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Vaccine 32 (2014) 7108–7114

Contents lists available at ScienceDirect

Vaccine

j o ur na l ho me page: www.elsev ier .com/ locate /vacc ine

P2-serotyped live-attenuated bluetongue virus without NS3/NS3axpression provides serotype-specific protection and enables DIVA

emke Feenstraa,b,∗, Mieke Maris-Veldhuisa, Franz J. Dausa, Mirriam G.J. Tackena,ob J.M. Moormanna,b, René G.P. van Gennipa, Piet A. van Rijna,c

Central Veterinary Institute of Wageningen UR (CVI), Department of Virology, P.O. Box 65, 8200 AB, Lelystad, The NetherlandsDepartment of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The NetherlandsDepartment of Biochemistry, Centre for Human Metabonomics, North-West University, South Africa

r t i c l e i n f o

rticle history:eceived 26 August 2014eceived in revised form 2 October 2014ccepted 16 October 2014vailable online 31 October 2014

eywords:luetongue virusISA vaccineerotypeIVAS3everse genetics

a b s t r a c t

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges.Vaccination is the most effective measure to control vector borne diseases; however, there are 26 knownBTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are availablefor a limited number of serotypes, but each of these have their specific disadvantages, including theinability to differentiate infected from vaccinated animals (DIVA).

BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but areimportant for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro.Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep isstrongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 withoutNS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is apromising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine.

Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, byexchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccineswith respect to reassortment between vaccine viruses.

Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAsare therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, wedeveloped an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibod-ies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTVchallenge.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Bluetongue virus (BTV), a Culicoides borne orbivirus in theamily Reoviridae, causes Bluetongue (BT) in ruminants, charac-erized by fever, oral and nasal erosions and discharge, oedema,

oronitis, anorexia and death, due to damage of the vascularndothelium [1,2]. At least 26 serotypes, hardly showing cross pro-ection, are known [3,4]. Historically, BT is endemic in regions with

∗ Corresponding author at: Central Veterinary Institute of Wageningen UR (CVI),epartment of Virology, Lelystad, The Netherlands. Tel.: +31 0 320 238 834.

E-mail addresses: femke2.feenstra@wur.nl, feenstra f@hotmail.comF. Feenstra).

ttp://dx.doi.org/10.1016/j.vaccine.2014.10.033264-410X/© 2014 Elsevier Ltd. All rights reserved.

temperate and tropical climate, and more recently with moder-ate climate, related to the presence of competent vectors [5]. InEurope, outbreaks of BTV serotypes 1, 2, 4, 8, 9 and 16 have beenreported and serotypes 2, 10, 11, 13 and 17 are endemic in the USA[6], causing large economic losses [7,8].

BTV has a 10 segmented double-stranded (ds) RNA genomeencoding seven viral proteins (VP1–VP7) and four non-structuralproteins (NS1-4) [9–11]. The outer capsid protein VP2, encoded bySeg-2, is the major serotype determining protein. Membrane asso-ciated NS3/NS3a, encoded by Seg-10, is important for virus release

and is an inhibitor of the cellular interferon response [12–16].

Vaccination is effective in controlling BT outbreaks [17]. Cur-rently marketed vaccines are either conventionally live-attenuatedor chemically inactivated and each has specific disadvantages,

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uch as incomplete protection and required re-vaccinations fornactivated vaccines, and residual virulence, reassortment, and hor-zontal and vertical vaccine spread for live-attenuated vaccines18–21]. Promising vaccine candidates have been described, butone of these are marketed yet [22]. In addition to efficacy andafety, the ability to differentiate infected from vaccinated animalsDIVA) is important to detect infections in vaccinated livestock [17].

Recently, we described the rational design of the next-eneration Disabled Infectious Single Animal (DISA) vaccine23]. BT DISA vaccine is based on live-attenuated vaccine virusTV6/net08 [24,25], without NS3/NS3a expression and is serotypedy exchange of immunodominant VP2. NS3/NS3a knockout results

n avirulence and strongly reduced viremia, whereas protections dependent on local replication. Here, we demonstrate thatxchange of only VP2 induces serotype-specific protection at nineeeks post vaccination in sheep, and we show the ability of DIVAith an experimentally developed NS3 ELISA.

. Material and methods

.1. Cell culture and viruses

BSR cells (a clone of BHK-21 cells [26]) were cultured in Dul-ecco’s modified Eagle’s medium (DMEM, Invitrogen), containing% fetal bovine serum (FBS), 100 IU ml−1 Penicillin, 100 �g ml−1

treptomycin and 2.5 �g ml−1 Amphotericin B.DISA vaccine for serotype 8 has been described previously [23].

riefly, it is based on live-attenuated BTV6/net08 with Seg-2 fromTV8/net07 generated using reverse genetics [27], and with a dele-ion in Seg-10 [28] leading to an NS3/NS3a negative phenotype.

DISA vaccine was produced by infection of BSR cells at low multi-licity of infection (MOI). When >50% of cells were immunostainedositive for BTV VP7 in a duplicate well using anti-VP7 monoclonalntibody (MAb) ATCC-CRL-1875, DISA vaccine was harvested byreeze thawing and centrifugation. Vaccine in clarified supernatantas used for vaccination.

Challenge virus BTV8/net07 [29] was isolated on eggs and pas-aged three times on BHK-21 cells (BTV8/net07/e1/bhkp3) [27].TV2 challenge virus (BTV-2/SAD2001/01) was isolated from sheepPirbright Institute, UK) and was grown for one passage in embry-nated chicken eggs, two passages on BHK-21 cells and threeassages on KC cells (BTV2/SAD01/01/e1/bhkp2/kcp3).

Virus titers were determined by endpoint dilution on BSRells and expressed as 50% tissue culture infectious dose per mlTCID50 ml−1).

.2. Animal experiment

All animal experiments were performed under the guidelines ofhe European Community and were approved by the Committee onhe Ethics of Animal Experiments of the Central Veterinary Insti-ute (permit number 2013.016). Sixteen female Blessumer sheepf 6–24 months old were obtained from a Dutch farm and wereree of BTV and BTV antibodies. Sheep were randomly allocatedn four groups of four animals. After one week of acclimatization,heep were vaccinated with 2 × 1 ml of 105 TCID50 ml−1 DISA vac-ine. Sheep were injected subcutaneously (s.c.) in the back betweenhe shoulder blades at both sides of the spinal cord. At 21 daysost vaccination (dpv), animals received a booster vaccination.t 84 dpv, one vaccinated and one non-vaccinated group were

nfected s.c. with 4 × 1 ml 105 TCID50 ml−1 BTV8/net07/e1/bhkp3

r BTV2/SAD01/01/e1/bhkp2/kcp2. At 21 days post challenge (dpc),ll sheep were sacrificed.

Body temperature and clinical signs were examined, and EDTAlood and serum were collected at indicated days (Figs. 1 and 2).

2 (2014) 7108–7114 7109

Clinical signs were scored according to the clinical score table forBluetongue in sheep (Table S1 [25]). Statistical differences in bodytemperature were calculated using a split plot ANOVA and maximaltemperatures were compared using a pairwise T-test with p < 0.05indicating significance.

2.3. Detection of BTV RNA

Samples of EDTA blood were examined for BTV RNA by panBTVreal-time reverse transcription (RT) PCR testing targeting Seg-1.After isolation of BTV-RNA using the MagNA Pure isolation robot(Roche) [30], Seg-1 RT PCR was performed using primers F-pan-S1, R-pan-S1, and probe P-pan-S1 [31] according to the all-in-onemethod for the panBTV Seg-10 RT PCR [30] (Table 1). Crossing point(Cp) values were calculated, and samples without Cp value show-ing increase of the OD640/530 were interpreted as 40 and negativesamples were set at 45. Statistical differences in Cp value were cal-culated using a split plot ANOVA and minimal Cp values of eachanimal were compared using a pairwise T-test with p < 0.05 indi-cating significant differences.

2.4. VP7 ELISA

The Bluetongue competition VP7 enzyme-linked immunosor-bent assay (VP7 ELISA) was used to detect VP7 antibodies in serumsamples according to the supplier’s instructions (ID.Vet). The per-centage of blocking was displayed as 100 minus value.

2.5. Experimental NS3 ELISA

Optimal dilutions of coated NS3 antigen, serum, mouse MAb33H7, and conjugated rabbit anti-mouse MAb were determined inadvance by a brief validation with positive and negative controlsera (not shown). E. coli produced BTV NS3 antigen was dissolved incoating buffer (100 mM bicarbonate/carbonate, pH9.6) and boundovernight at 4◦C to Nunc maxi sorp plates. After incubation for1 h at 37◦C with dilution buffer (PBS 0.1% Tween 20 and 5% FBS),100 �l of serum samples (1:2 in dilution buffer) were incubated incoated wells for 1 h at 37◦C. After washing, wells were incubatedwith 100 �l 1:1000 MAb 33H7 (Ingenasa, Madrid, Spain) for 1 h at37◦C. After washing, 100 �l 1:5000 rabbit anti-mouse MAb (DAKO,P0260) was added and incubation was continued for 1 h at 37◦C.After washing, wells were incubated at room temperature withTMB substrate (ID.Vet) for 10 min. Coloring was stopped by stopsolution (ID.Vet). OD450 was determined and percentage blockingwas displayed as 100 minus value. Seroconversion cut-off is 30%,based on the mean blocking percentage of negative samples plusthree times the standard deviation.

2.6. Serum neutralization test

Serotype-specific neutralizing antibody (nAb) titers were deter-mined by serum neutralization tests (SNTs) with serum from0, 21, 42, 84 and 105 dpv according to Haig [23,32] usingBTV8/net07/e1/bhkp3 and BTV2/SAD01/01/e1/bhkp2/kcp2.

3. Results

3.1. Temperature and clinical signs

After the first and booster vaccination, none of the animals expe-

rienced clinical signs or fever, indicating that the DISA vaccine isnon-pathogenic. At one day and four days after booster vaccina-tion (22 dpv and 26 dpv), two different sheep developed a slightlyelevated body temperature (not shown).

7110 F. Feenstra et al. / Vaccine 32 (2014) 7108–7114

Fig. 1. Two groups of four sheep were vaccinated s.c., and boosted three weeks later with 2 × 1 ml of 105 TCID50 ml−1 DISA vaccine, serotyped for BTV8. Nine weeks afterb ged s.c clinicw

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ooster vaccination, vaccinated sheep and unvaccinated control groups were challenalculated for the indicated days. (B) Clinical signs of animals were determined andith BTV8 died 13 dpc.

After challenge, sheep in both control groups showed elevatedody temperatures of >39.5 ◦C at 4 dpc (Fig. 1A). After four consec-tive days, temperature declined to normal in all control animals,xcept for one sheep challenged with BTV8. This sheep had feverf >40 ◦C for seven successive days, and eventually died due to BT-pecific clinical signs. The highest temperature measured in bothontrol groups was not significantly different.

Vaccinated sheep challenged with BTV8 did not show elevatedody temperature, whereas BTV2 challenge resulted in an elevatedody temperature of >39.5 ◦C for all animals. The period of elevatedody temperature lasted shorter and elevation was significantly

ower than for control animals challenged with BTV2.Sheep in both control groups showed mild clinical signs of 1

oint per animal from 5 dpc (Fig. 1B). For the BTV2 challenge, thisasted for 9 days and 2–4 animals showed clinical signs, related tohe upper respiratory tract. Sheep in the BTV8 challenge controlroup showed similar signs from 5–10 dpc. Problems in the upperespiratory tract became worse for one sheep and this animal diedt 13 dpc.

Vaccinated sheep challenged with BTV8 showed no clinicaligns. In contrast, BTV2 challenge of vaccinated sheep resulted inifficulties with breathing at 5 dpc for one animal, which lasted for

days and another sheep had problems in the upper respiratoryract at 6 and 10 dpc.

.2. Viremia by PCR testing

After the first vaccination, no Seg-1 PCR signal was detected,ndicating the absence of viremia of vaccine virus (Fig. 2A). Onend three days after booster vaccination, one animal in each of theaccinated groups showed a very weak PCR signal with a Cp valuef 40 (Fig. 2A). These sheep were not the same as the ones showinglightly elevated body temperatures.

After challenge with BTV2 or BTV8, all sheep in control groupshowed viremia by PCR starting at 3 dpc, and Cp values were <40 for–8 days. Both panBTV Seg-1 and Seg-10 PCR (not shown) were per-ormed. Cp values of positive samples were about 5 cycles higher in

c. with 4 × 1 ml virulent BTV8 or BTV2. (A) Average body temperature per group wasal score was cumulated for each group per day. Only one control animal challenged

the Seg-1 PCR compared to the Seg-10 PCR, but viremia of infectedanimals was detected from 3 to 21 dpc by both tests. No PCR signalwas detected in vaccinated animals challenged with BTV8. BTV2challenge of vaccinated sheep resulted in viremia at 4 dpc, with Cpvalues <40 for 1–5 days. Viremia was significantly lower (higher Cpvalues) than in the BTV2 challenge control group, suggesting par-tial protection against heterologous BTV2 challenge by vaccinationwith DISA vaccine for serotype 8.

3.3. Humoral immune responses

3.3.1. VP7 ELISAThe humoral immune response was initially examined for VP7

antibodies using an ELISA (Fig. 2B). All vaccinated animals showed>50% blocking at 7 dpv. Although percentage of blocking wasdeclining from 10 dpv, blocking was still >50% at 21 dpv. VP7 sero-conversion peaked one week post booster vaccination, but neverreached 100% and declined again. At 42 dpv, the first animal showeda blocking <50%, and at 84 dpv/0 dpc, five out of eight animalsshowed <50% blocking.

BTV challenge resulted in >96% blocking at 7 dpc for vacci-nated animals. Non-vaccinated control animals were slightly later(at 9 dpc). After BTV challenge, no obvious decline in blockingwas observed up to 21 dpc in both control and vaccinated groups.Although BTV8 challenge virus was undetectable by PCR in vacci-nated animals, it still strongly boosted the VP7 antibody response.

3.3.2. Differentiation of infected from vaccinated animals by NS3ELISA

Since DISA vaccine is not expressing NS3/NS3a, all animalsremained negative for NS3 Abs by an experimentally developedNS3 ELISA up to challenge, with blocking percentages <10%. At 11dpc, control animals challenged with BTV2 or BTV8 showed >30%

blocking, except for one sheep with 23% blocking. These blockingpercentages further increased rapidly to 52–86% and remained highuntil 21 dpc, the end of the experiment (Fig. 2C). In contrast, vacci-nated sheep challenged with homologous BTV8 remained negative

F. Feenstra et al. / Vaccine 32 (2014) 7108–7114 7111

Fig. 2. Two groups of four sheep were vaccinated s.c., and boosted three weeks later, with 2 × 1 ml of 105 TCID50 ml−1 DISA vaccine, serotyped for BTV8. Nine weeks afterbooster vaccination, vaccinated sheep and unvaccinated control groups were challenged s.c. with 4 × 1 ml virulent BTV8 or BTV2. (A) Viremia in whole blood samples of sheepwas determined using Seg-1 RT PCR. (B) Seroconversion was determined using BTV VP7 ELISA. ELISA signals are expressed as blocking percentage with >50% as thresholdfor seroconversion. (C) Presence of seroconversion for NS3 antibodies was examined using an in-house experimentally developed NS3 competitive ELISA. ELISA signals aree

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or NS3 Abs (blocking <18%) (Fig. 2C). In vaccinated sheep chal-enged with heterologous BTV2, blocking percentages for NS3 Absncreased slightly quicker than in control animals and reached evenlightly higher blocking percentages. Two animals showed >30%locking at 9 dpc. At 11 dpc, three animals showed >30% block-

ng, and at 14 dpc all four animals were seropositive for NS3 Abs61–84% blocking). The blocking percentage further increased andemained positive (66–83%) until the end of the experiment. Appar-ntly, DISA vaccine for serotype 8 is not protective for serotype 2,nd consequently replication of BTV2 challenge virus resulted innduction of NS3 Abs as detected by the NS3 DIVA test.

.3.3. Serum neutralization testsSNTs were performed using homologous (BTV8) and heterol-

gous (BTV2) virus, with sera from vaccinated/challenged sheep

ollected at 0, 21, 42, 84 and 105 dpv (Fig. 3). Sera of 0 dpc and 21pc of control animals were also examined. SNTs for both serotypes

and 8 were negative at 0 dpv for vaccinated groups and at 0 dpcor control groups.

At 21 dpv, nAb titers of 2–4 for serotype 8 were detected inall vaccinated sheep, except for one showing no detectable nAbs(Fig. 3A). The nAb titer increased to 6–24 in all animals three weeksafter booster vaccination (42 dpv), but declined at 84 dpv, withnAb titers of 2–16. Challenge with BTV8 led to high nAb titersof 16–128, specific for serotype 8, in both vaccinated and non-vaccinated sheep. As expected, challenge of non-vaccinated sheepwith BTV2 showed a very low cross neutralization titer for serotype8 of 0–8. Both challenge with homologous BTV8 or heterologousBTV2 led to high nAb titers against BTV8 in vaccinated sheep at 21dpc.

No nAb titers specific for serotype 2 were detected after primaryvaccination, and after booster vaccination only one sheep induceda nAb titer of only 2 at 42 dpv, which is likely a very low cross neu-tralization (Fig. 3B). As expected, BTV2 challenge of control sheepraised high nAb titers specific for serotype 2 (32–128), but BTV8

challenge of control animals led to a nAb titer of 4 in only one sheepat 21 dpc. Challenge of vaccinated sheep with homologous BTV8 didnot result in detectable nAbs against serotype 2. In contrast, BTV2challenge of vaccinated sheep resulted in serotype 2 specific nAb

7112 F. Feenstra et al. / Vaccine 32 (2014) 7108–7114

Fig. 3. Two groups of four sheep were vaccinated s.c., and boosted three weeks later, with 2 × 1 ml of 105 TCID50 ml−1 DISA vaccine, serotyped for BTV8. Nine weeks afterb ged sw v, 84 di

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ooster vaccination, vaccinated sheep and unvaccinated control groups were challenere determined using an SNT with serum samples collected at 0 dpv, 21 dpv, 42 dp

n BSR cells is indicated. Representative results are shown.

iters of 64–128, which is similar to titers found for control sheephallenged with BTV2.

. Discussion and conclusion

Recently, we have shown that a single dose of BT DISA vaccineith VP2 of serotype 8 protects against virulent BTV8 challenge,espite a low nAb titer at the day of challenge [23]. Previous stud-

es showed that protection at three weeks after a single vaccinations not serotype specific [25]. This might be due to the strong induc-ion of serotype non-specific cytotoxic T-lymphocyte (CTL) levelst 7–21 dpv [33–35]. Here, we show that protection is serotypepecific at nine weeks after prime–boost vaccination, since heterol-gous BTV2 challenge induced fever, clinical signs, viremia, anderoconversion for NS3 Abs, whereas vaccinated sheep are com-letely protected against homologous BTV8 challenge. Therefore, it

s important to challenge later than three weeks post vaccination totudy serotype-specific protection. Although DISA 8 vaccinated ani-als were not protected against BTV2 challenge virus, challenge did

trongly booster the BTV8-specific nAb response. This phenomenonas also been shown for AHSV life-attenuated vaccines [36].

Previously, protection of ‘serotyped’ BT vaccine candidates withoth VP2 and VP5 exchanged using reverse genetics has beenemonstrated [25,37]. Here, we show that serotype determin-

ng VP2 is sufficient to generate serotype-specific protection. The

ajority of nAbs are directed against VP2 [38] and single VP2 sub-

nit vaccines can be protective. However, addition of VP5 enhancesrotection of subunit vaccines [39–42]. Possibly, heterologous VP5

n replicating DISA vaccine will further enhance protection as well.

.c. with 4 × 1 ml virulent BTV8 or BTV2. Titers of nAbs against BTV8 (A) and BTV2 (B)pv and 105 dpv/21dpc. The serum dilution that prevented complete CPE formation

The VP7 Ab blocking percentage declined after both the first andsecond vaccination with the DISA vaccine, to about 50% at day ofchallenge. Further, the nAb titer against BTV8 was much lower afterDISA vaccination (vaccinated groups at 21 dpv) than after BTV infec-tion (control groups at 21 dpc). Although viremia was undetectablein the blood by PCR, BTV8 challenge induced a strong booster ofserotype 8 specific nAbs in vaccinated animals. Likely, replicatingDISA vaccine strongly triggers the cellular immune response lead-ing to serotype-specific protection, whereas the humoral immuneresponse is much lower than after BTV infection. Neutralizing anti-bodies against BTV are able to confer protection, although thenAb titer is not always correlated to protection [43]. There is onlylimited knowledge about the cellular immune response againstBTV, although CTLs have been shown to contribute to protec-tion [35,44] and BTV-specific T-helper cells have been identified[45]. Mainly NS1, but also VP2, seems to contain most CTL epi-topes [34,45,46]. We assume that the cellular immune responsedirected to VP2 is involved in serotype-specific protection. Inter-estingly, NS3/NS3a suppresses the induction of interferon [47], andthe absence of this suppression after DISA vaccination might resultin a stronger induction of the anti-viral immune response. Moreresearch is needed to elucidate the importance of the differentaspects of immune responses upon BTV infection, and in particularafter vaccination with DISA vaccine.

DIVA testing is a main advantage for control of disease in live-stock. In addition to the induction of a protective immune response,

the DISA vaccine also enables DIVA based on the NS3/NS3a antibodyresponse, as has been described in combination with inactivatedvaccines [48]. Importantly, the NS3 humoral response after BTVinfection is fast and lasts longer than the infectious period [48–52].

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or inactivated vaccines, very stringent downstream processingill be needed to remove non-structural proteins in order to enableIVA based on one of these proteins. Conventional live-attenuatedaccines do not have DIVA possibilities. We here demonstrateIVA potential for replicating DISA vaccine with an experimentallyeveloped NS3 cELISA. Still, this serological DIVA test needs exten-ive validation to determine diagnostic sensitivity and specificity.dditionally, serological DIVA can be combined with genetic DIVA,ince DISA vaccine is not detected by panBTV PCR testing on Seg-1053].

All known BT vaccines, including the here described DISAaccine candidate, induce serotype-specific protection, a major dis-dvantage to combat multi-serotype situations, as present in manyarts of the world. In South Africa, three penta-serotype, conven-ional live-attenuated BT vaccines (15 different BTV serotypes inotal) are used in a defined and strict order, to induce broad pro-ection (described in [54]). However, the safety of these cocktailss controversial due to under-attenuation, reversion to virulence,r reassortment events [19,20,55]. BT DISA vaccines for differenterotypes will contain the common vaccine backbone of nine iden-ical genome segments and differ only for Seg-2. Consequently, BTISA vaccines cannot reassort with one another and can thus safelye combined in multi-serotype cocktail vaccines. Further, DISA vac-ines will all contain mutated Seg-10 that harbors attenuation, DISAnd DIVA. BT DISA vaccines for other serotypes are currently underevelopment.

uthor contributions

Conceived and designed the experiments: FF, RGPvG, PAvR. Per-ormed the experiments: FF, MMV, FJD, MGJT. Analysed the data:F, PAvR. Wrote the paper: FF, PAvR. Reviewed drafts of the paper:JMM

cknowledgments

We would like to thank the CVI animal caretakers for excel-ent assistance and Phaedra Eblé for veterinary supervision of thenimal experiment. We thank Carmen Vela and Paloma RuedaIngenasa, Spain) for the generous gift of aliquots of NS3 directed

Abs.This study was funded by the Dutch Ministry of Economic Affairs

CVI-project 1600020-01) and by the Central Veterinary Institutef Wageningen UR (CVI-project 1600217-01).

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.vaccine.014.10.033.

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