Marylène Tignon
email: marylene.tignon@var.fgov.

Gábor Kulcsár

Andy Haegeman

Timea Barna

Katalin Fábián

Réka Lévai

Yves Van Der Stede

Attila Farsang

Robert Vrancken

Katinka Belák

Frank Koenen

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

Keywords: Classical swine fever virus (CSFV), DIVA marker vaccine, CP7E2alf, C-strain, oronasal vaccination, diagnostic tests

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

As detected by CSFV E2-specific antibody ELISA and virus neutralisation, seroconversion first occurred at 11 dpv in the C-strain vaccinated group and between 11 and 15 dpv in the CP7E2alf vaccinated group. Serological response was still observed at 98 dpv. CP7E2alf serological response remained negative using the CSFV E rns ELISA whereas seroconversion occurred in C-strain vaccinated group.

In conclusion, the primary replication site of CP7E2alf vaccine virus was found to be the tonsils similarly to C-strain and virulent field Introduction Classical swine fever (CSF) is a highly contagious and often fatal disease of domestic pigs and wild boar. Outbreaks of CSF usually cause important economic losses, and impair internal and international trade of pigs and pig products [START_REF] Vandeputte | Classical swine fever: the European experience and a guide for infected areas[END_REF][START_REF] Edwards | Classical swine fever: the global situation[END_REF]. The aetiological agent of CSF is classical swine fever virus (CSFV), a member of the Pestivirus genus of the Flaviviridae family [START_REF] Van Regenmortel | Virus Taxonomy: Classification and Nomenclature of Viruses[END_REF].

The effectiveness of modified live vaccines, such as the Chinese strain (C-strain), has been frequently reported in domestic pigs and in wild boar (Terpstra and Robijns, 1977;[START_REF] Chenut | Oral immunisation of swine with a classical swine fever vaccine (Chinese strain) and transmission studies in rabbits and sheep[END_REF][START_REF] Kaden | Oral immunisation of wild boar against classical swine fever: evaluation of the first field study in Germany[END_REF][START_REF] Kaden | Oral immunisation against classical swine fever (CSF): onset and duration of immunity[END_REF]Dewulf et al., 2004b;[START_REF] Dong | Marker vaccine strategies and candidate CSFV marker vaccines[END_REF]. Such vaccines induce protection shortly after administration. Prophylactic mass vaccination with modified live vaccines, combined with culling of infected pigs, successfully resulted in the eradication of the disease in most countries of the European Union (EU) [START_REF] Vandeputte | Classical swine fever: the European experience and a guide for infected areas[END_REF][START_REF] Greiser-Wilke | Vaccination against classical swine fever virus: limitations and new strategies[END_REF]. Currently, modified live vaccines are still in use where the disease is endemic in domestic pigs and for disease control in wild boar [START_REF] Von Rüden | Retrospective analysis of the oral immunisation of wild boar populations against classical swine fever virus (CSFV) in region Eifel of Rhineland-Palatinate[END_REF]. Therefore, strict restrictions on the international trade in pig products are implemented from countries using vaccination (Greiser-Wilke and [START_REF] Greiser-Wilke | Vaccination against classical swine fever virus: limitations and new strategies[END_REF]. The lack of serological discrimination between A c c e p t e d M a n u s c r i p t 6 naturally infected animals and animals vaccinated with a modified live vaccine hampers disease control relying on serology (van Oirschot, 2003;Pasick, 2004). To overcome this problem, marker vaccines which allow differentiation of infected from vaccinated animals (DIVA) have been developed.

The first subunit vaccine was developed in 1993 based on the expression of the E2 envelope protein of the Brescia strain within the baculovirus system [START_REF] Hulst | Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera[END_REF]. Subunit vaccines induce a DIVA serological response and reduce morbidity and mortality following subsequent challenge with a virulent CSFV field strain (Moormann et al., 2000). However, horizontal or vertical virus transmission is not completely prevented when challenge occurs in the first week post vaccination [START_REF] Dewulf | An E2 sub-unit marker vaccine does not prevent horizontal or vertical transmission of classical swine fever virus[END_REF](Dewulf et al., , 2004b)).

Moreover, the subunit vaccines are not applicable for CSF control in wild boar by oral immunization.

A new generation of live marker vaccines was developed based on the construction of chimeric pestiviruses (for review see [START_REF] Beer | Novel marker vaccines against classical swine fever[END_REF]. The E2 or E rns -coding region of CSFV or cytopathogenic bovine viral diarrhoea virus (BVDV) strain CP7 has been replaced by that of a heterologous strain (van Gennip et al., 2000;Reimann et al., 2004;Rasmussen et al., 2007;[START_REF] Wehrle | Chimeric pestiviruses: candidates for live-attenuated classical swine fever marker vaccines[END_REF]. One of these constructs, CP7E2alf, is based on the infectious full-length clone of BVDV strain CP7 in which the BVDV E2 gene is replaced by that of A c c e p t e d M a n u s c r i p t 7 CSFV strain Alfort/187 (Reimann et al., 2004). CP7E2alf proved to be completely safe in pigs following intramuscular inoculation. Neither viraemia, nor virus transmission to contact animals were detected.

Furthermore, immunogenicity and protective effectiveness have been demonstrated after intramuscular administration in pigs (Reimann et al., 2004;Koenig et al., 2007a) and oral vaccination in wild boar (Koenig et al., 2007b;[START_REF] Kaden | Oral vaccination against classical swine fever with a chimeric Pestivirus : comparative investigations of liquid and lyophilized virus[END_REF]. Additionally, the DIVA properties of this live vaccine have been described (Reimann et al., 2004). Nevertheless additional data regarding immunogenicity after oronasal administration, dose effect and persistence over a longer period are required to improve vaccine characterisation and application efficiency.

The aim of the present study was to evaluate the live marker

Materials and methods

Vaccine

The conventional C-strain vaccine ('Riems') was kindly provided by Dr. V. Kaden (Friedrich-Loeffler-Institut, Germany). The CP7E2alf marker vaccine (Reimann et al. (2004) was produced using Good

Manufacturing Practice by Fort Dodge Veterinaria (Spain).

Vaccines were diluted in saline solution to 10 4.5 50% tissue culture infective dose per ml (TCID50/ml) and virus titre was checked by back-titration (10 4.48 and 10 4.59 TCID50/ml for CP7E2alf and C-strain viruses respectively).

Animals

For the experiment, pigs were recruited twice from the same conventional breeding farm with an interval of six months. Each group was constituted by 46 Kahyb breed pigs (Hungarian landrace-typed hybrid), two months old and weighing approximately 12 kg. These animals were free of pestivirus antibodies, as tested by standard diagnostic procedures.

Experimental design

The study design, the administration dose and routes were similar to a previous experimental infection with a field isolate ('WIL-11722') (Tignon et al., 2008). Vaccination with CP7E2alf and C-strain were conducted separately with an interval of six months. Six month later, the second group of animals (n=46) was vaccinated with the C-strain modified live vaccine, except two pigs kept as negative controls. Vaccination and slaughtering protocols were conducted in the same way as previously described.

Clinical examination and sample collection

All the living animals were clinically monitored using a scoring system (Mittelholzer et al., 2000) based on the observation of ten parameters (breathing, liveliness, body tension, body shape, walking, skin, eyes, appetite, defecation and leftovers) and the rectal temperatures were recorded on a daily basis, before sample

Haematology

The total number and differentiation of peripheral blood leucocytes (PBLs) were determined in EDTA-treated blood samples by using a Sysmex E-4000 automatic haemocytometer (Toa Medical Electronics, Kobe, Japan). Counts below 10,000 leukocytes/µl were considered as leucopaenia. From 0 until 70 dpv, data were collected from 10 animals and later from the remaining animals.

Virological analysis

Virus isolation (VI) from the tonsil samples and from EDTA-treated blood was performed on semi-confluent monolayer of porcine kidney cells (PK15, ATCC-CCL33) (Dewulf et al., 2004a). After 48 hours, the cells were fixed with isopropanol and stained with International, Lissieu, France) were used for CSFV-specific genome detection. In addition, one BVDV-specific [START_REF] Letellier | Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus[END_REF] and one Pestivirus-specific (pan-pesti) [START_REF] Hoffmann | A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses[END_REF] rRT-PCR assays were used for CP7E2alf virus RNA detection. The rRT-PCR assays were performed using an ABI 7500 FAST machine (Applied Biosystems, Lennik, Belgium) and analyzed using the Sequence Detection Software 1.4. For CSFV-, BVDV-and pan-pestispecific rRT-PCRs, threshold cycle (Ct) values lower than 42 were considered positive.

Serological analysis

The CSFV E2 and E rns antibody responses were investigated with the Blocking percentages in the E rns ELISA were considered as positive when above 50%. The neutralising response was determined in the virus neutralisation test (VNT) according to the OIE manual [START_REF] Anonymous | OIE Manual of Standards for Diagnostic Tests and Vaccines. List A and B Diseases of Mammals, Birds and Bees[END_REF] using two viral strains: Alfort/187 and CP7E2alf.

For VNT, 50% neutralising doses (ND50) were expressed as Log10transformed values of neutralising antibody titres, with values greater than one regarded as positive.

Statistical analysis

The data were presented as medians with either the corresponding range of values or the 25 th and 75 th percentiles.

Results

Clinical examination

Clinical disease was not recorded in the two groups vaccinated with the CP7E2alf and C-strain using the referred scoring system. The maximal daily cumulative clinical score per animal observed was 2 and 5 in CP7E2alf and C-strain groups respectively. Median rectal temperatures remained below 40.5°C during the 98 dpv (Figure 1).

Haematology

The initial median PBL population before infection was 20.5 x 10 3 cells/µl (with range from 15.7 to 25.5 x 10 3 ) and 20.7 x 10 3 cells/µl (with range from 17.8 to 28.3 x 10 3 ) for the C-strain and CP7E2alf groups, respectively (Figure 2). Leucopaenia was not observed in either vaccinated group. PBL populations of both groups were

Vaccine virus detection in sequential tonsil samples

The CP7E2alf virus was only isolated on cell culture from the tonsils of two animals at 4 and at 5 dpv, respectively (Table 1). The marker

vaccine

Vaccine virus detection in sequentially collected blood samples

The VI and rRT-PCR assays revealed no viraemia in blood samples from the CP7E2alf vaccinated group (Table 2). In contrast, viraemia started between 3 and 8 dpv in the C-strain group, as detected by

Discussion

Vaccine virus distribution after CP7E2alf vaccination has previously been described up to 42 dpv (Reimann et al., 2004;Koenig et al., 2007a) but data for longer persistence in the tissues and blood are lacking. Similarly, although the persistence of serological response induced by C-strain vaccine has already been established for several years [START_REF] Kaden | Oral immunisation against classical swine fever (CSF): onset and duration of immunity[END_REF]van Oirschot, 2003), it has not been monitored for more than 42 days after CP7E2alf vaccination (Koenig et al., 2007a). Therefore, the presence of CP7E2alf and C-strain virus in the tonsils and blood and the host serological response were investigated during 98 dpv in this study to determine the vaccine virus persistence in tonsils and blood and further evaluate the duration of the CP7E2alf serological response.

As reported in the present study, the chimeric CP7E2alf virus was present in the tonsils early after oronasal vaccination. This result confirms other observations done after CP7E2alf intramuscular administration (Reimann et al., 2004;Koenig et al., 2007a). More importantly, the time point of detection in the tonsils was similar for CP7E2alf and C-strain which confirms previous reports (Lorena et al., 2001;[START_REF] Kaden | Classical swine fever virus Strain 'C'. How long is it detectable after oral vaccination?[END_REF]. This not only suggests that the tonsils are the initial replication site for CP7E2alf, as already described for Cstrain vaccine virus and for virulent CSFV strains (Mittelholzer et al., 2000;Tignon et al., 2008), but further confirms the CSFV-like to 63 dpv and more than 77 dpv respectively.

The viraemia observed after intramuscular CP7E2alf vaccination by Koenig et al. (2007a) was not confirmed on blood samples collected in this study. A different administration route and/or the nature of the used samples (blood instead of purified leucocytes) may explain this result. However, transient viraemia was detected in the C-strain group, as previously described (Lorena et al., 2001;Koenig et al., 2007a). Nevertheless, the number of viraemic animals detected by VI and rRT-PCR, as well as the duration of viraemia, was lower than those observed after infection with a virulent strain ('WIL-11722') in similar experimental conditions (Tignon et al., 2008).

The serological response observed after oronasal C-strain vaccination was consistent with early onset and intensity established by [START_REF] Kaden | Oral immunisation against classical swine fever (CSF): onset and duration of immunity[END_REF]. In contrast, a slightly delayed and reduced response with high variability between animals was observed in the CP7E2alf group that has not been described previously (Reimann et al., 2004;Koenig et al., 2007aKoenig et al., , 2007b;;[START_REF] Kaden | Oral vaccination against classical swine fever with a chimeric Pestivirus : comparative investigations of liquid and lyophilized virus[END_REF]. In those studies, domestic pigs or wild boars were vaccinated intramuscularly with 10 5.3 to 10 7 TCID50/ml or A c c e p t e d M a n u s c r i p t 21 orally with 10 5.75 to 10 6.5 TCID50/ml of CP7E2alf marker vaccine. This suggests that the reduced vaccine dose combined with oronasal delivery may be the determining factors for our results (Reimann et al., 2004;Koenig et al., 2007aKoenig et al., , 2007b;;[START_REF] Kaden | Oral vaccination against classical swine fever with a chimeric Pestivirus : comparative investigations of liquid and lyophilized virus[END_REF]. This is supported by a recent report of dose effect on the onset and intensity of the serological response after CP7E2alf vaccination [START_REF] Leifer | First efficacy data of the GMP-produced modified live marker vaccine prototype CP7_E2alf[END_REF]. Notwithstanding the delayed serological response, our data showed that neutralising and total antibody titres remained stable up to 77 dpv. At that time, the observed decrease of the mean serological response could mostly be explained by elimination of animals with higher serological response during the random slaughtering procedure. However, antibodies persisted to the end of the experiment, at least in one of the two remaining animals. Nonetheless, the present study demonstrated the immunogenic potential of CP7E2alf despite a suboptimal vaccine dose, such as it may occur where there is incomplete bait uptake by animals or where vaccine titre is reduced in the field or during the production process [START_REF] Kaden | Oral vaccination against classical swine fever with a chimeric Pestivirus : comparative investigations of liquid and lyophilized virus[END_REF]. Moreover, variations here observed in VNT titres using different CSFV VNT strains also indicated that cross-neutralisation of CP7E2alf with CSFV was influenced by the antigenicity of the BVDV backbone of the chimera and the homology with E2 [START_REF] Dekker | Six antigenic groups within the genus pestivirus as identified by cross neutralization assays[END_REF][START_REF] Van Rijn | Subdivision of the pestivirus genus based on envelope glycoprotein E2[END_REF], thus adjusting the conclusion of Koenig et al. (2007b) that E2 is the major or even the only pestiviral protein that induces It was not the purpose of the present study to directly evaluate the protective capacity of CP7E2alf, as this has already been demonstrated elsewhere (Reimann et al., 2004;Koenig et al., 2007b). The protective role of neutralising antibodies has been established elsewhere (Terpstra and Wensvoort, 1988;Suradhat et al., 2001). If it is generally accepted that vaccinated pigs with active neutralising antibody titres higher than 1.50 ND50 are protected against challenge (Terpstra and Wensvoort, 1988;review in Granges et al., 2007 andin Suradhat et al., 2007), the VNT titres observed in some of the CP7E2alf vaccinated animals of this experiment may not be conclusive based on this minimal protective VNT value. However, protection after vaccination has even been observed in the absence of neutralising antibodies (reviewed by Ganges et al., 2007 andSuradhat et al., 2007), indicating that other aspects of the immune response may also be involved.

Nevertheless, the late seroconversion and reduced VNT titres, obtained after vaccination with suboptimal dose, as well as differences in cross-reactivity observed in VNT, should be further investigated in regard to the protection, as well as the importance of cellular components in the immune response against CSFV infection. and CSFV strains can be easily achieved with RT-PCRs or rRT-PCRs targeting non-homologous regions [START_REF] Vilcek | Organization and diversity of the 3'-noncoding region of classical swine fever virus genome[END_REF][START_REF] Hoffmann | Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever[END_REF][START_REF] Haegeman | Characterisation of the discrepancy between PCR and virus isolation in relation to classical swine fever virus detection[END_REF][START_REF] Beer | Novel marker vaccines against classical swine fever[END_REF]. This is clearly demonstrated in the present study where CSFV-specific molecular detection methods (rRT-PCRs) targeting the CSFV 5'UTR region did not detect the CP7E2alf vaccine in tonsils and blood while BVDV-specific rRT-PCR did. By contrast, those rRT-PCR tests are not discriminatory for C-strain vaccine and other CSVF strains [START_REF] Hoffmann | Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever[END_REF]Tignon et al. 2008). Although certain RT-PCR and rRT-PCRs have been developed to differentiate between Cstrain and certain CSFV genotypes (Pan et al., 2008;[START_REF] Zhao | Evaluation of a multiplex real-time RT-PCR for[END_REF], none is currently available which is able to differentiate Cstrain from all CSFV genotypes.

In view of the construction of the chimeric vaccine virus, characterised by the presence of CSFV E2 but not CSFV E rns gene (Reimann et al., 2004), the CP7E2alf allows a serological differentiation from wild-type CSFV. The latter can be clearly seen in this study as the CP7E2alf vaccinated animals did not seroconvert

for CSFV E rns but did for CSFV E2, confirming previous studies (Reimann et al., 2004;Koenig et al., 2007aKoenig et al., , 2007b)). As expected, no serological DIVA was possible for C-strain vaccine, as it induced CSFV-specific E2, E rns and neutralising antibodies, in accordance A c c e p t e d M a n u s c r i p t 24 with previous descriptions [START_REF] Kaden | Oral immunisation against classical swine fever (CSF): onset and duration of immunity[END_REF][START_REF] Kaden | Classical swine fever virus Strain 'C'. How long is it detectable after oral vaccination?[END_REF] and similarly to the serological picture observed after infection with mildly to moderately virulent strains (Mittelholzer et al., 2000).

Conclusion

In conclusion, the initial replication of CP7E2alf and C-strain vaccines occurred in the tonsils, which is similar to the wild virus. The

vaccine

  CP7E2alf following oronasal administration to domestic pigs in comparison to the C-strain. The presence and persistence of vaccine virus was investigated in tonsils and blood samples by comparison with the C-strain. The serological response observed after oronasal vaccination was studied up to 98 days post vaccination (dpv) in the two vaccine groups and the DIVA properties of the vaccines were compared.

  pigs were housed in a biosecure unit in agreement with EU directive 91/630/EEC and its amendments 2001/88/EC and 2001/93/EC. Following a seven-day acclimatization period, the pigs were clinically inspected and blood sampled. On the first day (day 0), CP7E2alf live marker vaccine was administrated via the intranasal (1 ml) and the oral (1 ml) routes by means of a syringe to all animals of the first group (n=46), except two pigs kept as negative controls. The two non-vaccinated controls were slaughtered on the day of vaccination by an intravenous pentobarbital injection, followed by exsanguination. Subsequently, two randomly selected vaccinated animals were slaughtered on a daily basis between 1 and 8 dpv, at 13 and 18 dpv, and then weekly from 21 to 98 dpv.

  the first 8 days and subsequently bi-weekly until 98 dpv. Animals were considered clinically ill when their daily cumulative clinical score exceeded the value of 6. Pigs were considered febrile when rectal temperatures exceeded 40.5°C.Blood samples with addition of EDTA as anticoagulant and serum samples were collected from each animal in the group during the experiment. Samples were collected on slaughter days and additionally at 11 and 15 dpv. At necropsy, the tonsils of the two slaughtered animals were aseptically collected for virological examination. The samples were stored at -80°C until analysis.

  conjugated anti-CSFV immunoglobulins. Cultures were passaged one to three times. After RNA extraction with the Viral RNA kit (Qiagen Benelux B.V., Venlo, The Netherlands), the presence of viral RNA molecules was investigated in the tonsils and blood samples by real-time RT-PCR (rRT-PCR). The rRT-PCR described by Hoffmann et al (2005) and the commercial TaqVet CSF rRT-PCR kit (Laboratoire Service

  HerdChek CSFV ELISA (IDEXX Europe B.V., Schiphol-Rijk, The Netherlands) and PrioCHECK CSFV E rns ELISA (kindly provided by Prionics A.G., Zurich, Switzerland) respectively. Values obtained in the ELISAs were expressed as blocking percentages. Blocking percentages between 30 and 40% in the E2 ELISA were considered A c c e p t e d M a n u s c r i p t 12 as doubtful while those above 40% were considered positive.

  4 (P≤0.01), 5 (P≤0.05) and 7 dpv (P≤0.01), as well as at 28 (P≤0.001) and 63 dpv (P≤0.01) by the Wilcoxon-Mann-Whitney test.

  virus genome was detected in the tonsils of four more animals at 7, 18, 21 and 63 dpv by BVDV-specific rRT-PCR. However, CP7E2alf vaccine genome was not detected in the tonsils by the pan-pesti-specific rRT-PCR, nor could the vaccine virus be detected in the tonsils by the two CSFV-specific rRT-PCRs.The C-strain vaccine virus was isolated from the tonsils collected between 3 and 13 dpv except for one animal at 3 dpv and another at 8 dpv. CSFV-specific rRT-PCRs detected the vaccine virus genome in the tonsils from 3 until 77 dpv with only one of the two collected samples being positive at 3, 63, 70 and 77 dpv. At the end of the experiment (98 dpv) the C-strain vaccine virus was still present in one of the two remaining animals.

  majority of the animals (14 pigs) were viraemic for only one day but some others for up to three days (2 animals) (data not shown). C-strain vaccine RNA was detected by CSFV-specific rRT-PCRs in blood samples collected between 3 and 11 dpv with a mean duration of two days (data not shown). Four additional rRT-PCR positive samples were detected up to 42 dpv in the C-strain vaccine group.CSFV E2-specific antibody responseSeroconversion to CSFV E2-specific antigen was first observed on day 15 dpv (2 out of 26 animals) in the CP7E2alf group and on day 11 dpv (19 out of 28 animals) in the C-strain group(Figures 3A and 3B). At 42 dpv all animals but one had seroconverted in the CP7E2alf group within the range of 61.24 to 92.64 E2 blocking percents. Decrease of the median value observed after 77 dpv is mainly imputed to random slaughtering of strongly E2 positive animals. After 70 dpv, blocking percentages above 80% were detected in sera from 5 of the 8 remaining animals while at 91 dpv in only one of the 4 remaining animals. Decrease of antibody titre after 77 dpv was only observed in two animals, with the weakest positive reactions, which were slaughtered at 91 and 98 dpv.In the C-strain group, all animals presented seroconversion at 18 dpv within the range of 40.95 to 74.66 %. The E2 blocking percentage values of the two groups were significantly different A c c e p t e d M a n u s c r i p t 16 from 11 to 91 dpv with P≤0.001 from 11 to 70 dpv, P≤0.01 at 77 and 84 dpv and P≤0.05 at 91 dpv. Individual variations in the E2-specific antibody response, noticed by high range of E2 blocking percentages, were observed in the CP7E2alf group with some animals exhibiting delayed, low intensity or transient seroconversion. CSFV E rns -specific antibody response E rns blocking percentages in the C-strain and CP7E2alf groups were significantly different between 28 and 91 dpv, with P≤0.05 at 28, 84 and 91 dpv, P≤0.01 from 63 to 77 dpv and P≤0.001 from 35 to 56 dpv. No animal seroconverted in the CP7E2alf group and the E rns blocking percentages remained negative until 98 dpv (Figures 4A and 4B). Seroconversion to the CSFV E rns -specific antigen was first observed on 15 dpv (3 out of 26 animals) in the C-strain group within the range of positivity from 52.88 to 60.27 E rns blocking percents. At 35 dpv, 11 of the 20 remaining animals of the C-strain group were E rns positive for E rns (range of positivity from 50.24 to 75.73%).Proportion of E rns positive animals increased until 91 dpv when all the remaining animals presented seroconversion within the range of positivity from 51.78 to 87.31%). Individual variations in the E rns response were observed among animals in the C-strain group, with some transient positive responses. with the Alfort/187 strain, neutralising antibodies were first detected 13 days after CP7E2alf vaccination, in one of the 28 remaining animals (Figures5A and B). Most of the animals seroconverted between 18 and 35 dpv, with the last one at 49 dpv.At that time, the range of positive values extended from 1.00 to 2.51 ND50. Decrease of the median value observed from 77 dpv is mainly due to random slaughtering of strongly VNT positive animals. At 77 dpv, 4 animals of the 8 remaining had VNT titres above 2.00 ND50 while one was negative. At 91 dpv, the negative animal was still present along with one strongly positive pig. Reduction in the VNT titre after 77 dpv was only observed for one positive animal slaughtered at 84 dpv.Using the homologous strain in the VNT (CP7E2alf), the onset of neutralising antibodies was observed earlier, at 11 dpv, in three out of the 27 CP7E2alf-vaccinated animals (Figure5). Most of the animals (22/26 animals) presented neutralising antibodies at 18 dpv within the range of positivity from 0.70 to 2.20 ND50. The last animal seroconverted at 49 dpv. The median ND50 value increased from 11 dpv until 56 dpv and then decreased up to 98 dpv. At 77 dpv, 5 animals of the 8 remaining had VNT titres above 2.00 ND50 while at 91 dpv only one VNT positive animal remained. Reduction of VNT titres after 77 dpv was observed in the three low positive animals slaughtered at 91 and 98 dpv. VNT values obtained with Alfort/187 and CP7E2alf in the CP7E2alf group were significantly different A c c e p t e d M a n u s c r i p t 18 between 13 and 54 dpv (P≤0.001), at 63 dpv (P≤0.01) and between 70 and 91 (P≤0.05). As previously described for the E2 ELISA, individual variations were observed in the VNT response to vaccination in the CP7E2alf group. Some animals had a delayed seroconversion occurring later than 35 dpv. Others seroconverted with low neutralising titres while transient seroconversion was also observed.In the C-strain group, neutralising antibodies were observed from 11 dpv in 7 out of 28 animals (Figure5). At 18 dpv, neutralising antibodies were detected in all the animals of the C-strain group within the range of positivity from 1.00 to 2.20 ND50. The median ND50 value increased from 11 dpv until 98 dpv.VNT values obtained with Alfort/187 in the two vaccination groups were significantly different from 11 to 91 dpv, with P≤0.001 from 11 to 77 dpv, P≤0.01 at 84 dpv and P≤0.05 at 91 dpv. Comparing the mean VN titres obtained in the CP7E2alf group with the homologous strain and in the C-strain group with Alfort/187, significant differences were observed at 11 dpv (P≤0.05), between 21 and 28 dpv (P≤0.05) and at 84 dpv (P≤0.01).

  CP7E2alf chimera(Reimann et al., 2004). The specific design of our animal experiment allowed for the first time the evaluation of the persistence of CP7E2alf vaccine for an extended period. Hereby, it could be demonstrated that CP7E2alf vaccine virus persisted in the tonsils for a similar length to C-strain, up

  neutralising antibodies. Therefore, the crossreactivity of the marker vaccine with CSFV genotypes representative of field strains should be further evaluated.

  DIVA properties remains the major drawback of C-strain vaccination. Genetic differentiation between the chimera CP7E2alf

  marker vaccine induced an immune response observed up to 98 days after oronasal administration in domestic pigs and allowed the use of serological DIVA tests combining CSFV-specific E2 and E rns antibody detection. This study confirms the suitability of CP7E2alf as a interesting and practical marker vaccine candidate for the oral vaccination programmes to control classical swine fever in domestic pigs and wild boar.

Figure 1 :Figure 3 :Figure 4 :

 134 Figure 1: Evolution of rectal temperatures (lines) and clinical scores

Figure 5 :

 5 Figure 5: Proportion of animals positive in VNT (A) and range of

Acknowledgements

We would like to thank Prof. Sándor Belák and Dr Michael O'Connor for suggestions, discussions, and critical comments. We are also grateful to R. Debaugnies, M.-L. Denne, F. Jebbari and C. Thoraval at the VAR and the laboratory team at the DVMP for technical support and we address a special thanks to the technicians at the DVMP for excellent animal care. This study was funded by the European Union in the 6FP project CSFVACCINE & WILDBOAR (SSP1-501599). The animal experiments were conducted with the approval of the VAR and DVMP ethical committees.

Conflict of Interest


Johanna Zemke

Patricia König

Katrin Mischkale

Ilona Reimann

Dr Martin Beer
email: martin.beer@fli.bund.de

Novel BVDV-2 mutants as new candidates for modified live vaccines

Keywords: Bovine viral diarrhea virus type 2, pestivirus, vaccination, infectious pestivirus clone, pseudovirions, modified-live vaccine

Protection against Bovine viral diarrhea virus (BVDV) type 2 infection of commercially available vaccines is often limited due to marked genetic and antigenic differences between BVDV type 1 (BVDV-1) and 2 (BVDV-2). Therefore, the immunogenicity of selected BVDV-1 and -2 mutants derived from infectious full-length cDNA clones and their use as modified-live vaccine candidates against challenge infection with a virulent heterologous BVDV-2 field isolate were investigated. Deletion mutants of BVDV-1 and BVDV-2 lacking a part of the N pro gene (BVDV-1ΔN pro / BVDV-2ΔN pro ) were used as well as a packaged replicon with a deletion in the structural core protein encoding region (BVDV-2ΔCpseudovirions). The 25 calves used in this vaccination/challenge trial were allocated in five groups (n=5/group). One group received BVDV-1ΔN pro (1 shot), one group BVDV-2ΔN pro (1 shot), one group received both, BVDV-1ΔN pro and BVDV-2ΔN pro (1 shot), and one group was immunised with the BVDV-2ΔC pseudovirions (2 shots). The fifth group served as nonvaccinated control group. All groups were challenged intranasally with the BVDV-2 strain HI916 and monitored for signs of clinical disease, virus shedding and viremia. All tested BVDV vaccine candidates markedly reduced the outcome of the heterologous virulent BVDV-2 challenge infection showing graduated protective effects. The BVDV-2ΔN pro mutant was able to induce complete protection and a "sterile immunity" upon challenge. Thus it represents a promising candidate for an efficacious future live vaccine.

Introduction

The two species of Bovine viral diarrhea virus, BVDV-1 and BVDV-2, are grouped in the genus Pestivirus within the family of Flaviviridae [START_REF] Mayo | A summary of taxonomic changes recently approved by ICTV[END_REF] together with other important animal pathogens such as Classical swine fever virus (CSFV) and Border disease virus (BDV). The genome of these enveloped viruses consists of a positive-sense single stranded RNA of about 12.3 kb in size. Analysis revealed at least eleven genetic groups within BVDV-1 (BVDV-1 a-k; Vilcek et al., 2001) and two within BVDV-2 (BVDV-2 a-b; Becher et al., 1999a). Furthermore, a cytopathogenic (cp) and a non-cytopathogenic (ncp) biotype can be differentiated in both species with respect to their effects on cells in vitro.

BVD is spread worldwide and goes along with high economic losses in the cattle industry (Brownlie et al., 1984;[START_REF] Houe | Epidemiology of bovine viral diarrhea virus[END_REF]. Most postnatal infections with both BVDV species take a subclinical course or cause only mild disease which can go along with fever, respiratory symptoms and reproductive disorders such as reduced fertility, abortions, congenital defects or stillbirth. In contrast to the infection of immunocompetent animals, which normally leads to an immune response and as a result to elimination of the virus, infection of pregnant animals in the first trimester with an ncp strain of one of both BVDV species may lead to transplacental infection of the fetus and to the development and birth of persistently infected calves (PIs) [START_REF] Moennig | Pathogenesis of intrauterine infections with bovine viral diarrhea virus[END_REF].

In the 1980ies, a new acute severe form of BVD was observed in North America [START_REF] Carman | Severe acute bovine viral diarrhea in Ontario[END_REF][START_REF] Corapi | Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus[END_REF][START_REF] Corapi | Thrombocytopenia and hemorrhages in veal calves infected with bovine viral diarrhea virus[END_REF]. The course of disease was characterized by a hemorrhagic syndrome, associated with pronounced thrombocytopenia and a significant lethality rate [START_REF] Pellerin | Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities[END_REF][START_REF] Ridpath | Segregation of bovine viral diarrhea virus into genotypes[END_REF]. Subsequently, the causative strains were found to be genetic distinct from previous BVDV isolates and were typed as BVDV-2 [START_REF] Pellerin | Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities[END_REF][START_REF] Ridpath | Segregation of bovine viral diarrhea virus into genotypes[END_REF]Harpin et al., 1995). Up to now, the prevalence of BVDV-2 increased especially in North America (up to 50 % of BVDV isolates; Ridpath, A c c e p t e d M a n u s c r i p t 4 2005), but was also described in Europe [START_REF] Beer | Cytotoxic T-lymphocyte responses in cattle infected with bovine viral diarrhea virus[END_REF][START_REF] Letellier | Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 5' untranslated region[END_REF][START_REF] Falcone | Bovine viral diarrhea disease associated with a contaminated vaccine[END_REF] and other countries (Canal et al., 1998;[START_REF] Nagai | Nucleotide sequence homology to bovine viral diarrhea virus 2 (BVDV 2) in the 5' untranslated region of BVDVs from cattle with mucosal disease or persistent infection in Japan[END_REF].

Different approaches are adopted for disease eradication and various comprehensive control programs tackling the reduction of losses in different countries are pursued. In this context, especially in countries with a high prevalence, high cattle density and very active and quick trading, BVDV vaccination is a valuable tool of BVDV control. However, after vaccination with commercially available BVDV-1 vaccines, protection against BVDV-2 infection is often limited due to a marked genetic and antigenic heterogeneity between BVDV-1 and BVDV-2 (Becher et al., 1999a;Beer et al., 2000). In some cases clinical disease can be prevented, while viremia still occurs. But prevention of viremia is paramount for circumventing transplacental infection of the fetus and thus emergence of PI calves. Taking the heterogeneity and the prevalence data into account, the demand for vaccines that provide a reliable prophylaxis for both, BVDV-1 and BVDV-2, is strengthened (Beer et al., 2000;[START_REF] Fulton | Bovine viral diarrhoea virus antigenic diversity: impact on disease and vaccination programmes[END_REF]. In current vaccination programs modified-live vaccines are used as well as inactivated ones, but there are concerns about their safety and/or efficacy [START_REF] Becher | RNA recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease[END_REF][START_REF] Carman | Severe acute bovine viral diarrhea in Ontario[END_REF]. Modified-live vaccines are considered to be efficacious as immunogenic proteins are amplified through viral replication, and offer a long lasting protection, but their safety is questionable especially when being used during pregnancy [START_REF] Moennig | Pathogenesis of intrauterine infections with bovine viral diarrhea virus[END_REF] or in animals having contact to pregnant ones. The available inactivated vaccines are safe, but they have disadvantages concerning fetal protection, duration of protection and production costs, as high amounts of antigen and formulation with adjuvants are required (Beer et al., 2000;Beer and Wolf, 2003;[START_REF] Carman | Severe acute bovine viral diarrhea in Ontario[END_REF].

New developments aim at combining the immunogenicity of live attenuated vaccines with the safety of inactivated ones by the use of genetically engineered constructs like vector vaccines, DNA vaccines, subunit vaccines, and marker or deletion mutants (Bruschke et al., 1997; A c c e p t e d M a n u s c r i p t 5 [START_REF] Reimann | Packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response[END_REF]. Vaccination with pseudovirions e.g. already proved to be successful for BVDV-1. These pseudovirions are infectious virus particles derived from engineered mutants with a deletion in the structural protein region (replicons), and are produced through in transcomplementation by a helper cell line. They are safe through their inability to assemble new infectious virions when replicating in non-complementing cells so they are "defective in second cycle" (DISC; [START_REF] Reimann | Packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response[END_REF]Widjojoatmodjo et al., 2000). Another approach aims at attenuation through the deletion of the non-structural protein N pro , which is involved in the suppression of the host's innate immune system. Mutants were tested safe and efficacious for BVDV-1 [START_REF] Meyers | Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease[END_REF]P. König, unpublished data) and CSFV (Tratschin et al., 1998;[START_REF] Mayer | Establishment and characterisation of two cDNA-derived strains of classical swine fever virus, one highly virulent and one avirulent[END_REF]. Those mutants can be propagated on conventional cell lines as the autoprotease N pro is dispensable for viral growth in vitro.

In this study, selected genetically engineered BVDV deletion mutants derived from infectious full-length cDNA clones were characterized in vivo in a vaccination/challenge trial. A BVDV-1 and a BVDV-2 deletion mutant, each lacking a major part of the N pro gene (BVDV-1ΔN pro / BVDV-2ΔN pro ), as well as pseudovirions derived from a BVDV-2 core protein deletion mutant (replicon) were tested as modified live candidates against a heterologous, virulent BVDV-2 isolate for challenge infection.

Materials and Methods

Virus strains, deletion mutants and cells

The cytopathogenic BVDV-1b strain CP7 was isolated from a case of fatal Mucosal Disease as described previously [START_REF] Corapi | Monoclonal antibody analyses of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections[END_REF]. The CP7 cDNA full-length clone is based on the plasmid pA/BVDV and was kindly provided by G. Meyers (FLI Tübingen; Meyers et al The BVDV-2 strain 890 (Bolin and Ridpath, 1992) is an ncp strain belonging to the subgroup BVDV-2a and was isolated in the USA from a heifer that died of acute BVDV infection. The 890 full-length cDNA clone (p890FL) was constructed and assembled by K. Mischkale et al. as were the BVDV-2 mutants used in this study [START_REF] Mischkale | Characterization of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants[END_REF]. For receiving v890FLΔN pro (BVDV-2ΔN pro ) a deletion of 156 aa was set as described above for BVDV-1ΔN pro . The v890FLΔC replicon (BVDV-2ΔC) has a partial deletion of 43 aa (nt 986-1114) in the core protein encoding region.

The challenge strain, ncp BVDV-2a HI916, was isolated in Germany during an acute severe outbreak of BVD (kindly provided by G. Wolf, LMU München; Martin et al., 2005). BVDV-1b strain SE5508 was used in neutralisation assays as a heterologous BVDV-1 prototype virus.

Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% BVDV free foetal bovine serum and incubated at 37°C in a humidified atmosphere containing 5% CO 2 .

A diploid bovine esophageal cell line, KOP-R (RIE244, Collection of cell lines in veterinary medicine, CCLV, FLI Insel Riems) that is highly susceptible to BVDV infection was used for virus isolation and neutralisation assays. The challenge virus stock was produced on Madin Darby bovine kidney (MDBK) cells (RIE261; CCLV, FLI Insel Riems). Virus stocks of the BVDV-1 and BVDV-2 N pro deletion mutants were propagated on interferon-incompetent MDBK cells (kindly provided by G. Keil, FLI; RIE728; CCLV, FLI Insel Riems). Generation of v890ΔC_trans (BVDV-2ΔC pseudovirions) was carried out using a new wisent helper cell line (WT-2; P. König, unpublished data). Cells and cell culture media were routinely screened for the absence of BVDV and BVDV-specific antibodies (CCLV, FLI Insel Riems). In vitro

Animals and experimental design

25 conventionally reared female Holstein-Frisian calves were obtained from local farms after being tested negative for BVDV and BHV-1 (antibodies, antigen and genome). Their age ranged from 6 to 14 months. For the trial, animals were allocated into 5 different groups (n = 5 per group) and were housed in the Biosafety Level -3 facility of the FLI. For vaccination, virus stocks were diluted in cell culture medium to a final concentration of 5 x 10 5 tissue culture infective doses 50% (TCID 50 ) per ml. All calves were vaccinated intramuscularly with 2-5 ml containing approximately 1 x 10 6 TCID 50 per animal (see values of the backtitrations enclosed in brackets behind the assigned mutant below). One group received the BVDV-2ΔN pro (9.3 x 10 5 TCID 50 ), one the BVDV-1ΔN pro (9.3 x 10 5 TCID 50 ), and one group received a mixture of both N pro deletion mutants in a single application (BVDV-2ΔN pro & BVDV-1ΔN pro ; 1.3 x 10 6 TCID 50 ; approximately 6.5 x 10 5 TCID 50 of each mutant). An additional group was vaccinated with the BVDV-2ΔC-pseudovirions (first shot:

1.1 x 10 6 TCID 50 / second shot: 6.3 x 10 5 TCID 50 ) and the animals of the fifth group served as non-vaccinated controls.

Two different vaccination schemes were used: animals that received the BVDV-2ΔCpseudovirions were immunised twice, the shots given 25 days apart (day 0 and day 25).All other groups were immunised only once at day 25.

A c c e p t e d M a n u s c r i p t 8 Intranasal challenge infection with the BVDV-2 strain HI916 ensued 35 days after the last vaccination for all 25 animals with 2.3 x 10 6 TCID 50 in a volume of 2 ml (1ml per nostril) with the help of a nebulizer. All viral suspensions were backtitrated on KOP-R cells after inoculation to confirm infectious titres of the respective inoculum.

Clinical evaluation

Body temperatures were measured daily during the whole trial. Mean values of the BVDV-1ΔN pro group had to be calculated from four animals from day 39 on, as the behavior of one animal did not allow further measurement. The calves were examined for adverse reactions immediately after vaccination and challenge infection. Further clinical examinations were carried out during the sampling periods and comprised noting signs of clinical disease, focusing on respiratory and digestive disorders, and controlling general health status (depression, feed intake and behavior). Cumulative clinical scores were obtained using a defined scoring system, ranking signs from 0 (inconspicuous) to 4 (markedly abnormal). The clinical score was normalized to a cutoff line for mean group values at 2 points, above which we stated the score as raised. The cut-off was calculated from pre-vaccination signs and permanent basal symptoms like mild nasal discharge and sporadic coughing.

Samples

Nasal swabs and blood samples were taken daily over a period of 10 days after vaccination (after the first for the BVDV-2ΔC group) and for 14 days after challenge infection (16 days in the control group). Serum samples were obtained weekly throughout the course of the study.

Specimens were subjected to virological, hematological and serological investigations.

Hematological investigations (differential blood cell counts)

Blood samples were taken by jugular venipuncture and collected in sterile blood collection tubes (Monovette) with potassium EDTA as anticoagulant (Sarstedt, Nuembrecht, Germany).

Total white blood cell counts as well as thrombocyte counts were determined by size distribution analysis with an Abbott CellDyn 3700 analyzer.

Virus isolation

Virus isolation was done in cell culture, inoculating monolayers of KOP-R cells with 4 replicates per animal and specimen. After 3 to 4 days of incubation viral infection and replication was detected by indirect immunofluorescence staining of NS3 protein using mab mix WB103/105 (anti-NS3, CCpro, Germany). Binding was detected using ALEXA488 goatanti-mouse-IgG conjugate (Molecular Probes). Evaluation was carried out using an IX51 fluorescence microscope (Olympus, Hamburg, Germany). Furthermore, one blind passage of supernatants was performed after 3 to 4 days of inoculation.

For determination of nasal virus shedding, swab sticks containing a sterile cotton plug (Medical wire and equipment MW&E, Corsham, England) were used. Swabs were submerged in 1 ml of cell culture medium supplemented with 200 U/ml penicillin, 200 µg/ml streptomycin and 5 µg/ml amphotericin B (Sigma-Aldrich, Deisenhofen, Germany). Four replicates per sample (100 µl/replicate) were co-cultivated on KOP-R cells and analyzed as described above. Samples were stored at -70°C until further use. 5mL of EDTA blood per animal and day were treated with an ammonium chloride blood lysisbuffer (155 mM NH 4 Cl, 10 mM KHCO 3 , 1 mM EDTA/ pH 7.4). After haemolysis and centrifugation at 3200 rpm, pelleted leukocytes were washed twice with phosphate buffered saline without Ca ++ /Mg ++ (PBS-) and were re-suspended in 1 ml PBS-. KOP-R cell cultures were inoculated with 4 replicates per sample, containing approximately 3 x 10 6 A c c e p t e d M a n u s c r i p t 10 leukocytes/100 µl each, and tested for BVDV using immunofluorescence analysis as described above. Purified leukocytes were stored at -70°C until further use.

Serology

Sterile blood collection tubes (Monovette) with a clot activator (Sarstedt, Nuembrecht, Germany) were applied for the weekly serum sampling. After centrifugation at 3000 rpm, serum aliquots were stored at -20°C. For serological investigations all sera were inactivated at 56°C for 45 minutes.

A commercially available competitive NS3 antibody ELISA (PrioCHECK ® BVDV Ab) was used and samples were processed following the manufacturer's instructions (Prionics AG, Switzerland). In order to confirm results, we additionally tested all samples with a second antibody ELISA, primarily detecting E2 antibodies (HerdChek® BVDV Ab, IDEXX Europe B.V.), according to the manufacturer's instructions. Furthermore, sera from all animals were tested in a standard neutralisation assay (NA) against selected BVDV-1 and -2 strains (BVDV-1 SE5508, BVDV-2 890 and HI916). For all three virus strains, inactivated serum samples were serially diluted (log2 steps) in triplicates of 50 µl in 96-well plates using cell culture medium. 50 µl of a virus dilution (1000 TCID 50 /ml) was added per well and the plates were incubated at 37°C. After 2 h of incubation, 100 µl of a KOP-R cell suspension (2 x 10 4 cells/well) were added and the plates were incubated for 4 days. BVDV antibody-positive and antibody-negative sera were used as test controls. The virus titre was confirmed by backtitrations (log10 dilutions, 8 parallels). Neutralisation, i.e. absence of virus infection and replication, was detected by indirect immunofluorescence (see above). Titres were expressed as reciprocal of the highest dilution that caused 50% neutralisation (log 2 ND 50 ).

Sequence analysis

Total RNA from cells transfected with the full-length RNA of the BVDV-2ΔC mutant was extracted with the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. E2-encoding sequences were amplified using the Superscript III One-Step RT-PCR system (Invitrogen, Karlsruhe, Germany). After agarose gel electrophoresis, the E2-PCR product was further purified with the QIAex II Gel Extraction kit (Qiagen, Hilden, Germany). Sequencing of the E2 encoding region was carried out using the Big Dye® Terminator v1.1 Cycle sequencing Kit (Applied Biosystems). Nucleotide sequences were read with an automatic sequencer (3130 Genetic Analyzer, Applied Biosystems, Foster City, USA) and analyzed using the Genetics Computer Group (GCG) software version 11.1 (Accelrys Inc., San Diego, USA). Custom primers were used for sequencing (MWG Biotech, Ebersberg, Germany).

Results

Vaccination

Following first vaccination with the pseudovirions (day 0), the general condition of the animals in the BVDV-2ΔC group remained unaffected. The animals showed neither adverse reactions nor a temperature rise (Fig. 1) or clinical signs of disease. No leukopenia could be observed (data not shown). To the contrary, there was even a slight increase in mean leukocyte counts up to a maximum of 147 % on day 7. Neither shedding via nasal excretions nor pseudovirion viremia was detectable by virus isolation in cell culture (Table 1 andTable 2). Therefore, the group was not sampled following second vaccination. In the NS3-specific blocking ELISA, the pseudovirion-immunised animals developed only a marginal rise in the inhibition levels and remained negative until booster vaccination (Fig. 2). Neutralising A c c e p t e d M a n u s c r i p t 12 antibody titres against all three strains tested were not detectable after the first vaccination (Fig. 3).

After vaccination of all groups at day 25 (2 nd immunisation of the BVDV-2ΔC group), all animals stayed within the physiological temperature range (Fig. 1). One animal from the BVDV-1ΔN pro and one out of the BVDV-2ΔN pro group showed a small elevation in temperature for one (day 28) to two days (day 31, day 32) respectively, but did not develop fever. The BVDV-1ΔN pro group showed some clinical reaction post vaccination (p. vacc.).

They had slightly elevated mean clinical score values for 2 days (day 28 and day 35) due to mild respiratory symptoms (data not shown). None of the other groups had raised scores in this period of monitoring. Calves in the groups which received the different ΔN pro -mutants had a short and monophasic decline in their leukocyte counts, which were reduced up to 28% (mean group value BVDV-1ΔN pro & BVDV-2ΔN pro , day 29) p. vacc. (Fig. 4). In both, the BVDV-1ΔN pro and the BVDV-2ΔN pro group, up to 20% reduction of mean values was observed (days 29-30 and days 30-32). Thrombocyte counts also slightly decreased in all three sampled groups paralleling leukocyte counts (data not shown).

Vaccine virus was detected by isolation on cell culture in the nasal swab sample from one BVDV-2ΔN pro -immunised animal on one single day (day 32, 1 replicate after blind passage; Table 1). No nasal virus shedding could be observed in the other groups. In addition, animals from all three ΔN pro -groups groups had a very limited vaccine virus viremia (Table 2). Virus could be isolated from purified leukocytes from at least two animals per group for at least one day between day 29 and day 33 of the trial (BVDV-1ΔN pro : 3 animals, 2 days; BVDV-1ΔN pro & BVDV-2ΔN pro : 3 animals, 1 to 2 days; BVDV-2ΔN pro : 2 animals, 3 to 4 days). All animals vaccinated on day 25 with the N pro deletion mutants were scored positive in a NS3 blocking ELISA (Fig. 2) from day 14 after vaccination. As mentioned above, animals in the BVDV-2ΔC group stayed negative after first immunisation but showed a clear boost in were found in the BVDV-2ΔN pro group (Fig. 2). All control animals stayed seronegative during the vaccination period. Neutralising antibody titres (Fig. 3) were found in all immunised groups as soon as 14 days after vaccination except for the BVDV-2ΔCpseudovirion group. Testing sera against the heterologous BVDV-1 strain SE5508, highest pre-challenge titres with a mean value of up to 891 ND 50 were reached in the groups receiving BVDV-1ΔN pro (alone or in the mixed application). All other groups stayed basal in their titres (mean values ≤ 2 ND 50 ) prior to challenge (Fig. 3). Using the BVDV -2 challenge strain HI916 in a neutralisation assay, slightly different results were obtained. Highest titres after immunisation were reached in the group receiving the BVDV-2ΔN pro mutant (mean value 97 ND 50 ) followed by the BVDV-1ΔN pro & BVDV-2ΔN pro and the BVDV-1ΔN pro group (28 ND 50 /11 ND 50 ). The group vaccinated with the pseudovirion preparation (BVDV-2ΔC), which was clearly positive in the ELISA by day 7 after booster vaccination, however stayed at basal to non detectable neutralising titre levels (< 2 ND 50 ) until challenge infection. Similar values and trends were seen against BVDV-2 strain 890. Mean titres of 79 ND 50 were found in the BVDV-2ΔN pro vaccinated animals at the day of challenge. The graduation between the groups was similar to the one obtained against BVDV-1 HI916 (BVDV-1ΔN pro & BVDV-2ΔN pro 18 ND 50 ; BVDV-1ΔN pro 11 ND 50 ). In the BVDV-2ΔC group, neutralising antibodies ranged from very low to undetectable (mean values ≤ 2 ND 50 ) even against the parental BVDV-2 890 strain.

A c c e p t e d M a n u s c r i p t 14

Challenge infection

After challenge infection at day 60 of the trial, all control animals showed typical and clear signs of clinical disease. They had a biphasic rise in their body temperatures, a slight one at day 3, and a pronounced one at days 8 and 9 p. chall. with maximum mean group values of up to 41°C (Fig. 1). Simultaneously, a marked rise in clinical scores could be found peaking at days 8 to 10 (data not shown). Besides fever, all calves had marked respiratory symptoms (coughing and mucopurulent nasal discharge), depression with reduced appetite, and 2 animals showed watery diarrhea for 2 to 3 days. In contrast, clinical effects of the challenge infection were clearly reduced -or even absent -in the vaccinated groups. Vaccinated animals had an elevation in body temperature at day 7 p. chall. to different degrees (Fig. 1). Highest mean values were reached in the BVDV-1ΔN pro (40°C) and in the BVDV-1ΔN pro & BVDV-2ΔN pro (39.8°C) groups. The BVDV-2ΔC group stayed in the physiological temperature range, peaking at 39.4°C. Temperature reaction went along with moderated respiratory symptoms in the BVDV-1ΔN pro group (data not shown). The only animals that stayed completely unaffected regarding their body temperature and clinical signs were those vaccinated with BVDV-2ΔN pro .

After challenge infection, all control animals developed a severe leukopenia (Fig. 4). They had a bi-to triphasic decrease (days 3, 7 and 13 p. chall.) in leukocyte counts with maximum levels of 48 % reduction at day 7 after challenge. The vaccinated animals showed no or only a monophasic decline of the leukocyte numbers. A maximum decrease of about 12 % in the BVDV-1ΔN pro and the BVDV-2ΔC group was found at day 4. In addition, those animals quickly recovered to pre-infection counts (day 7 p. chall.). The group that received the mixed application of ΔN pro mutants (BVDV-1ΔN pro & BVDV-2ΔN pro ) had a mean drop of 20 % at day 5 p. chall. and of 24 % at day 10 p. chall., and these reduced leukocyte counts persisted till the end of the trial (day 89/day28 p.chall.) with a mean reduction of 20 % (Fig. 4). BVDV-A c c e p t e d M a n u s c r i p t 15 2ΔN pro vaccinated animals showed no decrease in leukocyte blood counts after challenge infection. Thrombocyte counts were not as heavily affected by BVDV-2 infection as expected. The control animals had a mean reduction to a maximum of 35 % at day 3 after test infection. All other groups showed no or only a monophasic less marked decrease (data not shown). No clinical effects like bloody diarrhoea, petechia or hematomas on injection/injury sites, which were described for the challenge virus strain, were observed. Thereafter counts notably increased in the controls (to mean values of 195 %), the BVDV-1ΔN pro group (175 %) and slightly in the BVDV-1ΔN pro & BVDV-2ΔN pro one (125 %) corresponding to severity of infection and disease (data not shown).

Performing virus isolation, we found a long and pronounced challenge virus viremia in the control animals for up to 11 days (day 62 -day 73; Table 2) while there was a clear reduction in duration (day 63 -day 68) and amount in all vaccinated groups (BVDV-2ΔC: 4 animals, 1 day; BVDV-1ΔN pro : all animals, 1 to 5 days; BVDV-1ΔN pro & BVDV-2ΔN pro : 2 animals, 1 to 3 days). In contrast, no challenge virus could be isolated from the leukocytes of the BVDV-2ΔN pro group. Furthermore, challenge virus was detectable in the nasal swab samples of all control animals from day 61 till day 71 (Table 1). Duration (day 62 -day 68) and levels of nasal virus shedding were again markedly reduced in the vaccinated animals (BVDV-2ΔC: all animals, 1 to 4 days; BVDV-1ΔN pro : 4 animals, 1 to 3 days; BVDV-1ΔN pro & BVDV-2ΔN pro : 2 animals, 1 to 2 days). No challenge virus could be recovered from the nasal swab samples of the BVDV-2ΔN pro -vaccinated animals.

From day 14 p. chall., all control animals scored positive in the NS3 blocking ELISA, while NS3 antibodies in all other groups were slightly boostered. Mean blocking values of 100 % were reached in the five groups at day 89 (Fig. 2). Infection with BVDV-2 HI916 also induced a boost in neutralising antibodies titres in the immunised groups detected by A c c e p t e d M a n u s c r i p t 16 neutralisation assays peaking at day 14 to day 28 p. chall. (Fig. 3). The controls developed detectable neutralising titres by day 14. Against BVDV-1 SE5508 maximum titres were reached in the BVDV-1ΔN pro and BVDV-1ΔN pro & BVDV -2 ΔN pro group with 3821 ND 50 and 1552 ND 50 respectively. The titres in the other three groups also increased, but mean values peaked at a markedly lower level (97 ND 50 and 16 ND 50 ), leaving a distinct gap between the two groups vaccinated with the BVDV-1 mutant and those receiving solely BVDV-2 (mutants or challenge strain). Tested against the BVDV-2 strain 890, sera of the BVDV-1ΔN pro animals featured maximum titres of 1552 ND 50 . Values of all other groups followed closely and at the term of the study end titres were very similar in all groups (588 ND 50 ). Highest neutralising titres against the BVDV-2 strain HI916 remained lower (274 ND 50 ; BVDV-1ΔN pro group), but all mean group values were again similar at the end of the trial (181 ND 50 ) with slightly lower values for the BVDV-2ΔC-immunised group (69 ND 50 ). In general, the conducted neutralising assays in this study showed that titres of BVDV-2 exposed animals against BVDV-2 strains were lower than those of the BVDV-1 vaccinated animals against the applied BVDV-1 strain.

Sequencing results of the E2 region of BVDV-2 ΔC

Due to widely differing results of the NS3-specific ELISA and the neutralisation assay for the BVDV-2ΔC-immunised group, we sequenced the region of the replicon encoding the E2 protein. The E2 protein is the major immunogen of BVDV and the predominant inducer of neutralising antibodies. We found one nucleotide change compared to the corresponding sequence of the parental full-length cDNA clone. It was located at nucleotide position 2736 referred to the full-length cDNA and leads to an amino acid change from leucine to histidine.

Discussion

In contrast to vaccines currently licensed in Europe, commercially available vaccines in the United States, where BVDV-2 is highly prevalent, include BVDV-2 strains (Ridpath, 2005).

Although data show a markedly lower prevalence in Europe [START_REF] Beer | Cytotoxic T-lymphocyte responses in cattle infected with bovine viral diarrhea virus[END_REF][START_REF] Beer | A new inactivated BVDV genotype I and II vaccine. An immunisation and challenge study with BVDV genotype I[END_REF], outbreaks of severe acute disease associated with hemorrhagic syndrome, reproductive losses and high lethality rates are reported. Thus, future vaccines will prove advantageous to also mediate a stable protection against BVDV-2 strains [START_REF] Becher | Impfung gegen BVD/MD: Gegenwärtiger Stand und Perspektiven, 19. Arbeits-und Fortbildungstagung des AVID[END_REF]Beer et al., 2000). In order to test and compare new potential BVDV-2 vaccine candidates in vivo we experimentally vaccinated cattle and subsequently challenged them with a recent, virulent German BVDV-2 field strain (HI916), which allowed graduated classification between the different vaccinated groups. The results of this study show that clinical symptoms, leukopenia, viremia and nasal virus shedding after experimental infection of calves could be clearly reduced or even prevented with all three tested BVDV mutants compared to the markedly affected control animals. However, thrombocytopenia and hemorrhagic syndrome described after field-infection with BVDV-2 HI916 (Martin et al.;2005), could not be observed.

In this study, a BVDV-2 replicon construct [START_REF] Mischkale | Characterization of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants[END_REF] with a deletion in the core protein region could be successfully complemented in trans with the help of a newly established helper cell line, expressing the BVDV-1-PT810 structural proteins C to E2 (König, unpublished data). Infectious particles, so called pseudovirions, could be generated in sufficient amounts to characterise BVDV-2ΔC in vivo. Following immunisation with BVDV-2ΔC-pseudovirions neither nasal virus shedding nor pseudovirion viremia was detectable as it was previously described for a BVDV-1ΔC mutant [START_REF] Reimann | Packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response[END_REF].

These in vivo results clearly indicate that pseudovirions are defective in second cycle (DISC) and therefore neither horizontally nor vertically transmissible after vaccination. Serological A c c e p t e d M a n u s c r i p t 18 responses after immunisation with pseudovirions resembled those of inactivated vaccines (Beer and Wolf, 2003), showing low NS3 antibody levels after the first application and a clear and quick boost effect after the second one 25 days later. Unexpectedly, these antibodies had very low to non-existent neutralising abilities against any of the tested BVDV-1 and -2 strains, which is most likely the reason for the reduced protective effect in comparison to the previously published data from immunisation trials using BVDV-1ΔC [START_REF] Reimann | Packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response[END_REF].

Therefore, we sequenced the E2 encoding region of the replicon cDNA and found a single nucleotide change at nucleotide position 2736 leading to an amino acid change in a highly immunogenic region (leucine to histidine; aa 109 of the p890FL E2). For CSFV it was demonstrated by van Rijn ( 2007) that the local variability by one or more aminoacids in the E2 region may lead to differences in affinity, avidity and in cross-neutralisation. Therefore, we speculate that the altered E2 of BVDV-2ΔC could entail a lack of neutralising E2 antibodies and thus less efficient protective qualities. Possible functional defects of the mutated BVDV-2ΔC E2 protein could be masked and functionally complemented in the pseudovirion particles by the structural proteins of a BVDV-1 strain provided by the helper cell line. In the animal, the non-replicative BVDV-1 E2 protein from the putative hybridenvelopes did not induce detectable levels of neutralising antibodies against the tested BVDV-1 strain. However, BVDV-2ΔC had clear protective effects, which underlines the importance of cell-mediated responses to Pestivirus infection as it was described previously [START_REF] Beer | Cytotoxic T-lymphocyte responses in cattle infected with bovine viral diarrhea virus[END_REF][START_REF] Larsson | Bovine virus diarrhoea virus induces in vitro a proliferative response of peripheral blood mononuclear cells from cattle immunized by infection[END_REF][START_REF] Kimman | Cellular immune response to hog cholera virus (HCV): T cells of immune pigs proliferate in vitro upon stimulation with live HCV, but the E1 envelope glycoprotein is not a major T-cell antigen[END_REF][START_REF] Pauly | Classical swine fever virus-specific cytotoxic T lymphocytes and identification of a T cell epitope[END_REF].

As previously described, the non-structural N pro protein interferes with the host's innate immune response by interacting with interferon regulatory factor-3 (IRF3) and targeting it for proteasomal degradation [START_REF] Hilton | The NPro product of bovine viral diarrhea virus inhibits DNA binding by interferon regulatory factor 3 and targets it for proteasomal degradation[END_REF]. Knocking out this immunosuppressive function through deletion of an essential part of the genome aims at attenuating BVDV strains making them future vaccine candidates with enhanced safety. CSFV mutants with a deletion in the A c c e p t e d M a n u s c r i p t 19 N pro gene already showed to be attenuated [START_REF] Mayer | Establishment and characterisation of two cDNA-derived strains of classical swine fever virus, one highly virulent and one avirulent[END_REF]. BVDV-1ΔN pro mutants also proved to be highly attenuated even in pregnant animals and to mediate complete protection against a heterologous BVDV-1 challenge [START_REF] Meyers | Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease[END_REF]König et al., unpublished data). In our study, we could show that in vivo both, the BVDV-1 and the BVDV-2 N pro deletion mutant provided clinical protection against challenge infection. Interestingly, lower homologous titres of neutralising antibodies were necessary to gain a "sterile immunity" than it was described for previous studies (Bolin [START_REF] Carman | Severe acute bovine viral diarrhea in Ontario[END_REF]Beer et al., 2000). It turned out that a neutralising titre of 64 ND 50 to 128 ND 50 against the challenge strain prior to infection was sufficient to prevent systemic challenge virus infection, irrespective to the titres against the reference BVDV-1 strain SE5508. In all three groups NS3-specific antibodies could be detected as soon as 14 days after vaccination indicating effective replication of all mutants. The group immunised with BVDV-1ΔN pro had high neutralising antibodies against the BVDV-1 strain, which were apparently less effective in neutralising BVDV-2 strains in vitro, and this group was less protected against challenge infection than were the others. In contrast to the results of Beer et al. (2000), there was a marked difference in the titres developed against BVDV-1 and BVDV-2 when both N pro deletion mutants were simultaneously applied. A possible explanation could be the more efficient infection and higher replication levels of the cp BVDV-1ΔN pro , subsequently leading to interference with BVDV-2ΔN pro infection and replication. This assumption is supported by our findings that reisolated virus from leukocyte samples was characterized as BVDV-1 by selective immunofluorescence staining. Nevertheless, only the BVDV-2ΔN pro group developed a complete protective immune response inducing a "sterile immunity" against heterologous BVDV-2-challenge infection.

The BVDV-2 strain 890 served as basis for the first approach generating this kind of BVDV-2 deletion mutants as it offers many options for virulence studies. This advantage was used in A c c e p t e d M a n u s c r i p t 20 the presented proof-of-principle study to investigate in detail how effectively the two applied mutation strategies, deleting a structural protein or a protein acing as an immunosuppressing modulator, attenuate such an isolate in vivo. Obtained results could then be more reliably transferred to less virulent strains that would be used for further developments. As infectious virus from the constructed BVDV-2 full-length cDNA clone (p890FL) is still moderately virulent in cattle [START_REF] Mischkale | Characterization of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants[END_REF], it could be concluded from this trial that all tested BVDV-2 deletion mutants are further attenuated. It should be additionally beneficial to the safety of future BVDV-2 deletion mutants to originate from a cytopathogenic full-length cDNA clone, since cp viruses are the standard for attenuated BVDV-1 live vaccine preparations [START_REF] Fulton | Bovine viral diarrhoea virus antigenic diversity: impact on disease and vaccination programmes[END_REF]van Oirschot et al., 1999;Beer and Wolf, 2003). If cytopathogenic vaccine viruses would be able to reach the fetus, abortion could be induced in sporadic cases, but the possibility of induction of a persistent infection would be excluded, as there are no data that indicate reversion of a cp strain to an ncp one in vivo (Ridpath et al., 2005). [START_REF] Meyers | Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease[END_REF] have shown that prevention of transplacental infection with BVDV-1 and -2 ΔN pro mutants requires a further elimination of the RNase activity of E RNS .

To investigate this safety aspect, vaccination studies with BVDV-2 N pro deletion mutants in pregnant animals have to be carried out. Reversions or recombinations of the assessed ncp BVDV-2ΔN pro as well as for prospective cp BVDV-2 ΔN pro mutants with a second strain, as described previously (Becher et al., 1999b;Meyers et al., 1992), cannot be excluded by our present results. At least for BVDV-2ΔC these events are highly unlikely as discussed for BVDV-1 replicons that were thoroughly analysed in vitro [START_REF] Reimann | Trans-complementation of autonomously replicating Bovine viral diarrhea virus replicons with deletions in the E2 coding region[END_REF][START_REF] Reimann | Packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response[END_REF]. Both issues, that would require extensive double infection studies in vitro as well as in vivo, were not addressed in the setup of this first trial, which served as promising basis for future developments.

  The CP7 N pro deletion mutant (BVDV-1ΔN pro ) is lacking 156 aminoacids (aa) of the non-structural N pro gene (nucleotide (nt) 422-889;Reimann et al., unpublished data).

  the BVDV-2ΔC replicon was transfected by electroporation into the trans-complementing helper cells. Infected, resp. transfected cell cultures were freeze/thawed once. All virus stocks were subsequently cleared from cell debris through low speed centrifugation, titrated on KOP-R cells and tested for sterility and absence of mycoplasma. All virus preparations were stored at -70°C until use.

  A c c e p t e d M a n u s c r i p t 11

  ELISA positive as soon as 7 days after their second vaccination. Mean blocking values between 91 % and 95 % were reached in the different vaccination groups prior to challenge infection. Slightly lower mean inhibition values (81 %)

  candidates tested for safety and efficacy markedly reduced the outcome of the heterologous BVDV-2 challenge infection in cattle while showing graduated protective effects with regards to clinical symptoms, nasal virus shedding and viremia. The BVDV-2ΔNpro mutant provided complete protection leading to a "sterile immunity" against the highly virulent BVDV-2 challenge infection, facilitating its possible use as a future efficacious vaccine candidate.Furthermore, the results of this study implicate further investigations, such as (i) consecutive vaccination with BVDV-1ΔNpro and BVDV-2ΔNpro to test for enhanced protection, (ii) construction and immunogenicity testing of a BVDV-2ΔC mutant without the observed amino acid exchange in the E2 region and (iii) development of a cp BVDV-2 full-length cDNA clone including corresponding deletion mutants.

  ., Hehnen, H.R., Wolfmeyer, A., Poll, G., Kaaden, O.R.Wolf, G., 2000. A new inactivated BVDV genotype I and II vaccine. An immunisation and challenge study with BVDV genotype I. Vet.Microbiol. 77 [1-2], 195-208. Beer, M. and Wolf, G., 2003. [Vaccines against infection with bovine viral diarrhea virus/mucosal disease (BVDV/MD): a short overview]. Berl Munch.Tierarztl.Wochenschr. 116 [5-6], 252-258. Bolin, S.R. and Ridpath, J.F., 1992. Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves. Am.J.Vet.Res. 53 [11], 2157-2163. Bolin, S.R. and Ridpath, J.F., 1995. Assessment of protection from systemic infection or disease afforded by low to intermediate titers of passively acquired neutralizing antibody against bovine viral diarrhea virus in calves. Am.J.Vet.Res. 56 [6], 755-759. Brownlie, J., Clarke, M.C.Howard, C.J., 1984. Experimental production of fatal mucosal disease in cattle. Vet.Rec. 114 [22], 535-536. Bruschke, C.J., Moormann, R.J., van Oirschot, J.T.van Rijn, P.A., 1997. A subunit vaccine based on glycoprotein E2 of bovine virus diarrhea virus induces fetal protection in sheep against homologous challenge. Vaccine 15 [17-18], 1940-1945. Canal, C.W., Strasser, M., Hertig, C., Masuda, A.Peterhans, E., 1998. Detection of antibodies to bovine viral diarrhoea virus (BVDV) and characterization of genomes of BVDV from Brazil. Vet.Microbiol. 63 [2-4], 85-97.

  .F., 2005. Practical significance of heterogeneity among BVDV strains: impact of biotype and genotype on U.S. control programs. Prev.Vet.Med. 72 [1-2], 17-30. Tratschin, J.D., Moser, C., Ruggli, N.Hofmann, M.A., 1998. Classical swine fever virus leader proteinase Npro is not required for viral replication in cell culture. J.Virol. 72 [9], 7681-7684. van Oirschot, J.T., Bruschke, C.J.van Rijn, P.A., 1999. Vaccination of cattle against bovine viral diarrhoea. Vet.Microbiol. 64 [2-3], 169-183. van Rijn, P.A., 2007. A common neutralizing epitope on envelope glycoprotein E2 of different pestiviruses: implications for improvement of vaccines and diagnostics for classical swine fever (CSF)? Vet.Microbiol. 125 [1-2], 150-156. Vilcek, S., Paton, D.J., Durkovic, B., Strojny, L., Ibata, G., Moussa, A., Loitsch, A., Rossmanith, W., Vega, S., Scicluna, M.T.Paifi, V., 2001. Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch.Virol. 146 [1], 99-115. Widjojoatmodjo, M.N., van Gennip, H.G., Bouma, A., van Rijn, P.A.Moormann, R.J., 2000.Classical swine fever virus E(rns) deletion mutants: trans-complementation and potential use as nontransmissible, modified, live-attenuated marker vaccines. J.Virol. 74[START_REF] Fulton | Bovine viral diarrhoea virus antigenic diversity: impact on disease and vaccination programmes[END_REF],2973-2980. Wolfmeyer, A., Wolf, G., Beer, M., Strube, W., Hehnen, H.R., Schmeer, N.Kaaden, O.R., 1997. Genomic (5'UTR) and serological differences among German BVDV field isolates. Arch.Virol. 142 [10], 2049-2057. of vaccine (A) /challenge (B) virus: Highly susceptible KOP-R cell cultures were inoculated with 4 replicates of nasal swab fluids (100 µl / replicate) and after 3-4 days virus replication was verified by immunofluorescence staining. Results were scored according to the number of positive inoculations (grey underlay) out of the 4 replicates (0 = no BVDV isolation 4 = all inoculations BVDV positive). A first result was confirmed after one blind passage of the supernatants. Samples that were only detected positive after passaging are highlighted in light grey.

  (A) / challenge (B) virus: Highly susceptible KOP-R cell cultures were inoculated with 4 replicates of purified leukocytes (3 x 10 6 leukocytes / replicate) and after 3 to 4 days virus replication was verified by immunofluorescence staining. Results were scored according to the number of positive inoculations (grey underlay) out of the 4 replicates (0 = no BVDV isolation 4 = all inoculations BVDV positive). A first result was confirmed after one blind passage of the supernatants. Samples that were only detected positive after passaging are highlighted in light grey.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Development of NS3-specific antibodies after vaccination with the indicated

Figure 3 :

 3 Figure 3: Neutralising antibody titres against BVDV-1 SE5508 (A), the BVDV-2 890 wild

Figure 4 :

 4 Figure 4: Blood leukocytes were counted after vaccination(s) (day 0 and day 25) ( ) and after

  Figure 1

Table 2 :

 2 Virus isolation from purified leukocytes

	Page 33 of 40

Acknowledgements

The authors would like to thank the laboratory staff, especially Birgit Goerl and Doreen Reichelt, for excellent technical assistance, as well as the animal keepers for their committed professional work. Furthermore, we are very grateful to Georg Wolf (Veterinary Faculty, LMU Munich) for providing the BVDV-2 isolate HI916.

This study was financially supported by Intervet Schering-Plough Animal Health (The Netherlands).

Conflict of interest statement None