivirals in a control/eradication strategy.