Target determinationExpression and purification of capripoxvirus target protein

As an antigenic target for capripoxvirus, we used the homolog of the vaccinia virus C-type lectin-like protein A34 (LSDV ORF 123). The expression and purification of the antigen were performed as previously described [28]. This recombinant antigen was successfully used in ELISA to detect antibodies against capripoxvirus in sheep, goats and cattle [28]. After the initial proof of concept, the protein, based on the corresponding sequence of LSDV NI-2490 (NC_003027), was outsourced for production to GenScript, Inc. (Piscataway, NJ, USA).

Expression and purification of RVFV nucleoprotein

The plasmid pET 32a-RVFV NP for expression of the RVFV nucleocapsid (NP) protein was used to transform BL21a cells (Thermo Fisher Scientific, Waltham, MA, USA). The transformation was confirmed by DNA amplification using the T7 promoter and T7 terminator primers. The thermocycler conditions used were: (1X) 95 °C, 10 min; (30X) 95 °C, 30 s; 55 °C, 30 s; 72 °C, 1 min 30 s; (1X) 72 °C, 2 min; hold at 4 °C. Cells were grown in Luria–Bertani (LB) broth medium (Thermo Fisher Scientific, Waltham, MA, USA) containing carbenicillin (Merck KGaA, Darmstadt, Germany), and protein expression was induced with 0.3% L-arabinose and 1 mM IPTG (Merck KGaA, Darmstadt, Germany) for five hours. The initial expression test was performed at time points T0h, T1h, T2h, T3h, T4h, and T5h. Upon mechanical lysis of the cells, the recombinant NP was purified by an Invitrogen Probond kit (Thermo Fisher Scientific, Waltham, MA, USA) which is based on affinity purification using a nickel column to bind His-tagged proteins. Since we wanted a native conformation of the protein, we followed the commercial protocol described by the manufacturer under native purification conditions (10 mM imidazole for binding, 20 mM for washing, and 250 mM imidazole in the elution). For buffer exchange, an Amicon concentrator (Merck KGaA, Darmstadt, Germany) with a MWCO of 3 kDa was used to exchange the original buffer with storage buffer (50 mM Tris HCl, 150 mM NaCl, 25 mM Sucrose, and protease inhibitor pills, pH 8.0).

RVFV nucleoprotein quantification

Pierce BCA for protein quantification was used following the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA).

Western blot

The proteins were analysed by SDS-PAGE. Antibody binding in both goat and cattle positive sera, was confirmed by Western blot.

The preparation of proteins for the SDS-PAGE gels and Western blots followed standard procedures. The protein preparations (20 μL) were heated at 80 °C for 10 min in 4X LDS sample buffer (Thermo Fisher Scientific, Waltham, MA, USA). The samples were loaded onto an SDS-PAGE (NUPAGE 10% (v/v)) gel (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to a 0.2 μm PVDF membrane (Thermo Fisher Scientific, Waltham, MA, USA). The membrane was probed for 1 h at room temperature with anti-penta His antibody (1:1000) (Merck KGaA, Darmstadt, Germany), washed three times with PBS containing 0.5% (v/v) Tween 20 (PBS-T) and probed for 1 h with diluted (1:5000) goat anti-mouse antibodies conjugated to horseradish peroxidase (Merck KGaA, Darmstadt, Germany). For detection, ECL substrate (GE Healthcare, Chicago, IL, USA) was used according to the manufacturer’s instructions.

RVFV NP ELISA

100 ng per well of RVFV NP purified antigen in carbonate/bicarbonate buffer pH 9.6 vf = 50 μL was shaken overnight at 4 °C in a 96 well plate (Polysorb, Nalgene). The plate was washed 3X with 1XPBS-T and blocked with 50 μL of protein-free blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA), hereon known as BB1, for 30 min at 37 °C. The diluted sera were added and incubated for 90 min at 37 °C. The plate was washed 3X with 1XPBS-T, and an HRP-conjugated secondary antibody (Merck KgaA, Darmstadt, Germany), diluted 1:10 k, was added and incubated for 45 min at RT. The plate was washed 3X with 1XPBS-T, and 100 μL of TMB per well was added and incubated in the dark for 10 min. 100 μL of stop solution was added per well and the plate was read at 450 nm.

Virus neutralization test

The virus neutralization tests were carried out as previously described [28].

Capripox serum samplesExperimental and field sera

Four types of reference population sera were used in this study: (a) bovine LSD-positive sera (n = 77) obtained from naturally LSDV-infected animals from Bulgaria (NDRVMI, Sofia, Bulgaria) and the Republic of North Macedonia (School of Veterinary Medicine, University “Ss Cyril and Methodius”, Skopje, Republic of North Macedonia). These samples were confirmed LSD-positive by VNT; (b) bovine LSD-negative sera (n = 59) from countries where the disease is historically not present, namely France (provided by IDVet, Montpellier, France), Austria (provided by AGES, Austria), and the Republic of North Macedonia (sera collected before 2010) (School of Veterinary Medicine, University “Ss Cyril and Methodius”, Skopje, Republic of North Macedonia); (c) Capripox-positive sheep and goat sera (n = 27) from experimentally infected animals from Pirbright (UK), LCV (Mali) [29] and AHI (Ethiopia) [30], which were confirmed positive by VNT; and (d) Capripox-negative sheep and goat sera (n = 181) from Austria (provided by AGES, Austria), where the disease is historically not present. See Supplementary Table 1.

Specificity control sera

Parapoxvirus-positive sera were used to determine cross-reactivity. They consisted of 12 orf naturally infected goat samples (provided by Stéphane Bertagnoli, Ecole Vétérinaire de Toulouse, Toulouse, France), 4 pseudocowpox (PCP)-positive cattle sera from Zambia (provided by Maureen Ziba from CVRI, Zambia), and one bovine papular stomatitis (BPS) (provided by AGES, Austria). See Supplementary Table 1.

Sera from longitudinal studies

We used three sets of samples from longitudinal studies. The first set comprised samples collected at 0, 6, 12, 18, 20, 23, 26, and 30-days post-infection (DPI) from two cattle that were experimentally infected with a virulent South African LSDV Neethling strain [31]. The second set consisted of samples collected at 0, 7, 14, 21, 28, 35, 42, 49, and 56 DPI from four goats experimentally infected with GTPV Oman 84. This was part of a larger GTPV study with experimentally infected Ethiopian goats [30]. The third set consisted of samples from a single sheep produced by LCV (Mali) [29]. The sheep was experimentally infected with SPPV Algeria/93 Djelfa and samples were collected at 0, 7, 14, 21, and 28 DPI.

RVF serum samplesSera

Four types of reference populations of RVF sera were used in this study: (a) bovine RVF-positive sera (n = 6) obtained from Zambia (provided by Maureen Ziba from CVRI, Zambia), field-infected cattle from Côte d’Ivoire (provided by Dr. Emmanuel Couacy-Hymann, Laboratoire Central de Pathologie Animale, Bingerville), and commercially available bovine RVF-positive serum samples (IDVet, Montpellier, France). These samples were confirmed to be positive by cELISA (IDvet, Montpellier, France); (b) bovine negative sera (n = 56) from countries where the disease is historically not present, namely France (provided by IDVet, Montpellier, France), Austria (provided by AGES, Austria) and, the United Kingdom (the Pirbright Institute); (c) RVF-positive sheep and goat sera (n = 13) from a goat experimental infection in Teramo, Italy (provided by Chiara Pinoni from Diagnostica e Sorveglianza Malattie Esotiche, Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G.Caporale"), and sheep and goat field infections collected during routine screening at the Laboratoire Vétérinaire de Kinshasa, DRC. These samples were confirmed to be positive by cELISA (IDvet, Montpellier, France); and (d) negative sheep and goat sera (n = 180) from Austria (provided by AGES, Austria), where the disease is historically not present. All the samples were confirmed to be negative by cELISA (IDvet, Montpellier, France). See Supplementary Table 1.

Luminex multiplex serological assayCoupling of target antigens to beads

The viral target antigens were covalently coupled to polystyrene carboxyl magnetic beads, in regions 34 and 63 (Bio-Rad, Hercules, California, USA) following a modified protocol based on Anderson et al. [32]. Briefly, 1.2 ×106 beads placed into a 1.5 ml Eppendorf tube were washed, vortexed and sonicated once with 100 μL of sterile water and 3 times with 100 μL of activation buffer (0.1 M sodium phosphate (monobasic), pH 6.2). Separation was performed via a magnetic separator stand (Thermo Fisher Scientific, Waltham, MA, USA). After a final round of vortexing and sonication, the beads were resuspended in 80 μL of activation buffer. 10 μL of freshly made 50 mg/mL Sulfo-NHS (Thermo Fisher Scientific, Waltham, MA, USA) and 10 μL of 50 mg/mL EDC (Merck KGaA, Darmstadt, Germany) diluted in activation buffer were added to the microspheres. After a 20-min incubation in the dark on a plate shaker, the beads were magnetically separated and resuspended in 500 μL of coupling buffer (0.1 M PBS pH 7.4) followed by vortexing and sonication. After two rounds of washing with coupling buffer, vortexing and sonication the beads were resuspended in 100 μL of coupling buffer. 10 μg of purified RVFV NP, or A34-CaPV antigens in 300 μL of coupling buffer were added to the corresponding resuspended carboxyl magnetic beads (regions 63 for RVFV or 34 for A34-CaPV). Per bead type, the final volume was 400ul in coupling buffer. The beads were incubated for 2 h on a plate shaker at 200 rpm at room temperature in the dark, vortexing at 10-min intervals. They were then magnetically separated, washed twice with 1000 μL of wash buffer (0.1 M PBS, 0.05% Tween 20 pH 7.4), followed by vortexing and sonication. After the magnetic separation of the beads, the microspheres were resuspended once in 250 μL of BB1, then vortexed and sonicated as described above. Then the beads were separated via the magnetic separator, resuspended in 100 μL of BB1 and stored at 4 °C in the dark. The number of microspheres recovered after the coupling reaction was counted with a haemocytometer.

Luminex multiplex assay

The filtered microplate (Merck KGaA, Darmstadt, Germany) was pre-wetted by filling each well twice with 50 μL of wash buffer (10X PBS, 0.05%Tween 20) and once with 50 μl of BB1. The liquid was removed through the filter at the bottom of the plate via a vacuum manifold (Merck KGaA, Darmstadt, Germany). A 1000 coupled beads per antigen per sample or control in a final volume of 5 μL of BB1 each were added to 100 μL of serum diluted 1:500 in BB1 in the case of cattle samples and 1:2000 in BB1 in the case of sheep or goat samples. The plate covered with its lid and wrapped in aluminium foil was incubated overnight at 4 °C on a horizontal orbital microplate shaker set at 300 rpm. The liquid was then removed with the vacuum manifold. 150 μL of wash buffer were added to each well and the liquid was removed with the vacuum manifold. This was repeated 4 times. 50 µl of diluted species-specific (anti-ruminant) biotin-conjugated secondary antibody (Merck KGaA, Darmstadt, Germany), hereon known as SGB, diluted at 1:10 k in BB1 for bovine samples and 1:40 k in BB1 for sheep or goat samples, was added to each well. The plate was incubated for 30 min in the dark, at room temperature on a horizontal orbital microplate shaker set at 300 rpm. The liquid was then removed with the vacuum manifold and washed 5 times with 150 μL of wash buffer. 50 µl of diluted streptavidin–PE diluted 1:250 in BB1 was added to each well. The plate was incubated for 10 min in the dark, at room temperature on a horizontal orbital microplate shaker set at 300 rpm. The liquid was removed with a vacuum manifold and the wells were washed 5 times with 150μL of reading buffer (0.01 M PBS pH 7.4). 50 µl of reading buffer was added to the wells. The plate was shaken at 300 rpm for 1 min. Further, 75 μL of reading buffer was added to each well (final volume 125 μL). The plate was read in the BioPlex 200 Luminex machine (Bio-Rad, Hercules, CA, USA) (50 beads/region and the median value obtained for each reaction event per bead set using a volume of 50 μL/sample).

Unless otherwise stated, all samples were analysed in duplicate and average readings calculated. For all samples, the multiplex assay mean fluorescence intensity (MFI) data were corrected for background levels by subtracting the no-serum-added control signal from the serum-added signals.