Development of a competitive ELISA for NS3 antibodies as DIVA test accompanying the novel Disabled Infectious Single Animal (DISA) vaccine for Bluetongue
Introduction
Bluetongue (BT) has been listed as a notifiable disease to the World Organisation for Animal Health (OIE) [1]. BT is a non-contagious 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 identified [5], [6], [7], [8], [9]. BT is endemic in temperate and (sub)tropical climate zones [10], however, has expanded to areas with a milder climate related to competent vectors [11], [12], [13].
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-1–10) encoding structural proteins VP1–VP7, and non-structural proteins NS1–NS4 [16], [17], [18]. VP7 protein is the major serogroup specific protein used in ELISAs to detect BTV antibodies (Abs). VP2 is the major target for serotype specific 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 [22], [23], [24], [25], [26], [27]. 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 [28], [29], [30], [31], [32]. Consequently, BT DISA vaccines potentially enable specific detection of BTV infected animals based 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 specificity were compared with a commercial BTV cELISA detecting VP7 Abs, and the DIVA potential was studied in combination with BT DISA vaccine.
Section snippets
Epitope mapping on NS3
BSR cells (a clone of BHK-21 cells; [35]) were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 5% foetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 2.5 μg/ml Amphotericin B.
Plasmids with full length mutated cDNA of Seg-10 for run-off 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
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 36–44 – represent independent epitopes of two groups of two MAbs. Transient expression of mutant ΔD(S2reposition) expressing NS3/NS3a up to amino acid (aa)
Sensitivity
Challenge controls were used to study NS3 seroconversion after BTV infection. Note that these naïve 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 naïve sheep infected with BTV2 or 8 (n = 12) were positive for NS3 Abs (31–90%),
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 [30], [31], [32], [39]. NS3 related expression from mutated Seg-10 has been reported [37], [40], which makes an
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 1–24 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 Thür, IVI Switzerland, and sera for BTV27 (BTV-n) and EHDV6 were kindly provided by Emmanuel Breard, ANSES, France. Field sera from South African sheep
References (49)
- et al.
The epidemiology of bluetongue
Comp Immunol Microbiol Infect Dis
(1994) - et al.
A DIVA system based on the detection of antibodies to non-structural protein 3 (NS3) of bluetongue virus
Vet Microbiol
(2009) - et al.
A European field strain of bluetongue virus derived from two parental vaccine strains by genome segment reassortment
Virus Res
(2008) - et al.
Risk factors associated with the occurrence of undesired effects in sheep and goats after field vaccination with modified-live vaccine against bluetongue virus serotypes 2, 4 and 16
Vet Microbiol
(2010) - et al.
Live attenuated bluetongue vaccine viruses in Dorset Poll sheep, before and after passage in vector midges (Diptera: Ceratopogonidae)
Vaccine
(2005) - et al.
Viraemia and clinical disease in Dorset Poll sheep following vaccination with live attenuated bluetongue virus vaccines serotypes 16 and 4
Vaccine
(2010) - et al.
Vaccines against bluetongue in Europe
Comp Immunol Microbiol Infect Dis
(2008) - et al.
Bluetongue virus serotype 6 in Europe in 2008-Emergence and disappearance of an unexpected non-virulent BTV
Vet Microbiol
(2012) - et al.
VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA
Vaccine
(2014) - et al.
Application of bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2
Vaccine
(2015)
Diva vaccines that reduce virus transmission
J Biotechnol
Bluetongue virus with mutated genome segment 10 to differentiate infected from vaccinated animals: a genetic DIVA approach
Vaccine
Epizootic hemorrhagic disease virus serotype 6 experimentation on adult cattle
Res Vet Sci
Bluetongue virus RNA detection by RT-qPCR in blood samples of sheep vaccinated with a commercially available inactivated BTV-8 vaccine
Vaccine
Rapid detection and quantitation of bluetongue virus (BTV) using a molecular beacon fluorescent probe assay
J Virol Methods
Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus
J Virol Methods
Terrestrial animal health code
The epidemiology of bluetongue
Rev Sci Tech Off Int Epizoot
Bluetongue
Climate change and the recent emergence of bluetongue in Europe
Nat Rev Microbiol
Identification of the serotype-specific and group-specific antigens of bluetongue virus
Onderstepoort J Vet Res
Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2
PLoS ONE
Genetic characterization of Toggenburg Orbivirus, a new bluetongue virus, from goats, Switzerland
Emerg Infect Dis
Novel bluetongue virus serotype from Kuwait
Emerg Infect Dis
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2021, Journal of Immunological MethodsCitation Excerpt :The results are presented in the Table 4. Non-structural proteins have always served as a suitable candidate for functional studies and diagnostics, especially to understand viral dynamics and to differentiate vaccinated from infected animals (Barros et al., 2009; Tacken et al., 2015). The antibodies against NS proteins are generated later than structural proteins in infective cycle, however they tend to fade off earlier (Barros et al., 2009; Tacken et al., 2015).
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2020, Veterinary MicrobiologyCitation Excerpt :DISA vaccines are fully replicative, but non-transmittable owing to a deletion in Seg-10. Because of the lack of NS3/NS3a, a cELISA for anti-NS3 antibody has been developed for DIVA (Tacken et al., 2015). In the present study, four recombinant tagged viruses were rescued with HA tags introduced into VP2 and their potential use for preparing inactivated labeled vaccines was explored.
Diagnostic DIVA tests accompanying the Disabled Infectious Single Animal (DISA) vaccine platform for African horse sickness
2018, VaccineCitation Excerpt :Most likely, essential and specific aa residues of these epitopes are also located adjacent to the 77aa region (101–120). Surprisingly, LD on AHSV-NS3/NS3a (PPYNFASAP) harbours one out of five nonconserved epitopes, whereas LD is the most immunogenic region of BTV-NS3/NS3a [35]. On the other hand, deletion of LD (9aa deletion) resulted in the same weaker staining as the 77aa deletion compared to entire NS3/NS3a, suggesting that many epitopes of Abs in the polyvalent α-NS3 rabbit serum are deleted in these in-frame deletion NS3/NS3a proteins.
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2017, Veterinary MicrobiologyCitation Excerpt :Similarly, engineered DISA vaccines have been created including a virus that lacks NS3 but induces protective immunity to BTV. Furthermore, animals vaccinated with this engineered virus may be distinguished from infected ones (DIVA) on the basis of their serological responses to NS3 (Feenstra et al., 2014, 2015; Tacken et al., 2015). Virus-like particle (VLP) vaccines have been shown to be promising vaccine candidates against BTV (Roy et al., 1994; Stewart et al., 2010).