Vaccine 33 (2015) 55395545

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Development of a competitive ELISA for NS3 antibodies as DIVA test

accompanying the novel Disabled Infectious Single Animal (DISA)
vaccine for Bluetongue
Mirriam G.J. Tacken a , Franz J. Daus a , Femke Feenstra a,b , Ren G.P. van Gennip a ,
Piet A. van Rijn a,c,
a

Department of Virology, Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
c
Department of Biochemistry, Centre for Human Metabonomics, North-West University, Potchefstroom, South Africa
b

a r t i c l e

i n f o

Article history:
Received 12 July 2015
Received in revised form 4 September 2015
Accepted 5 September 2015
Available online 19 September 2015
Keywords:
Bluetongue
DIVA
Disabled Infectious Single Animal vaccine
ELISA
Non-structural protein 3

a b s t r a c t
Recently, we have developed a novel vaccine for Bluetongue named BT Disabled Infectious Single Animal
(DISA) vaccine. Due to the lack of non-essential NS3/NS3a protein, BT DISA vaccine is a replicating vaccine,
but without the inherent risks of live-attenuated vaccines, such as residual virulence or reversion to virulence by mutations, reassortment with eld virus, horizontal spread by vectors and vertical transmission.
The immune response induced by BT DISA vaccines is rapidly induced, highly protective and serotype
specic which is dependent on the immunodominant and serotype determining VP2 protein. The BT
DISA vaccine platform provides the replacement of exclusively VP2 from different serotypes in order to
safely formulate multivalent cocktail vaccines. The lack of NS3/NS3a directed antibodies by BT DISA vaccination enables differentiation of infected from vaccinated animals (DIVA principle). A highly conserved
immunogenic site corresponding to the late domain was mapped in the N-terminal region of NS3. We
here established an NS3-specic competitive ELISA (NS3 cELISA) as serological DIVA test accompanying
BT DISA vaccines. To this end, NS3 protein missing putative transmembrane regions was produced in
large amounts in bacteria and used as antigen in the NS3 cELISA which was investigated with a variety
of sera. The NS3 cELISA displayed a high sensitivity and specicity similar to the commercially available
VP7-specic cELISA. Results of previously performed vaccination-challenge trials with BT DISA vaccines
clearly demonstrate the DIVA system based on the NS3 cELISA and BT vaccine free of NS3 protein.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Bluetongue (BT) has been listed as a notiable disease to the
World Organisation for Animal Health (OIE) [1]. BT is a noncontagious disease of ruminants caused by bluetongue viruses
(BTV) transmitted by Culicoides biting midges [2,3]. BTV causes economic losses by morbidity and mortality but mainly by restrictions
on movement and trade of animals and animal products [4]. Today,
27 BTV serotypes have been identied [59]. BT is endemic in temperate and (sub)tropical climate zones [10], however, has expanded
to areas with a milder climate related to competent vectors [1113].

Corresponding author at: Department of Virology, Central Veterinary Institute

of Wageningen UR, PO Box 65, 8200 AB Lelystad, The Netherlands,
Tel.: +31 320238 686; fax: +31 320238 668.
E-mail address: piet.vanrijn@wur.nl (P.A. van Rijn).
http://dx.doi.org/10.1016/j.vaccine.2015.09.020
0264-410X/ 2015 Elsevier Ltd. All rights reserved.

BTV, the prototype virus species of genus Orbivirus, family

Reoviridae [14,15], is a non-enveloped, multi-layered virus particle that contains a ten-segmented genome of double stranded
RNA (Seg-110) encoding structural proteins VP1VP7, and nonstructural proteins NS1NS4 [1618]. VP7 protein is the major
serogroup specic protein used in ELISAs to detect BTV antibodies (Abs). VP2 is the major target for serotype specic neutralising
Abs (nAbs) [19,20]. BTV infection also raises non-neutralizing Abs
against NS3/NS3a [21].
Vaccination is the most effective measure to control BT. Currently available live-attenuated and inactivated BT vaccines have
inherent advantages and disadvantages [2227]. The replicating
BT Disabled Infectious Single Animal (DISA) vaccine exhibits all
advantages of live-attenuated vaccine without the inherent risks of
live-attenuated vaccines by the deletion of non-essential NS3/NS3a
as previously discussed [2832]. Consequently, BT DISA vaccines
potentially enable specic detection of BTV infected animals based

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M.G.J. Tacken et al. / Vaccine 33 (2015) 55395545

on NS3 Abs according to the principle of Differentiation of Infected

from Vaccinated Animals (DIVA) [33,34].
We here describe the development of a competitive ELISA for
BTV NS3 Abs (NS3 cELISA). The diagnostic sensitivity and specicity
were compared with a commercial BTV cELISA detecting VP7 Abs,
and the DIVA potential was studied in combination with BT DISA
vaccine.
2. Material & methods
2.1. Epitope mapping on NS3
BSR cells (a clone of BHK-21 cells; [35]) were cultured in Dulbeccos modied Eagles medium (DMEM; Invitrogen) containing
5% foetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin and 2.5 g/ml Amphotericin B.
Plasmids with full length mutated cDNA of Seg-10 for runoff RNA transcription were synthesized by Genscript Corporation
(Piscataway NJ, USA), or were constructed according to standard
procedures. Seg-10 of strain KUW (BTV26) was based on accession
number JN255162. BTV1 based viruses with mutated Seg-10 were
rescued using reverse genetics [36]. The following BTV mutants
were previously described; MutAUG1 + 2 [28], StyI-rev1, StyI-rev2
and BsiWI-rev1 [37], BTV1(S10)25 [38], and D(S2reposition) [39].
Monoclonal antibody (MAb) against BTV-VP7 was produced by
hybridoma ATCC-CRL-1875 (American Tissue Culture Collection).
MAb 32H2, 32B6, 33H7, 31E9 and 32F1 against BTV-NS3 (generous
gifts from Ingenasa, Spain), and rabbit -mouse serum conjugated
to horseradish peroxidase (Dako P0260) were used to map epitopes
on NS3 according to standard procedures [40,41].
2.2. NS3 production and purication
Open reading frame (ORF) encoding NS3 from BTV8/
NET2006/04 (Genbank accession number AM498060) was codonoptimised for bacterial expression and synthesized (Genscript
Corporation (Piscataway NJ, USA), and cloned in pET-51b(+)Ek/LIC
vector and transformed to Nova Blue GigaSinglesTM competent
cells (Novagen) and checked by sequencing. Similarly, expression
plasmid with an in-frame deletion from nucleotide position 369 to

568 of Seg-10 corresponding to amino acid (aa) codons 117 to 183

(NS3TM) was constructed (Fig. 1).
Escherichia coli strain BL21 (DE1) with pET-51b(+)Ek/LIC-NS3
or with pET-51b(+)Ek/LIC-NS3TM were grown in 400 ml of LB
medium (supplemented with 100 g/ml Ampicillin) at 37 C to an
optical density at 600 nm of approximately 0.6. Protein expression was induced by adding Isopropylthio--d-galactoside (IPTG)
to 1 mM nal concentration. After 4 h at 37 C, bacteria were harvested by centrifugation at 4000 g for 15 min and stored at 20 C.
His-tagged NS3 proteins were puried with NiNTA resinpacked columns according to the instructions of the supplier
(Qiagen). The eluted fractions with puried NS3 proteins were
pooled and repeatedly washed with PBS with 150 mM NaCl using
3 K Amicon ultra plus lter devices (Merck Millipore Ltd.). The
protein concentration was estimated according to the Bradford
procedure using bovine serum albumin as standard, and analysed
by SDSPAGE according to standard procedures. His-tagged NS3
proteins were identied by westernblot analysis using commercial
-His MAb conjugated to horseradish peroxidase (1:1000)(Roche)
or NS3 directed MAbs.

2.3. NS3 competitive ELISA

First, dilutions of puried NS3TM antigen, MAb 33H7, and rabbit -mouse serum conjugated to horseradish peroxidase (Dako
P0260) were optimized in a chessboard format with positive and
negative sera (not shown). Approximately 150 ng/well puried
NS3TM in coating buffer (100 mM bicarbonate/carbonate, pH 9.6)
was bound into maxi sorp plates (Nunc) overnight at 4 C. Coated
wells were incubated for 1 h at 37 C with PBS containing 0.1%
Tween 20 and 5% FBS to reduce non-specic binding. Sera were
diluted (1:2) in PBS 0.05%, Tween 20, 0.5% FBS, and 100 l was
added to wells and incubated for 1 h at 37 C. Unbound serum Abs
were removed by washing with 0.05% Tween 80. Then, wells were
incubated for 1 h at 37 C with 100 l MAb 33H7 (1:1000). After
washing, wells were incubated with 100 l conjugated rabbit mouse serum (1:4000) for 1 h at 37 C. After washing wells were
incubated for 10 min at room temperature with TMB substrate
(ID.VET), and colouring was stopped by stop solution (ID.VET). Optical density at 450 nm was determined for wells without adding

Fig. 1. Schematic representation of Seg-10 and the encoded NS3/NS3a proteins. Calpactin p11 binding domain (P), late domain (LD), two transmembrane regions (TM1 and
TM2) with a conserved glycosylation sites in-between (NLG149 ), the VP2 binding domain (BD) are indicated. The topological orientation is indicated by intracellular (IC) and
extracellular (EC). Putatively expressed NS3 related proteins are indicated by boxes, whereas untranslated sequences are shown by lines. Numbers indicate the amino acid
position, and numbers between brackets indicate the nucleotide position. Seg-10 NS3D(S2reposition) was used to map epitopes on NS3 (Supplemented data A), and Seg-10
mutant C was incorporated in BT DISA vaccines. NS3TM protein was used as antigen, and contains an in-frame deletion encompassing TM1, EC, and TM2. Streptavidin
tag (Strep) and His10 tag (His) were linked in-frame to the N- and C-terminus, respectively, and are indicated by striped boxes.

M.G.J. Tacken et al. / Vaccine 33 (2015) 55395545

serum (ODmax ), and serum sample (ODsample ). The percentage of

blocking was calculated as (ODmax ODsample )/ODmax 100%. The
threshold for seroconversion was calculated as the mean blocking
percentage of negative sera plus three times the standard deviation.
2.4. Sera
Sera were collected from animal experiments as previously
described [3032,4244]. Reference sera for BTV serotype 124,
and sheep serum for BTV26 (KUW) were provided by the Pirbright Institute, UK. Goat serum for BTV25 (TOV) was from IVI,
Switzerland. Goat serum for the proposed BTV27 (BTV-n), and
bovine EHDV6 sera were from ANSES, France. Bovine sera for
other EHDV serotypes were from unknown source. Field sera from
suspected sheep were collected in 2007/2008 during the BTV8 outbreak prior vaccination was taken place. Randomly collected sheep
sera from BT-endemic South Africa. The VP7 cELISA (ID.VET) was
performed according to the instructions of the supplier (ID.VET)
using a cut-off of 50%.
3. Results
3.1. Epitope mapping on NS3
To map epitopes on NS3/NS3a, BTV1 viruses expressing mutant
NS3/NS3a proteins were studied by IPMA with NS3 MAbs (Fig. 1).
MAbs 32B6 and 33H7 recognize the PPRY motif, whereas MAbs
32F1 and 31E9 recognize motif PSAP (Supplemented data A).
Apparently, the closely located PPRY and PSAP motifs in the late
domain (LD) PPRYAPSAP aa position 3644 represent independent epitopes of two groups of two MAbs. Transient expression of
mutant D(S2reposition) expressing NS3/NS3a up to amino acid
(aa) position 88, including the LD, was immunostained with all ve
MAbs (Supplemented data A). This indicates that all ve epitopes,
including MAb 32H2, are located on the N-terminal 88 aa sequence
of NS3/NS3a.
BTV1(S10)25 and BTV1(S10)26 were not immunostained by
NS3 MAbs (Supplemented data). The N-terminal 88 aa sequence
contains many differences (Fig. 2). Differences between NS3 of
BTV8 and TOV/KUW are clustered in the aa regions 1621, 2934
and 4548, including a mutation in the PPRY motif. Transiently
expressed NS3/NS3a proteins of BTV25 and 26 were immunostained with BTV serum, indicating that NS3/NS3a proteins of these
BTV strains share epitopes (not shown).
3.2. Production of NS3 antigen
We hypothesized that NS3 protein is toxic for bacteria caused by
the hydrophobic transmembrane regions TM1 and TM2. Therefore,
a codon-optimised His10 -tagged NS3 lacking an internal region

5541

encompassing the putative region of TM1 and TM2 but containing

the immunogenic LD was constructed (NS3TM) (Fig. 1). NS3TM
expression in E. coli BL-21 was strongly induced by IPTG (Fig. 3A).
Fractions with puried protein of about 23 kD corresponding with
the calculated MW of non-glycosylated His-tagged NS3TM were
pooled, and concentrated to a nal concentration of 0.2 mg/ml. The
protein was identied by Western blotting with -His-tag MAb
(Fig. 3B and C), or NS3 MAb (not shown). In summary, approximately 2 gram NS3TM per 400 ml E. coli culture was produced
resulting in about 1 gram puried NS3TM.
3.3. Development of the NS3 competitive ELISA
Initial studies showed that MAb 33H7 was the most promising
MAb to develop a competitive ELISA for NS3 Abs in combination
with conjugated rabbit -mouse serum to detect bound MAb (NS3
cELISA). The amount of coated NS3TM per M96 well, dilutions of
MAb 33H7 and conjugated rabbit -mouse serum were optimized
in a chessboard format with 1:2 diluted BTV serum (not shown).
The threshold of the NS3 cELISA was determined with pre-sera (day
0) of previously animal experiments [3032]. The mean blocking
percentage of these pre-sera (n = 44) was about 10%. The threshold
of 20% was calculated as the mean blocking percentage plus three
times the standard deviation.
3.4. Differentiation infected from vaccinated animals (DIVA)
Longitudinal sera of previously performed vaccination/challenge trials were tested and compared with VP7 cELISA
results [30,31] (Fig. 4). As expected, sera of vaccinated sheep
(n = 20) remained negative by the NS3 cELISA up to the day of
challenge (Fig. 4). Revaccination did not induce NS3 directed
Abs, however, did increase the VP7 Ab response (Fig. 4B) [30]. In
conclusion the NS3 cELISA did not detect sheep vaccinated with
DISA vaccine.
Protected sheep at 21 dpv negative by PCR [31] seroconverted for NS3 Abs after BTV challenge (Fig. 4A). The increase of
NS3 Abs was reciprocally correlated to the level of VP7 Abs at the
time of challenge (21 dpv/0 dpi), and correlated to the level of protection. Revaccinated sheep that were completely protected against
homologous BTV8 did not result in NS3 seroconversion, although a
very small increase of NS3 Abs was measured (Fig. 4B). No protection against heterologous BTV2 was observed after revaccination,
and indeed resulted in NS3 seroconversion.
Apparently, the NS3 ELISA can detect heterologous BTV
infections in animals repeatedly vaccinated with DISA vaccine. Furthermore, homologous BTV challenge of DISA vaccinated sheep was
detected by the NS3 cELISA but not by PCR testing. Thus, the NS3
cELISA is very sensitive and detected BTV replication that has been
unnoticed by PCR testing.

BTV8
1-24
TOV
KUW
BTV-n

:
:
:
:
:

*
20
*
40
*
60
MLSGLIQRFEEEKMKHNQDRVEELSLVRVDDTISQPPRYAPSAPMPSSMPTVALEILDKA :
......q.....k.....d.v.......................m............... :
...............RTES.T........GERL.L..G......A.PA..S......... :
................SDQK........AG.R..L..G......A.PA............ :
.VIL.............EQKT.......AG.R..L..G......A.PA............ :

60
60
60
60
60

BTV8
1-24
TOV
KUW
BTV-n

:
:
:
:
:

*
80
*
100
*
120
MSNTTGATQTQKAEKAAFASYAEAFRDDVRLRQIKRHVNEQILPKLKSDLSGLKKKRAII :
..................................................s......... :
............T.....................................G.M....T.. :
....................................................M....... :
.......................................D............M....... :

120
120
120
120
120

Fig. 2. Alignment of the N-terminal 120 amino acids of NS3. Completely conserved aa residues of BTV124 are indicated by dots, and aa positions showing variation (>5 out of
83 full length NS3 aa sequences) are indicated by small symbols. Differences compared to BTV8-NS3 are indicated by capitols for Toggenburg Orbivirus (TOV; BTV25), Kuwait
strain (KUW; BTV26), and BTV-n (BTV27). The late domain (PPRYAPSAP) is underlined.

5542

M.G.J. Tacken et al. / Vaccine 33 (2015) 55395545

Fig. 3. Production and purication of NS3TM. Proteins were separated by SDSPAGE and stained by CBB (A); prior to IPTG induction (1); 4 h post IPTG induction (2); 4 h
without IPTG induction (3). Proteins in pooled eluate of puried NS3TM was stained with CBB (B), or identied with anti-His MAbs by Western blotting (C). MW (kDa) of
reference proteins are indicated. Arrows indicate the position of His-tagged NS3TM.

4. Sensitivity
Challenge controls were used to study NS3 seroconversion after
BTV infection. Note that these nave sheep were infected at day
21 and day 84 of the vaccination/challenge experiment presented
in Fig. 4A and B, respectively. Four out of 16 sheep seroconverted
(>20%) at 9 dpi, and all were positive for NS3 Abs at 11 dpi. These
sheep seroconverted for VP7 Abs at 7 dpi (day 28 and day 91, respectively). At 14 and 21 dpi, all nave sheep infected with BTV2 or 8
(n = 12) were positive for NS3 Abs (3190%), and VP7 Abs (9298%).
Reference sera for BTV serotypes 124 were tested positive for
NS3 Abs (4589%), except for reference serum for serotype 4 (Fig. 5).
However, a well-dened sheep serum at 42 dpi with reference BTV4
was positive (BTV4(2), Fig. 5). Sera for BTV2527 were negative (2
to 12%). BTV25 and BTV27(2) were also negative by the VP7 cELISA,
whereas BTV26 and BTV27(1) were positive. An experimental indirect ELISA for BTV NS3 Abs (NS3 iELISA) was also positive for BTV26
and BTV27(1) (Supplemented data B).
4.1. Specicity
Pre-sera of sheep were negative for NS3 Abs, as described above.
EHDV VP7 positive sera of unknown origin for serotype 1 and 2
showed blocking of 53% and 28% by the NS3 cELISA (Fig. 5). However, sera for EHDV1, 5 and 8 were also positive for BTV VP7 Abs
indicating that these animals were also BTV infected. Sera of ve
cattle experimentally infected with EHDV6 were positive by the
EHDV VP7 cELISA [44] and were negative for BTV VP7 Abs (threshold 50%) (Fig. 5). Two out of ve sera were positive by the BTV NS3
cELISA indicating cross reaction of EHDV6 induced Abs. Four out of

ve of these EHDV6 sera were also positive by the NS3 iELISA for
BTV NS3 Abs (Supplemented data B) conrming cross reaction of
EHDV6 induced Abs.

4.2. Evaluation of the NS3 cELISA

Previously, cattle naturally or experimentally infected by
BTV8/net06 were monitored by PCR and VP7 cELISA [43]. These
cattle remained VP7 ELISA positive, whereas PCR positivity was
detected up to approximately 170 dpi. Here, these cattle were NS3
cELISA positive (maximum of 7090%) for at least 216 dpi which
was the end of the experiment. Naturally infected cattle has arrived
at the CVI at >50 dpi, and became negative for NS3 Abs after another
150 days. Thus, NS3 Abs were temporarily detected for at least
200 dpi, which is much longer than the infectious period and longer
than the PCR positive period, but shorter than for VP7 Abs.
Sera of BTV8 suspected sheep in the Netherlands in 2007/2008
were tested positive by the VP7 cELISA. Note that vaccination was
not taking place in this period. Of 139 sera, 127 sera were positive
by the NS3 cELISA (2087% in the NS3 cELISA), whereas 12 sera
were negative (720%) (Table 1A). Based on the difference in onset
of seroconversion, we suggest that VP7 positive/NS3 negative test
results could be expected early after infection, see Fig. 4.
A set of 96 sheep of unknown status regarding health and vaccination were randomly sampled in South Africa. Eight sera were
positive in both the VP7 and NS3 cELISA. Sera of 39 sheep were VP7
seropositive but negative for NS3 Abs (Table 1B), which are likely
from sheep infected or vaccinated a longer time ago, and could be
therefore negative for NS3 Abs. One serum was negative by the VP7

M.G.J. Tacken et al. / Vaccine 33 (2015) 55395545

5543

100
90
80
70
60
50
40
30
20
10
0
-10

VP7 cELISA (%)

100
90
80
70
60
50
40
30
20
10
0
-10
14
21
28
days post vaccinaon

35

42

100

100

90

90

80

80

70

70

60

60

50

50

40

40

30

30

20

20

10

10

VP7 cELISA (%)

-10

NS3 cELISA (%)

NS3 cELISA (%)

-10
0

14

21

28

35

42 49 56 63 70
days post vaccinaon

77

84

91

98 105

Fig. 4. Seroconversion for VP7 and NS3 Abs determined by cELISAs. Groups of four sheep were vaccinated with DISA vaccine and challenged with virulent BTV8 indicated with
open and lled arrows, respectively. The mean blocking percentage per group is shown for the VP7 cELISA and the NS3 cELISA by solid and dashed lines, respectively. The
threshold of the VP7 and NS3 cELISA is 50% and 20% blocking, respectively. (A) Groups were vaccinated at day 0 (0 dpv) with BT DISA vaccine 8 containing VP2 of serotype
8 and based on BTV1 (blue), BTV6 (green), and BTV8 (red), with an equal amount of inactivated not-adjuvanted BTV8 (purple) or with lysate as negative control (black). At
21 days post vaccination, all groups were infected with virulent BTV8. (B) Two groups were vaccinated twice at day 0 (0 dpv) and 21 dpv with BT DISA vaccine 8. At 84 dpv,
one group was infected with homologous virulent BTV8 (red) and one with heterologous virulent BTV2 (blue). Two groups served as challenge controls for virulent BTV8
(purple), and virulent BTV2 (dark blue).

Fig. 5. Diagnostic sensitivity and specicity of the NS3 cELISA. Reference sera for the serum neutralization tests for serotype 124, for recently discovered BTV serotypes 2527,
and for available EHDV sera were tested in the NS3 cELISA (lled bars) and VP7 cELISA (open bars). The percentage of blocking [(ODmax ODsample )/ODmax 100%] was
calculated. Test results were interpreted positive (+) or negative () using a cut-off of 50% for the VP7 cELISA (solid line), and 20% for the NS3 cELISA (dashed line).

5544

M.G.J. Tacken et al. / Vaccine 33 (2015) 55395545

Table 1
NS3 cELISA test results of eld sera. (A) VP7-positive sera from Dutch sheep that were
suspected to be infected by BTV in 2007/2008 were tested in the NS3 cELISA. Since
these sera were selected for VP7 Abs, no VP7 negative sera were tested (nd). (B)
South African sheep were randomly sampled and tested in the VP7 and the NS3
cELISA.
A
Suspected

VP7

(n = 139)
NS3

127
12

nd
nd

B
Random

VP7

(n = 96)
NS3

8
39

1
48

cELISA and positive for NS3 Abs, whereas 48 out of 96 sera were
negative by both cELISAs.
5. Discussion
The late domain (LD) of NS3/NS3a is highly conserved and
very immunogenic, and therefore important for the DIVA test
accompanying DIVA vaccine in which this part is deleted (DIVA:
Differentiation Infected from Vaccinated Animals). The novel Disabled Infectious Single Animal (DISA) vaccine lacks NS3/NS3a
expression by deletion of LD, and restore of LD expression is therefore excluded [3032,39]. NS3 related expression from mutated
Seg-10 has been reported [37,40], which makes an NS3 iELISA
detecting all NS3 Abs less suitable as DIVA test. A previously
published DIVA system based on a NS3 iELISA and inactivated BT
vaccine is dependent on the purity of BT vaccine with respect to
NS3 protein [21]. The here developed NS3 cELISA is also suitable
as DIVA test in combination with any BT vaccine free of NS3/NS3a
protein or in which LD is not expressed. In addition, a cELISA for
NS3 Abs is preferred as DIVA test, since such cELISA can be used
regardless the variety of ruminant species.
Furthermore, vaccination with live-attenuated or inactivated
vaccines could also result in PCR positivity [36,45,46], but panBTV
PCR tests based on Seg-10 [7,43,4749] will not detect BT DISA
vaccines according to a genetic DIVA approach [30,38].
The NS3 cELISA is not detecting NS3 Abs against BTV serotypes
2527 (Fig. 5), but is suitable to monitor DISA vaccinated populations for pathogenic BTV serotypes 124. Further, EHDV6 infection
can also lead to cross reacting Abs resulting in false positive test
results with the NS3 cELISA. Thus, the NS3 cELISA is not 100% sensitive and not 100% specic/selective for Abs directed against BTV
NS3. BT and EHD are both notiable diseases [49], and positive
results with the NS3 cELISA could be easily differentiated for BTV
and EHDV infections with specic VP7 based cELISAs.
Obviously, VP7 is more abundantly and earlier expressed than
NS3 in infected animals, and consequently VP7 Abs are earlier
detected than NS3 Abs (Fig. 4). However, panBTV PCR tests are
preferred to conrm acute BTV infections. Furthermore, BTV infection in DISA vaccinated animals that were unnoticed with a Seg-1
based panBTV PCR test were detected with the NS3 cELISA (Fig. 4)
[30,31]. These results indicate a high diagnostic sensitivity of the
NS3 cELISA, and show that the NS3 cELISA is an excellent tool to
monitor DISA vaccinated populations for BTV circulation.
NS3 Abs are detected in cattle for >200 dpi, which is much longer
than the infectious period [1] and longer that the period of PCR positivity [43]. On the other hand, this period is shorter than for VP7
directed Abs, which are likely lifelong present after BTV infection.

Taken together, positive results with the NS3 cELISA strongly suggest BTV infection, but EHDV infection cannot be excluded. Testing
for VP7 Abs will be needed to distinguish between EHDV and BTV
infections. Negative results suggest non-infectiousness of the animal, although an acute BTV infection, or a previous BTV infection
cannot be excluded. Testing of paired serum samples of at least
one week apart will differentiate between an acute or old infection.
Alternatively, PCR testing will detect acute BTV infections.
The NS3 cELISA can be further improved by use of conjugated
MAb and a modied NS3 antigen to detect BTV2527 infections.
The ultimate ELISA format should be validated with well-dened
sera raised against more serotypes of both BTV and EHDV, and for
different ruminant species, in particular cattle, sheep and goat.
Summarizing, the NS3 cELISA and Seg-10 based panBTV PCR
tests in combination with BT DISA vaccine is a suitable DIVA system
to adequately control BT outbreaks, and to safely allow transport
and trade of vaccinated livestock.
Acknowledgements
The authors thank dr. Carmen Vela and dr. Paloma Rueda (Ingenasa, Spain) for MAbs directed against NS3 of BTV. Reference sera
for BTV serotype 124 and BTV26 (KUW) serum were kindly supplied by Carrie Batten (the EU reference Institute for Bluetongue,
The Pirbright Institute United Kingdom). Serum for BTV25 (TOV)
was a generous gift from Barbara Thr, IVI Switzerland, and sera
for BTV27 (BTV-n) and EHDV6 were kindly provided by Emmanuel
Breard, ANSES, France. Field sera from South African sheep were
shipped by Christiaan Potgieter, Deltamune, South Africa. This
research was funded by the Dutch Ministry of Economic Affairs
(CVI-project 1600013-01 and 1600020-01).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.vaccine.2015.09.
020.
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