Introduction

Bovine viral diarrhea virus (BVDV) is an enveloped single-stranded RNA virus belonging to the pestivirus genus of the family Flaviviridae (Newcomer 2021). BVDV infection can result in a wide spectrum of clinical disease ranging from subclinical infection to fatal disease. Economic losses associated with viral infection are due to decreased fertility and milk production, slow fetal growth, diarrhea, enteritis, respiratory symptoms, abortion, teratogenesis, immune dysfunction, concurrent infections, occurrence of persistently infected (PI) calves that spread the virus in the herd, leading to deterioration in herd performance and increased animal mortality (Brodersen 2014) (Khodakaram-Tafti and Farjanikish 2017). The effects of BVDV are estimated to generate a global loss of USD 687 per head of cattle (Richter et al. 2017).

The virus is classified according to its genotype into BVDV-1, BVDV-2 and BVDV-3 (HoBi-like). Each genotype is further divided into different sub-genotypes (Al-Kubati et al. 2021; Tian et al. 2021). Cytopathic (CP) and non-cytopathic (NCP) variants are found in nature (Chi et al. 2022), the latter being responsible for the emergence of persistently infected animals (PI), which represent the main reservoir of the virus(Garoussi et al. 2019).

The BVDV genome encodes for a single polyprotein, which is digested by proteases to generate structural and non-structural proteins. Non-structural proteins (NS) are Npro, S2, NS3, NS4A, NS4B, NS5A, and NS5B. Structural proteins are Erns, E1, E2, and capsid protein C, which are located on the outer surface of the virus (Chi et al. 2022).

Various programs have been implemented to eradicate and control the disease (Moennig and Becher 2018). These programs primarily focus on identifying PI animals (Moennig and Yarnall 2021) and are based on knowledge of the regional epidemiological situation, including seroprevalence, vaccination, and PI prevalence (Antos et al. 2021; Greiser-Wilke et al. 2003). Serological tests for mass testing for antibodies and antigens of the virus have been chosen for routine screening of herds (Edmondson et al. 2007). Detection of antibodies in cattle is a valuable way of determining an animal’s immune status and any previous exposure to BVDV (Lanyon et al. 2014). A positive antibody test in an unvaccinated individual indicates that the animal has been previously exposed to BVDV and is not a PI animal. A positive result in a pregnant female suggests it may carry a PI fetus.

Recent studies have shed light on the prevalence of BVDV in South American countries, as well as the impact of the virus in Chile (Alocilla and Monti 2022) (Pinior et al. 2017). According to the study, the herd prevalence of BVDV in South America is approximately 70% and the prevalence of PI animals is 2.3%, highlighting the importance of infection in one of the largest cattle producing areas in the world (Richter et al. 2019). Published studies showed that the predominant genotype worldwide is BVDV-1 and the most common sub-genotype is BVDV-1b (Yeşilbağ et al. 2017). In Chile, the predominant genotype is BVDV-1, with the sub-genotypes BVDV-1a, b, and j being the most recurrent (Donoso et al. 2018).

There are different techniques for the detection of this pathogen, such as polymerase chain reaction (PCR), Enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (Dubovi 2013). ELISAs have been a valuable tool for monitoring and mass detection of BVDV, being one of the most widely used techniques due to their ease of implementation, rapidity, sensitivity, and specificity (Edmondson et al. 2007). Most commercially available ELISA kits for the detection of antibodies against BVDV are based on the recognition of antibodies against the NS3 protein (Hanon et al. 2017), using the protein from different virus subtypes to ensure the identification of antibodies produced by BVDV infection. However, the NS3 protein is found only in the early stages of virus replication and may not be found in NCP variant viruses (Brodersen 2014), which is a disadvantage and can lead to false negative determinations.

This work proposes the use of the E2 protein, catalogued as the most immunogenic protein of BVDV (Nelson et al., 2012; Patterson et al. 2012), in an indirect ELISA for the detection of antibodies using the recombinant E2 protein (rE2) of BVDV-1b, d and e subtypes. This ELISA has the advantage of covering the most prevalent strain worldwide and the most prevalent in southern Chile, in addition to detecting antibodies against cytopathic and non-cytopathic BVDV, thus contributing to the detection and control of the disease.

Materials and methods

rE2 plasmid construction

The DNA sequence of E2 protein from BVDV sub-genotypes 1b (Acc Number: EF101530.1), 1d (Acc Number: KT951841.1), and 1e (Acc Number: LT968777.1) were modified by adding the signal peptide sequence of human albumin preprotein (NP_000468.1) to the E2 N-terminal end. Different epitope tags were added to the C-terminus of each E2 variant for protein detection (E2-1e/E-tag, E2-1b/HA-tag, and E2-1d/Strep-tag), followed by 6xHis tag for purification (Fig. 1A). A 5 amimoacid spacer separated both tags. Codon sequences were optimized for protein expression in Chinese Hamster Ovary cells (CHO, ATCC, CCL-61). The DNA sequences were synthesized by GeneScript (Hong Kong, China) and inserted into the expression vector pCI-Neo, generating two expression vectors. pCI-Neo-V1 encodes for E2-1e and the Green Fluorescent Protein (GFP); and pCI-Neo-V2 encodes for E2-1b and E2-1d. In both vectors, an Internal Ribosome Entry Site (IRES) allowed the coexpression of proteins from a single transcriptional unit driven by the early/immediate cytomegalovirus promoter (pCMV).

Electrophoresis and Western blot analysis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions was performed on 12% polyacrylamide gel according to the standard method (Laemmli 1970). The gels were stained with Coomassie Brilliant Blue (Merck, Catalog N° B7920). For Western blot assays, samples were transferred to a 0.2 μm nitrocellulose membrane (GE Healthcare Life Science, Catalog N°. 10600001) in a semi-dry transfer system (Bio-Rad, USA). After blocking with skimmed milk 5% in Tris-buffered saline, rE2 proteins were identified using the appropriate combination of primary and Alexa fluor-conjugated secondary antibodies. E2-1e was detected with goat anti E-Tag polyclonal antibody (Novus Biological, Catalog N° NB600-518) / Alexa Fluor 790 AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch, Catalog N° 705-655-147). E2-1b was detected using mouse anti HA-Tag monoclonal antibody (Thermofisher, Catalog N°26183) / Alexa Fluor 680 AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch, Catalog N° 715-625-150). E2-1d was detected with rabbit anti Strep II-Tag polyclonal antibody (Novus Biological, Catalog NBP2-41073) / Alexa Fluor 790 AffiniPure donkey Anti-Rabbit IgG (Jackson ImmunoResearch, catalog 711-655-152) All rE2 variants were detected using mouse anti His-Tag monoclonal antibody (Biolegend, Catalog N° 652502) / Alexa Fluor 790 AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch. Catalog N° 715-655-150). Results were imaged using the Odyssey infrared imaging system (LI-COR Biosciences, USA) at 680–790 nm as needed.

Stable rE2-producing cell line

CHO cells were transfected with the DNA fragment encoding E2-1b and E2-1d using 1 µg DNA/1.5µL Lipofectamine ratio (Invitrogen, Catalog N° 11668-027). Stably transfected cells were selected with 0.5 mg/mL of G-418 (Sigma-Aldrich, Catalog N° A1720). Supernatants of CHO clones were analyzed for E2 expression using anti-His tag antibodies in Western blot assay. E2-1b and E2-1d were detected with anti-HA-tag and anti-Strep-Tag antibodies, respectively. The DNA fragment encoding E2-1e and GFP was transfected in the CHO clone expressing E2-1b and E2-1d, and cells were cultured with 0.5 mg/mL of G-418 for 20 days. Stably transfected cells were sorted by flow cytometry in the Cell Sorter BD FACSAria III (BD Biosciences, USA) to select high-GFP expression clones and seeded in 96-well plates. Clone´s supernatants were analyzed by Western blot to detect E2-1e and E2-1b with anti-E-tag and anti-HA-Tag antibodies, respectively.

rE2 production and purification

Selected CHO E2 expressing clone was adapted to growth in the serum-free medium Ex-Cell ACF CHO (Merck, Catalog N° C9098), supplemented with 4 M L-Glutamine (Gibco, Catalog N° 35050-061), 1% (v/v) Penicillin–Streptomycin antibiotics (Gibco, Catalog N° 10378016) and 1.2 mg/mL of G-418 (Santa Cruz animal Health, Catalog N° sc-29065 A). Suspension cells were cultivated for three days in a CO2 incubator at 5% CO2, 37 °C, 95% humidity, and 160 rpm (Hangzhou Allsheng Instrument orbital shaker, model OS-200). Afterward, the cells were sub-cultured every 2–3 days (duration of one passage) in the log phase at 1.0–2.0 × 106 viable cells/mL.

rE2 proteins were purified using immobilized metal affinity chromatography (IMAC). 1000 mL of cell supernatant were centrifuged at 10,000 rpm for 20 min and supplemented with Tris 50 mM pH 8.0, NaCl 300 mM, and Imidazole 5 mM. The solution was applied to a column containing 25 mL of Ni-charged Chelating Sepharose Fast Flow matrix (GE Healthcare Bioscience, Catalog N° 17-0575-01) previously equilibrated with the equilibrium buffer (Tris 50 mM pH 8.0, NaCl 300 mM) plus Imidazole 5 mM. The column was washed with equilibrium buffer with Imidizole 40 mM. E2 protein elution was performed with an elution buffer containing 400 mM Imidazole. Protein detection and collection were performed in the ÄKTA™ Start chromatography station (GE Healthcare Biosciences, USA) using the UNICORN™ 1.1 start control software. The eluate was pooled, concentrated, and dialyzed against PBS (8 g/L NaCl, 0.2 g/L KCl, 1.09 g/L Na2HPO4, 0.2 g/L KH2PO4, pH 7.2) in a dialyzing membrane of 3 kDa (Thermo Fisher Scientific, Catalog N° 11455909). rE2 concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Catalog N° 23225) and the purity was determined by densitometry using the ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). The mixture of purified rE2 proteins was aliquoted and stored at -20 °C or -80 °C until use.

rE2 deglycosylation with PNGase-F

Purified rE2 proteins (10 µg) were mixed with 1 µl of Glycoprotein Denaturing Buffer 10X in a final volume of 10 µl. The sample was denatured by heating at 90 °C for 10 min. After cooling for 5 min at room temperature, the reaction mix was supplemented with 2 µl of NP-40 10% and digested with 1 µl of PNGase-F (New England Biolabs, Catalog N° P0704L) (500 000 units/ml) overnight at 37 °C. The deglycosylated and glycosylated (without PNGase-F) rE2 proteins were analyzed by SDS-PAGE and Western blot as previously described.

Cattle serum sample

A total of 296 serum samples were collected from cattle herds located in southern Chile in Aysén, Ñuble, and Biobío regions, which are relevant regions of cattle existence in the country, holding 16.4% of the national cattle (Instituto nacional de estadística. INE 2021). One hundred and sixteen serum samples were used to determine the ELISA cut-off and 180 samples were used to validate the ELISA. Samples were tested for anti-BVDV antibodies using a commercial IDEXX BVDV Total Ab (IDEXX, Catalog N° 944000) to detect cattle anti-BVDV antibodies. A pool of positive and negative IDEXX-tested serum samples were used as negative and positive control sera to optimize the rE2-ELISA method.

Development and optimization of rE2 based Indirect ELISA

The amount of coating antigen, blocking agent, serum dilution, and conjugate were evaluated to establish the optimal conditions for the indirect ELISA. The amount of rE2 protein was serially diluted in a two-fold from 10 to 2.5 µg/mL. BSA 3% p/v, skimmed milk 5% p/v, casein p/v 5%, and horse serum 2.5% v/v solutions were evaluated as blocking agents. BVDV-positive and negative control serum were applied directly or diluted 2, 5, 10, or 25 times. HRP-labeled rabbit anti-bovine IgG (USBiological, Catalog N° 11903-01Z) was also diluted at 1:5000, 1:10000, 1:15000, and 1:20000.

Briefly, the Nunc™ Maxisorp ELISA plates (Nalgene, Catalog N° 442404) were coated with 100 µL/well of purified rE2 antigens in carbonate-bicarbonate buffer pH 9.6 at 4 °C overnight. After washing with PBS plus 0.05% Tween 20 (PBST) 5 times, the unbound sites in the wells were blocked with 200 µL/well blocking buffer for 2 h at 37 °C. Following blocking and washing, 100 µL/well of serum samples diluted in dilution buffer (1% of skimmed milk in PBST) were added to the wells in duplicate and the plate was incubated for 90 min at room temperature (RT) (18–26 °C). Next, the plates were washed and incubated with 100 µL/well of secondary antibody for 1 h at RT, followed by washes with PBST. The colorimetric reaction was developed by incubating the plates with 100 µL/well of TMB reagent (TMB 1-Step™, Thermo Fisher Scientific, Catalog N° 34018) for 10 min at RT. The reaction was stopped with 50 µL/well of 2.5 N H2SO4, and absorbance at 450 nm was measured using the Synergy™ HTX Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). The conditions selected for antigen coating concentration, blocking agent, conjugate and sample dilution were determined by the highest ratio of absorbance 450 nm values between the positive and negative controls (P/N).

Serum samples evaluation with rE2 based Indirect ELISA for BVDV antibodies detection

100 µL/well of sample dilution buffer were added to rE2-coating Nunc™ Maxisorp ELISA plates, then 25 µL/well of serum samples were added and incubated in a humidity chamber for 90 min at room temperature (18–26 °C). Then, the liquid content of each well was removed, and each well was washed with 300 µL of wash solution 3 times. Next, 100 µL/well of the conjugated antibody were added and incubated for 1 h at RT in a humidity chamber protected from light. Plates were washed again 3 times and 100 µl of TMB reagent were added and incubated for 10 min at RT. The reaction was stopped by adding 50 µL/well of 2.5 N H2SO4. The measurement was performed at 450 nm with the Synergy™ HTX Multi-Mode Microplate Reader.

Determination of the cut‑off value

The cut-off value was determined using 56 positive and 60 negative serum samples previously analyzed with the commercial IDEXX BVDV Total Ab ELISA kit. The same sera were evaluated with the established rE2-ELISA, and then the cut-off value was determined by receiver operating characteristic (ROC) curve analysis (Unal 2017). The cut-off was selected considering the highest true positive rate and the lowest false positive rate. Using the cut-off value, the test sensitivity (fraction of true positives to all with disease) and specificity (fraction of true negatives to all without disease) were determined.

Validation of rE2‑ELISA

To evaluate the potential clinical application of the optimized rE2-ELISA, 180 bovine serum samples were tested and the diagnosis with IDEXX BVDV Total Ab ELISA kit was compared. The positive and negative diagnosis was determined using the previously established cut-off value. Samples were assessed in duplicate. The results were compared with those obtained with the IDEXX kit to establish the true positives (TP), true negatives (TN), false positives (FP), and false negatives (FN) results.

To analyze intra-assay variation, 3 replicates of 11 different samples were run in one assay. To analyze inter-assay variation, 11 different samples were run in two independent assays. The coefficient of variation (CV) in both cases was calculated using the formula (Jaedicke et al. 2012) :

$$\:\text{C}\text{V}=\frac{\text{S}\text{t}\text{a}\text{n}\text{d}\text{a}\text{r}\text{d}\:\text{d}\text{e}\text{v}\text{i}\text{a}\text{t}\text{i}\text{o}\text{n}}{\text{m}\text{e}\text{a}\text{n}}\:\text{x}\:100$$

To confirm the ability of the rE2-ELISA to detect antibodies to a specific sub-genotype, 20 additional serum samples obtained from naturally infected cattle at the School of Veterinary Medicine were evaluated. The sub-genotype of BVDV that infected 7 of the 20 animals had been previously determined. This determination involved RNA extraction from serum samples, reverse transcription to cDNA, polymerase chain reaction (PCR) amplification of the 5’ untranslated region (UTR), followed by sequencing and phylogenetic analysis (Hugues et al. 2023). Six samples were from cattle infected with the BVDV-1e sub-genotype, one was from cattle infected with 1-b, and the remainder were unknown.

Results

Obtention of CHO clone for co-production of E2-1b, E2-1e and E2-1d

CHO cells were transfected with the pCINeo-V2 vector (Fig. 1A), and clones expressing E2-1b (HA-tag) and E2-1d (Strep-tag) proteins were obtained by G-418 antibiotic selection. The CHO-F78 clone was selected and co-transfected with the pCINeo-V1 vector for E2-1e expression (Fig. 1A). Fluorescence intensity was used to select a high-producing clone (Fig. 1B). Since the E2-1e and GFP genes are expressed from a bicistronic messenger RNA, it is expected that the more GFP is detected, the more E2-1e is produced. The non-specific expression of E2 protein variants was verified by SDS-PAGE and Western blot using anti-His tag/anti-mouse 680 nm (Fig. 1C). In addition, sixteen high-producing clones were checked by SDS-PAGE and Western blot using anti-E tag/anti-goat 790 nm (green band) and anti-HA tag/anti-mouse 680 nm (red band) for E2-1e and E2-1b coexpression, respectively (Fig. 1D). Bands around 60 kDa were detected under reducing conditions, corresponding to 1e and 1b variants of rE2 protein. CHO-A42 clone was selected for co-production of E2-1b and E2-1e because it showed higher expression of both proteins (Fig. 1D) and higher replication rate in culture.

Fig. 1
figure 1

Generation of CHO stable clones coexpressing three variants of E2 protein. (A) Representation of the E2-1e/GFP transcription unit from the pCINeo-V1 vector and E2-1b/E2-1d transcription unit from pCINeo-V2 vector. CMV: early/immediate cytomegalovirus promoter, E2: chimeric protein, IRES: internal ribosome entry site, GFP: green fluorescent protein (B) CHO clones transfected with pCINeo-V2 and co-transfected with pCINeo-V1. Transfected cells were obtained by a double selection process (G-418 antibiotic and GFP expression). (C) Analysis of E2 expression by SDS-PAGE and Western blot. The E2 variants were detected with anti-His antibody. (D) Analysis of 16 clones expressing E2-1e (green band) and E2-1b (red band) by SDS-PAGE and Western blot (WB). Relative quantification was performed by the Image Studio software from the Licor Odyssey System. MW: Molecular weight marker AccuRuler RGB plus

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Expression and purification of E2 protein variants

The purification process was carried out by IMAC and yielded 86% purity with some protein losses in the wash step at 40 mM Imidazole (Fig. 2A and B). Under reducing conditions, rE2 proteins showed a banding pattern higher than expected (≈ 60 kDa instead of 47 kDa, Figs. 1C, and 2B). Although E2 proteins are comprised of 382–388 amino acids with a theoretical molecular mass of 47.3–47.7 kDa, all variants have three potential sites for N-glycosylation (Sequence analysis on the NetNGlyc Server, http://www.cbs.dtu.dk/services/NetNGlyc/): at positions 135, 204, and 248. E2-1d and E2-1e have an additional N-glycosylation site at position 316, and E2-1e has a fifth potential N-glycosylation site at position 231. Adding glycans to those sites could increase E2 molecular mass, altering its relative mobility in SDS-PAGE. Therefore, the glycosylation status was evaluated by E2 deglycosylation with PNGase-F. The Western blot under reducing conditions showed deglycosylated protein bands of around 47 kDa as predicted (Fig. 2C, WB panel). These results confirm that the high molecular weight band pattern of rE2 in SDS-PAGE and Western blot was due to glycosylation.

Fig. 2
figure 2

Purification and deglycosylation analysis of E2 protein. (A) Chromatogram of protein purification using immobilized metal affinity chromatography (IMAC). UM: Unbound material, W: wash, E: elution. (B) SDS-PAGE and Western blot of purification samples. MW: Molecular weight marker AccuRuler RGB plus, IS: initial sample. (C) SDS-PAGE and Western blot of rE2 N-deglycosylation assay using PNGase-F, D: N-deglycosylated rE2, G: glycosylated rE2. Western blot analyses were performed with mouse anti-His Tag antibody

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rE2 indirect ELISA parameter setting and receiver operating characteristic curve analysis

The optimized parameters for indirect ELISA based on rE2 protein of different subtypes for detecting antibodies against BVDV were: protein coating concentration, blocking solution, dilution of secondary antibody, and serum samples dilution (Fig. 3). According to the results, the coating antigen concentration of 10 µg/mL showed the highest Positive/Negative (P/N) ratio, but a similar ratio was obtained by coating with 2.5 µg/mL of rE2 (Fig. 3A). Hence, the 2.5 µg/mL concentration was chosen because smaller quantities of protein were needed for the following experiments A comparison of the blocking solutions indicated that the maximum P/N ratio was found when using skimmed milk 5% solution, so was selected as the blocking agent (Fig. 3B). Secondary antibody dilution analyses showed that the 1/5000 dilution better differentiated positive from negative samples (Fig. 3C). Different dilutions of the serum samples were tested and the results showed that the best performing dilution was 1/25 (Fig. 3D). However, the 1/5 sample dilution was chosen because it simplifies the experimental procedure by allowing the sample to be diluted on the ELISA plate and also provides a high P/N ratio. With these results, the optimized parameters for the rE2 indirect ELISA were determined to be 2.5 µg/mL of rE2 per well for plate coating, skimmed milk as blocking solution, conjugated antibody dilution of 1/5000, and serum sample dilution of 1/5.

Fig. 3
figure 3

Determination of the optimal working conditions for the rE2-ELISA. (A) Concentration of the coating antigen. (B) Blocking solution. (C) Conjugate dilution. (D) Sample dilution. The selected condition for each parameter tested is highlighted in red

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The ROC analysis was performed by evaluating 60 negative and 56 positive serum samples. It showed that by selecting a cut-off absorbance of 0.63 OD at 450 nm (Fig. 4B), the diagnostic specificity of the technique is 98.33% with a sensitivity of 92.86% (Fig. 4A).

Fig. 4
figure 4

Selection of the cut-off value for the rE2-ELISA. (A) Receiver Operating Characteristic (ROC) curve analysis. Each point on the curve represents the true positive rate (or sensitivity)/true negative (specificity) pair corresponding to a unique decision threshold. (B) Plot of positive and negative sera evaluated by rE2-ELISA. Distribution of results obtained when evaluating positive and negative sera by rE2-ELISA, which were previously tested with a commercial ELISA kit for antibodies against BVDV (Mann-Whitney Test, **** p < 0,0001). The cut-off value (OD 450 value of 0.63) is drawn with a dotted line

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Validation of the BVDV rE2ELISA

Serum samples collected from cattle herds located in southern Chile were tested for anti-BVDV antibodies using IDEXX system and the optimized rE2-ELISA. The diagnosis showed that 103 BVDV positive and 63 BVDV negative samples were detected by both methods, with a positive coincidence rate of 95.37% and a negative coincidence rate of 87.5% (Table 1). Kappa statistical analysis showed that the level of agreement with the commercial ELISA BVDV Total Ab (IDEXX) was 0.836.

Table 1 Comparative analysis of rE2-ELISA with the commercial IDEXX total ab ELISA kit for detection of cattle anti-BVDV antibodies. TP: true positive, FP: false positive, FN: false negative, TN: true negative
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The intra- and inter-assay precision of the absorbance units obtained with the rE2 ELISA among the eleven serum samples tested showed mean CVs of 2,771% and 17,149%, respectively. These results met the acceptance criteria for immunoassay validation, CV < 20% (Fda and Cder 2018).

Serum samples from animals naturally infected with bovine viral diarrhea virus subtypes 1b, 1e, and unknown were provided by the Faculty of Veterinary Medicine, Universidad de Concepción. The known BVDV sub-genotype in some of the samples tested was previously determined by viral 5’-UTR PCR amplification sequencing and phylogenetic analyses (PMID 37679808).

Fig. 5
figure 5

Recognition of recombinant E2 proteins in the rE2 ELISA format by sera from cattle exposed to different BVDV subtypes. The cut-off value (0.63) for positive anti-E2 antibodies is represented by the horizontal line

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Nineteen of the 20 cattle serum samples exposed to natural BVDV infection recognized the recombinant protein mixture in the rE2-ELISA format (Fig. 5). Only serum N°5 showed an OD value below the cut-off and its sub-genotype is unknown. All serum samples genotyped as 1b and 1e recognized the protein mixture with strong signal intensity, ranging from 0.966 to 1.579. Similarly, serum samples of unknown sub-genotypes showed high absorbance values (between 1.356 and 1.769). These results demonstrate that the rE2 protein mixture was recognized by antibodies from animals naturally infected with BVDV and contains antigenic regions similar to the native viral E2 protein.

Discussion

BVDV is prevalent in cattle populations worldwide and causes a wide range of clinical manifestations, including diarrhea, respiratory disease, immunosuppression, growth retardation, sudden drop in milk production, abortion, reduced reproductive efficiency, and early death (Khodakaram-Tafti and Farjanikish 2017). Infection with BVDV results in significant economic losses, either directly through decreased productive performance of cattle herds or indirectly through the cost of control programs (Pinior et al. 2017). In Chile, according to the 2021 sanitary report of the regulatory agency Servicio Agrícola Ganadero (SAG), 53.4% of the animals with obligatory reporting pathologies correspond to Bovine Viral Diarrhea. Considering the lack of epidemiological control systems for BVD in South America, one of the largest cattle producers in the world, this study aimed to develop a high throughput diagnostic system for BVD that takes into account the genotypic variants distributed in the world and in Chile in the serodetection of this pathology.

The gold standard test for the serological diagnosis of bovine viral diarrhea is the virus neutralization test (VNT). However, it requires at least 6 days to obtain a result, cell culture, working with the live virus in appropriate facilities, and constant monitoring of serum and cells to avoid viral contamination (Larska et al. 2013). This makes the test expensive and challenging to use in epidemiologic control programs. Alternatively, the mass screening of antibodies against BVDV using ELISA is the most widely used method. It is characterized by its speed, sensitivity, and capacity to process a large number of samples. Commercial kits for detecting antibodies to BVDV use different antigens, including the whole virion antigens (Howard et al. 1985; Hsien-Jue Chu et al. 1985; Cho et al. 1991), which is expensive to produce and requires a high level of biosafety, and the NS3 protein, which fails to detect neutralizing antibodies (Brodersen 2014).

To overcome these limitations, an indirect ELISA was successfully developed to detect serum antibodies against bovine viral diarrhea virus using recombinant E2 proteins as antigens. The E2 transmembrane protein functions as a viral attachment protein for BVDV entry into host cells. E2 is considered the main antigenic viral protein (Donis 1995; Patterson et al. 2012), and neutralizing antibodies detection against E2 protein is a useful diagnostic tool for identifying animals infected with BVDV. In collaboration with the association of Angus farmers in Coyhaique, Chile, our group identified two sub-genotypes of the BVDV virus, 1d and 1e, with high prevalence in the country’s south (Hugues et al. 2023). We used these two sub-genotype sequences and the high prevalence worldwide sub-genotype 1b to generate a cell line that simultaneously produces three antigenic E2 proteins.

In the development of protein-based ELISA systems for virus detection, surface glycoproteins are good candidate antigens because they tend to be the main inducers of neutralizing antibodies and therefore the host is likely to produce antibodies against these proteins (Spencer et al. 2007). However, since these proteins are post-translationally modified with the addition of sugars, the correctly folded protein can only be produced in a eukaryotic system. The glycosylation patterns of E2 have been reported to influence the induction of immune responses (Wang et al. 2015). Previous studies have shown that serum antibodies do not recognize BVD E2 without N-glycans in indirect ELISA (González Pose et al. 2021). The CHO cell expression system was selected for rE2 production and glycosylation was confirmed (Fig. 2C). Recombinant antigen expression in a system that closely mimics native glycosylation must contribute to the sensitivity of the rE2-ELISA.

After determining the appropriate concentration and dilution of antigen, secondary antibody, and blocking agent, 180 serum samples were tested using this new rE2-ELISA. The optimal cut-off value was 0.63, resulting in a diagnostic sensitivity of 92.86% and specificity of 98.33%. Other ELISA systems using E2 protein as antigen have shown lower sensitivity and specificity values than the method established in this study. E2 protein sub-genotype 2, obtained in a eukaryotic expression system, gives sensitivities of 91.07% and specificities of 92.02% (Behera et al. 2015). In another study, E. coli expressed E2 protein, sub-genotype 1a, showed a sensitivity of 91.7% and a specificity of 96.7% (Wang et al. 2023). Including E2 sub-genotypes with the highest prevalence worldwide and those with the highest prevalence in the south of our country could increase the diagnostic power of the developed ELISA system.

The κappa value demonstrated a high correlation between the rE2-ELISA results and the ELISA BVDV Total Ab (IDEXX) (κ = 0.836) (Landis and Koch 1977). The IDEXX Kit is based on detecting antibodies against the NS3 protein (Hanon et al. 2017), found only in the initial stages of viral replication after BVDV infection and induces the production of anti-NS3 antibodies. While the viral protease NS3 is continuously expressed in CP BVDV-infected cells with a potential role in the induction of apoptosis, this protein is only expressed early in cell infection or not at all in NCP BVDV infections (Brodersen 2014). Therefore, the five “false positive” serum samples detected by the rE2 ELISA may have anti-E2 but not anti-NS3 antibodies if these animals were infected with an NCP BVDV strain, giving a false positive result compared to IDEXX. On the other hand, the nine “false negative” serum samples not detected by the rE2 ELISA may contain antibodies to NS3 as a result of the strong humoral immune response elicited by this highly conserved protein (Abad Shapouri et al. 2022). In contrast, the immune response to E2 may be weaker due to its lower conservation and antigenic variability (Mirosław and Polak 2020).

Of 180 samples analyzed, 103 (57.2%) were positive for anti-BVDV antibodies, corresponding with a high seroprevalence of BVDV infection, which coincides with the results reported previously (Alocilla and Monti 2022). There is a lack of information related to the causes of the spread, maintenance, or prevalence of infection in cattle under Chilean productive conditions, making it difficult to develop control and eradication strategies. In fact, in Chile, there is currently no official control or eradication program implemented by the regulatory agency SAG. Using rE2-ELISA in Chile, where there is not any vaccination program, would be an economical and effective tool for BVDV diagnostic, contributing to the control and eradication of the disease. The selection of E2 as the antigen, combined with the inclusion of variants prevalent worldwide and in Chile, makes the rE2-ELISA system a powerful diagnostic tool for serodiagnosis of CP and NCP variants of BVDV, applicable in a wide range of regions.

Conclusions

This work established a prototype indirect ELISA for antibody detection against BVDV based on 3 variants of the structural protein E2, demonstrating a sensitivity of 92.86% and a specificity of 98.33%. During the validation assay, the rE2-ELISA also exhibited high accuracy (Kappa > 0.8) compared with the commercial kit IDEXX BVDV Total AB (IDEXX). The results support that the developed indirect ELISA kit can become a valid alternative for BVDV-infected animal identification.