Rapid Quantification of Bluetongue Virus-Neutralizing Antibodies Using Bioluminescent Reporter-Expressing Viruses

Academic Editor: François Meurens Received: 3 October 2025 Revised: 22 October 2025 Accepted: 27 October 2025 Published: 29 October 2025 Citation: Jiménez-Cabello, L.; Utrilla-Trigo, S.; Calvo-Pinilla, E.; Nogales, A.; Ortego, J. Rapid Quantification of Bluetongue Virus-Neutralizing Antibodies Using Bioluminescent Reporter-Expressing Viruses. Vaccines 2025,13, 1102. https://doi.org/10.3390/ vaccines13111102 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Rapid Quantification of Bluetongue Virus-Neutralizing Antibodies Using Bioluminescent Reporter-Expressing Viruses Luis Jiménez-Cabello * , Sergio Utrilla-Trigo , Eva Calvo-Pinilla , Aitor Nogales and Javier Ortego Centro de Investigación en Sanidad Animal (CISA), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Valdeolmos, 28130 Madrid, Spain; ser[email protected] (S.U.-T.); [email protected] (E.C.-P.); [email protected] (A.N.); [email protected] (J.O.) *Correspondence: [email protected]; Tel.: +34-916202300 Abstract Bluetongue virus (BTV) is the causative agent of the significant livestock disease Bluetongue (BT), which causes severe economic losses associated with its considerable impact on the health and trade of ruminants. Background/Objectives: BTV infection and vaccination against the virus typically result in the induction of antibodies with the capacity to neutralize viral infection. Classic neutralization approaches resemble the methodology applied for neutralizing antibodies (NAbs) quantification. To improve long-standing and new-generation methodologies for the quantification of NAbs or evaluation of antivirals, we offer here the development of a new luciferase-based microneutralization approach as a proof-of-concept. Methods: Central to this innovative approach is the recently generated set of replication-competent reporter-expressing recombinant BTV, where the NanoLuc luciferase protein expression serves as a quantifiable readout for viral replication. After evaluating a set of heat-inactivated serum samples with neutralizing activity (measured via SNTs), these were incubated with 100 PFU of NLuc-expressing rBTV of serotype 1, 4 or 8 and Vero cells were infected with the serum–virus mixture. Then, the luminescent signal was measured at 48 h post-infection. Results: Using the proposed NLuc-based assay and the luminescent signal in the supernatant, we could detect neutralizing activity as soon as 48 h post-infection. Importantly, we were able to observe a strong correlation between NAbs titers measured by classic microneutralization assay and by our bioluminescent approach (BTV-1 Spearman r = 0.932901; p-value < 0.0001; BTV-4 Spearman r = 0.8070192; p-value < 0.0001; BTV-8 Spearman r = 0.9983; p-value < 0.0001). In addition, the NLuc-based assay displayed a serotype-specific character potentially equivalent to classic SNT methods. Conclusions: In summary, our reporter-based microneutralization assay provides a rapid and suitable method to quantify BTV-neutralizing antibodies in serum samples of natural hosts after vaccination or infection, with a serotype-specificity equivalent to classic SNT methods. Keywords: Bluetongue virus (BTV); neutralizing antibodies; reverse genetics; reporter gene; luciferase 1. Introduction Bluetongue (BT) is an arthropod-borne disease that affects wild and domestic ruminants [ 1 , 2 ]. The Bluetongue virus (BTV) (family Sedoreoviridae), which causes BT and is transmitted by Culicoides biting midges [ 3 , 4 ], is composed of a three-layered protein capsid harboring a double-stranded RNA (dsRNA) segmented genome [ 5 – 7 ]. The ten Vaccines 2025,13, 1102 https://doi.org/10.3390/vaccines13111102 Vaccines 2025,13, 1102 2 of 18 dsRNA segments encode for seven structural proteins (VP1–VP7) and five nonstructural proteins (NS1–NS5) [ 5 , 6 , 8 – 12 ]. Protein VP2 is the most exposed virion protein as it forms trimers located in the outer capsid layer [ 7 ]. This protein acts during the onset of cell infection through its interaction with the cellular receptor, allowing virus entry into the host cell [ 13 – 15 ]. Consequently, VP2 is the main target of virus-neutralizing antibodies (NAbs) and the determinant of virus serotype [ 16 – 18 ]. To date, more than 30 serotypes of BTV have been described, and due to the high genetic variability of protein VP2 among serotypes (especially in antigenically relevant regions [ 19 , 20 ]), neutralization among BTV serotypes is scarce [ 21 – 23 ]. As conventional vaccine approaches and most next-generation vaccines base their protective potential against BTV on the presentation/expression of protein VP2 [ 18 , 24 , 25 ], quantification of NAbs serves as a measure of vaccine efficacy. In this sense, a classic serum neutralization test (SNT) is considered as the gold standard for the detection and quantification of NAbs responses in BTV-exposed and -vaccinated animals [ 7 , 26 ]. Nonetheless, NAbs quantification using a plaque reduction neutralization test (PRNT) and a microneutralization assay are labor-intensive and time-consuming as they involve a plate incubation period of five days, and may lead to misinterpreted or inconsistent results [26,27]. Alternative methodologies for NAbs quantification have been developed against different viruses, such as pestiviruses, SARS-CoV-2, Rift Valley fever virus or Influenza virus [ 28 – 32 ]. These approaches exploit reverse genetics (RG) systems to generate fluorescent or luminescent viruses that allow us to avoid the utilization of fixative agents and offer an easier and more homogenous measurement than classical cell-based approaches, which rely on a reduction in virus-induced cytopathic effect (CPE). Similarly, reporter viruses have been confirmed as reliable tools to facilitate antiviral drug testing [ 33 , 34 ] as they enhance the efficiency and accuracy of antiviral compound identification compared to traditional methods. In this sense, genetically engineered reporter viruses permit rapid screening of large compound libraries, which boosts high-throughput drug screening [35–37]. Diverse RG systems exist for orbiviruses such as BTV, African Horse Sickness virus (AHSV) or Epizootic Hemorrhagic Disease virus (EHDV) [ 38 – 41 ]. Recently, we developed a whole-plasmid-based RG system for the rescue of recombinant BTV [ 42 ]. By using this RG system, we successfully recovered a recombinant BTV expressing reporter luminescent (NanoLuc luciferase, NLuc) gene [ 42 ]. Thus, this proof-of-concept study aims to demonstrate the feasibility of a methodology for the rapid and easy quantification of serotype-specific NAbs using luminescent reporter BTV as an alternative to a classical SNT. In addition, we also study the possibilities of these NLuc-expressing BTVs for the in vitro evaluation of antivirals against this viral pathogen. 2. Materials and Methods 2.1. Cell and Viruses Green monkey kidney cells (Vero) (ATCC catalog no. CCL-81) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 1% PSG (penicillin, 100 units/mL; streptomycin 100 µ g/mL; l-glutamine, 2 mM) at 37 ◦C in air enriched with 5% CO2. BTV serotype 1 (ALG2006/01) (BTV-1), BTV serotype 4 (SPA2004/02), BTV serotype 8 (BEL/2006), rBTV-1/NLuc, rBTV-4/Luc and rBTV-8/NLuc were used in the experiments. Generation of rBTV-1/NLuc, rBTV-4/Luc and rBTV-8/NLuc has previously been described [ 42 ]. Vero cells were used for the generation of virus stocks and virus titration was performed via plaque assay, as previously described [ 42 ]. This research was performed in the Animal Health Research Center (CISA, INIA-CSIC), a biological security facility (BSL3). Vaccines 2025,13, 1102 3 of 18 2.2. Microneutralization Assay Two-fold or four-fold dilutions of heat-inactivated sheep, cow or mice sera (56 ◦ C for 30 min) were incubated with 100 PFU of BTV-1, BTV-4 or BTV-8 for 1 h at 37 ◦ C and 4 ◦ C O/N. Vero cells (96-well plate format, 5 × 10 4 cells/well, triplicates) were then infected with the serum–virus mixture for 1 h at 37 ◦ C, 5% CO 2 . Then, the serum–virus mixture was removed and 150 µ L of fresh growth medium were added and incubated for 5 days at 37 ◦ C, 5% CO 2 . Thereafter, Vero cells were fixed with paraformaldehyde 4% and visualized with 2% crystal violet-PBS. The neutralization titer (named neutralization dilution 50, ND 50 , to differentiate from the neutralization titer calculated by NLuc signal) is defined as the reciprocal of the highest dilution that shows ≥50% inhibition of virus infectivity. 2.3. NLuc-Based Microneutralization Assays NLuc microneutralization assays were performed as previously described [ 43 , 44 ]. Two-fold or four-fold dilutions of heat-inactivated sheep, cow or mice sera (56 ◦ C for 30 min) were incubated with 100 PFU of rBTV-1/NLuc, rBTV-4/NLuc, or rBTV-8/NLuc for 1 h at 37 ◦ C and at 4 ◦ C, O/N. Vero cells (96-well plate format, 5 × 10 4 cells/well, triplicates) were then infected with the serum–virus mixture for 1 h at 37 ◦ C, 5% CO 2 . Then, the serum–virus mixture was removed and 150 µ L of fresh growth medium were added and incubated for 72 h at 37 ◦ C. At 48 or 72 h post-infection (h.p.i.), NLuc activity in the cell culture supernatants was quantified using Nano-Glo luciferase substrate (Promega, Madrid, Spain) and a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). The luminescent values of virus-infected cells in the absence of antibody were used to calculate 100% viral infection. Cells in the absence of viral infection were used to calculate the luminescence background. Triplicate wells were used to calculate the average and SD of neutralization using Microsoft Excel software. The 50% neutralization titer (NT 50 ) was determined by use of a sigmoidal dose–response curve (GraphPad Prism, v8.0.1 software). GraphPad Prism software was used to perform Spearman correlation analysis between NAbs titers obtained by classic or NLuc-based microneutralization assays. Bland–Altman plots were made with GraphPad Prism software. 2.4. Antiviral Assays Monolayers of Vero cells in 96-well plate format (5 × 10 4 cells/well, triplicates) were infected with 100 PFU of rBTV-1/NLuc. After 90 min of viral adsorption, the medium was replaced with 150 µ L of medium (containing only 2% FBS) supplemented with two-fold serial dilutions (starting concentration, 125 µ M) of ATA, and the cells were then incubated at 37 ◦ C, 5% CO 2 . At 48 h.p.i., NLuc activity in tissue culture supernatants was measured using Nano-Glo luciferase substrate (Promega) and FLUOstar Omega microplate reader (BMG Labtech). The luminescent values of virus-infected cells in the absence of ATA were used to calculate 100% viral infection. Cells in the absence of viral infection were used to calculate the luminescence background. Triplicate wells were used to calculate the average and SD of the inhibition using Microsoft Excel software. The 50% inhibition concentration (IC 50 ) was determined by the use of a sigmoidal dose–response curve (GraphPad Prism, v8.0.1 software). 3. Results 3.1. Use of Luminescent BTVs for Determination of BTV-NAb Titers By implementing a whole-plasmid RG system for BTV, we previously engineered and characterized recombinant BTVs of serotypes 1, 4 and 8 expressing a reporter luminescent (NLuc) gene [ 42 ]. The reporter gene was fused to the C-terminal end of segment 5 (S5) of BTV by means of the porcine teschovirus-1 (PTV-1) 2A cleavage sequence (Figure 1A). Vaccines 2025,13, 1102 4 of 18 S5 encodes non-structural protein 1 (NS1) and, consequently, expression of the reporter gene only occurs during the replicative cycle of BTV. Reporter-expressing BTVs were used to study the dynamics of viral infection in vitro and in vivo , and the expression of the NLuc reporter gene constituted a valid surrogate for virus replication [ 42 ]. Now, we have used these luminescent recombinant BTVs of serotype 1 (rBTV-1/NLuc), serotype 4 (rBTV4/NLuc) and serotype 8 (rBTV-8/NLuc) for the rapid quantification of NAbs against BTV-1, BTV-4 and BTV-8, respectively, using an NLuc-based microneutralization assay (Figure 1B), as has previously been performed for other RNA viruses [30–32]. Figure 1. (A) Representation of the recombinant BTV/Luc viruses used in this study. NLuc reporter gene was included as fusion proteins by means of a 2A picornavirus peptide fused to the 3 ′ end of segment 5 of BTV-1. (B) Schematic representation of the methodological procedure to measure virus infection by means of luminescent signal in supernatants of Vero cells infected with reporter viruses. An SNT to determine antibody-mediated virus inhibition requires the use of secondary approaches to detect the presence of the virus. To overcome this additional step and validate our NLuc-based microneutralization assay, rBTV-1/NLuc, rBTV-4/NLuc and rBTV-8/NLuc viruses were used to evaluate the neutralizing activity of serum samples obtained from BTVexposed or -vaccinated animals. First, we tested if virus neutralization could be tracked by luminescent signal using two serum samples from cow (obtained after experimental infection, Neutralizing Dilution 50 (ND 50 ) = 1:160) or sheep (obtained after immunization with a recombinant vaccine [ 45 ], ND 50 = 1:240) reactive to BTV-1 or BTV-4, respectively, and a mouse serum hyperimmune against BTV-8 (ND 50 = 1:400), as we lacked a natural BTV host serum reactive to BTV-8. To do so, two-fold dilutions of these heat-inactivated serum samples were pre-incubated with 100 PFU of rBTV-1/NLuc, rBTV-4/NLuc and rBTV-8/NLuc viruses, and Vero cells were infected with the virus–serum mixture. As a control, we included a non-reactive serum against BTV, as well as mock-infected cells and viruses in the absence of antibodies as internal controls. At 48 or 72 h.p.i., supernatants were collected, and the luminescent signal was measured. In our luciferase-based microneutralization assay, higher NLuc activity indicated higher viral replication and, therefore, lower neutralization levels. The negative serum sample did not neutralize rBTV-1/NLuc, rBTV-4/NLuc or rBTV-8/NLuc, as luminescent signals equivalent to the virus-infected cells without serum (internal control) were observed at 48 and 72 h.p.i. even at the lower serum dilution evaluated (Figures 2–4). In contrast, we observed that the serum obtained from a BTV-1-infected cow induced a dilution-dependent Vaccines 2025,13, 1102 5 of 18 reduction in the percentage of NLuc activity at 48 h.p.i. (Figure 2A) and 72 h.p.i. (Figure 2B). Similarly, we observed that the tested serum that was reactive to BTV-4, obtained from a BTV-4-vaccinated sheep [ 45 ], also neutralized the reporter rBTV-4/NLuc at 48 h.p.i. (Figure 3A) and 72 h.p.i. (Figure 3B), as indicated by the antibody-dilution-dependent detection of luminescent activity. Luminescent signals from supernatants of Vero cells infected with rBTV-8/NLuc also indicated that the mouse serum hyperimmune against BTV-8 neutralized the reporter virus at both times post-infection (Figure 4A,B). Figure 2. A luciferase-based microneutralization assay for the identification of NAbs against BTV-1. Vero cells were infected (100 PFU) with rBTV-1/NLuc pre-incubated with 2-fold serial dilutions (starting dilution of 1:500) of a BTV-1-exposed cow serum sample. At 48 (A) or 72 (B) h.p.i., NLuc activity in culture supernatants was quantified using a microplate reader, and the percentage of inhibition was calculated using sigmoidal dose–response curves. A negative serum obtained from a naïve cow was used as the control. Mock-infected cells and viruses in the absence of antibodies were used as internal controls. The percentage of luminescent activity was normalized to Vero cell infection with 100 PFU of rBTV-1/NLuc in the absence of serum (100% of luminescent activity) and Vero cells in the absence of virus (0% of luminescent activity). Data show the means ± SD of the results determined in triplicate. The starting dilution was established considering preliminary data. It is worth noting that these sera against BTV-1, BTV-4 or BTV-8 did not show crossneutralizing activity against the heterologous serotypes evaluated here, as determined via a classic SNT. Importantly, the three tested serum samples did not exhibit a seruminduced reduction in NLuc activity when their neutralizing activity was assessed against heterologous NLuc-expressing BTV serotypes, which mildly reflects the serotype-specificity of our NLuc-based microneutralization test. Using the sigmoidal dose–response curves, we could calculate the neutralizing titer 50 (NT 50 ) as a measure of virus neutralization based on the NLuc signal. The NT 50 values of the cow and sheep sera against rBTV-1/NLuc or rBTV-4/NLuc, respectively, were higher at 48 h.p.i. (cow serum NT 50 = 1:8356; sheep serum NT 50 = 1:8994) than at 72 h.p.i. (cow serum NT 50 = 1:3214; sheep serum NT 50 = 1:5929), which is intrinsically related to less viral replication (and lower viral protein expression) at early times of infection. In the case of rBTV-8/NLuc, we could estimate NT 50 values of 1:4365 (48 h.p.i) and 1:1137 (72 h.p.i.) against BTV-8. Despite the differences between the NT 50 estimated through NLuc measurement at two and three days of Vero cells infection, our luciferase-based microneutralization assay was able to determine the presence of homologous NAbs as early Vaccines 2025,13, 1102 6 of 18 as 48 h.p.i. Therefore, we selected the measurement of NLuc activity at 48 h.p.i. to confirm its suitability as a rapid alternative method to quantify NAbs in further studies. Figure 3. A luciferase-based microneutralization assay for the identification of NAbs against BTV-4. Vero cells were infected (100 PFU) with rBTV-4/NLuc pre-incubated with 2-fold serial dilutions (starting dilution of 1:25) of an inactivated-BTV-4-vaccinated sheep serum sample. At 48 (A) or 72 (B) h.p.i., NLuc activity in culture supernatants was quantified using a microplate reader, and the percentage of inhibition was calculated using sigmoidal dose–response curves. A negative serum obtained from a naïve sheep was used as the control. Mock-infected cells and viruses in the absence of antibodies were used as internal controls. The percentage of luminescent activity was normalized to Vero cell infection with 100 PFU of rBTV-4/NLuc in the absence of serum (100% of luminescent activity) and Vero cells in the absence of virus (0% of luminescent activity). Data show the means ±SD of the results determined in triplicate. The starting dilution was established considering preliminary data. Figure 4. A luciferase-based microneutralization assay for the identification of NAbs against BTV-8. Vero cells were infected (100 PFU) with rBTV-8/NLuc pre-incubated with 2-fold serial dilutions (starting dilution of 1:25) of a BTV-8 hyperimmune mouse sera sample. At 48 (A) or 72 (B) h.p.i., Vaccines 2025,13, 1102 7 of 18 NLuc activity in culture supernatants was quantified using a microplate reader, and the percentage of inhibition was calculated using sigmoidal dose–response curves. A negative serum (from a mockinfected mouse) was used as a control. Mock-infected cells and viruses in the absence of antibodies were used as internal controls. The percentage of luminescent activity was normalized to Vero cell infection with 100 PFU of rBTV-8/NLuc in the absence of serum (100% of luminescent activity) and Vero cells in the absence of virus (0% of luminescent activity). Data show the means ± SD of the results determined for triplicates. The starting dilution was established considering preliminary data. 3.2. NAbs Quantification by Luminescence Activity Correlates with NAbs Titer Determined via Classic SNT Despite the fact that luminescent signal in the supernatants of reporter BTV-infected cells indicated virus neutralization in the previous experiment, we could not establish a correlation between a classic SNT and an NLuc-based assay as we evaluated one positive sample for each BTV serotype. Therefore, we applied this rapid methodology to conduct a screening of NAbs in a set of BTV-positive sheep sera samples kindly provided by the Central Veterinary Laboratory (LCV Algete, Spain). For BTV-1-positive serum samples (estimated via classic microneutralization test, see Table 1), we detected NT 50 values above the lower dilution tested (1:10), with some of them reaching titers of 1:640 (Figure 5A). BTV1-negative serum samples (ID: 31, 33, 34, 35, 36, 37, 40 and 41) displayed high percentages of NLuc activity at the lowest dilution evaluated (1:10), and therefore did not show evidence of neutralization against rBTV-1/NLuc (Figure 5A). Table 1. Neutralization titer values of field sera against recombinant BTV-1 and rBTV-1/NLuc. Sheep ID BTV-1 (ND50) rBTV-1/NLuc (NT50) Sheep ID BTV-1 (ND50) rBTV-1/NLuc (NT50) 1 100 100 22 320 340 2 320 640 23 160 160 3 100 160 24 100 100 4 320 1600 25 25 25 5 25 40 26 25 25 6 100 100 27 25 25 7 400 160 28 40 25 8 160 100 29 100 100 9 80 640 30 100 100 10 640 400 31 10 25 11 320 640 32 25 40 12 640 400 33 0 0 13 320 640 34 10 25 14 160 640 35 0 5.5 15 80 400 36 0 0 16 160 160 37 0 5.5 17 640 640 38 320 340 18 40 100 39 40 40 19 40 40 40 10 5.5 20 400 400 41 0 0 21 640 400 ND 50 refers to NAbs titer quantified by classic microneutralization assay. NT 50 refers to NAbs titer quantified by NLuc-based assay. ND 50 and NT 50 values under the lowest dilution applied were assigned a value of 0. ND 50 and NT50 were determined in triplicate and the mean value is shown. NT50 CV = 1.1576. ND50 CV = 1.3069. It is worth noting that most serum samples were collected from BTV-4-exposed animals, as all the animals (except for sheep 32) had high titers of NAbs against this BTV serotype (estimated via classic microneutralization test, see Table 2). Interestingly, we Vaccines 2025,13, 1102 8 of 18 observed that all the serum samples, except for the serum from sheep 32, showed NT 50 values of over 1:40 against rBTV-4/NLuc (Figure 5C). Figure 5. Identification of NAbs against BTV in a collection of serum samples. Vero cells were infected (100 PFU) with rBTV-1/NLuc (A), rBTV-4/NLuc (C) or rBTV-8/NLuc (E) pre-incubated with 4-fold serial dilutions (starting dilution of 1:10) of sera samples. At 48 h.p.i., NLuc activity was quantified using a microplate reader. A negative sheep serum was used as the control. Mock-infected cells and viruses in the absence of antibodies were used as internal controls. The percentage of neutralization was normalized to Vero cell infection with 100 PFU of each recombinant BTV in the absence of antibodies (100% of luminescent activity) and Vero cells in the absence of virus (0% of luminescent activity). (B,D,F) Correlation between neutralization titers values obtained via SNT or NLuc signal against wild-type BTV-1 and rBTV-1/NLuc (B), wild-type BTV-4 and rBTV-4/NLuc (D) or wild-type BTV-8 and rBTV-8/NLuc (F), as calculated by Spearman’s rank order correlation. Confidence intervals of 95% are indicated by gray dotted lines. ND 50 refers to NAbs titer quantified by classic microneutralization assay. NT50 refers to NAbs titer quantified by NLuc-based assay. The set of sera showed neutralizing activity against BTV-1 and BTV-4, which is in line with the co-circulation of both serotypes in some regions of Spain. However, there is no evidence of BTV-8 circulation in 2023 [ 46 ], when the samples were collected. As expected, most serum samples did not show evidence of neutralizing activity against BTV-8 by either luminescent signal determination or a classic SNT (Figure 5E). However, sera from sheep 28, 29, 30 and 31 showed NT 50 values between 40 and 80 (Figure 5E). This could imply that our NLuc-based microneutralization test is not completely serotype-specific. Nonetheless, these four sera showed neutralizing capacity, as measured using a classic Vaccines 2025,13, 1102 9 of 18 microneutralization test (see Table 3), which might indicate that our bioluminescent assay could have an equivalent sensibility to traditional methodologies. Table 2. Neutralization titer values of field sera against recombinant BTV-4 and rBTV-4/NLuc. Sheep ID BTV-4 (ND50) rBTV-4/NLuc (NT50) Sheep ID BTV-4 (ND50) rBTV-4/NLuc (NT50) 1 1600 1600 22 400 2560 2 1280 2560 23 1600 2560 3 1280 2560 24 1600 2560 4 400 640 25 100 640 5 400 640 26 400 2560 6 640 2560 27 400 640 7 400 2560 28 640 2560 8 400 640 29 400 2560 9 400 640 30 400 2560 10 1280 2560 31 100 160 11 1280 2560 32 10 40 12 1280 2560 33 1280 2560 13 1280 2560 34 1280 2560 14 1280 2560 35 1280 2560 15 1280 2560 36 1280 2560 16 1280 2560 37 1280 2560 17 1280 2560 38 1280 2560 18 1280 2560 39 1280 2560 19 1280 640 40 1280 2560 20 25 640 41 1280 2560 21 1280 2560 ND 50 refers to NAbs titer quantified by classic microneutralization assay. NT 50 refers to NAbs titer quantified by NLuc-based assay. ND 50 and NT 50 were determined in triplicate and the mean value is shown. NT 50 CV = 0.5343. ND50 CV = 0.4347. To assess whether there is correlation between classic neutralization and the proposed bioluminescent assays, we determined the neutralization titers of the whole set of field sera against non-reporter wild-type BTV of serotype 1, 4 and 8 via classic microneutralization assay. NAbs titers of field sera against wild-type BTV-1, BTV-4 and BTV-8 and NT 50 values against rBTV-1/NLuc, rBTV-4/NLuc and rBTV-8/NLuc are gathered in Tables 1–3, respectively. Notably, we observed a strong correlation between neutralization titers against BTV-1 and rBTV-1/NLuc (Spearman r = 0.9329; 95% confidence intervals (CIs) = 0.8770–0.9639; p-value < 0.0001) (Figure 5B), and between BTV-4 and rBTV-4/NLuc (Spearman r = 0.8070; 95% CI = 0.6643–0.8930; p-value < 0.0001) (Figure 5D). Additionally, the data provided by our NLuc-based assay using rBTV-8/NLuc correlated with data gathered via classic microneutralization assay (Spearman r = 0.9983; 95% CI = 0.9967–0.9991; p-value < 0.0001) (Figure 5F). We also assessed the agreement between these two quantitative measurement methods for each serotype using Bland–Altman plots (Figure 6). On average, we observed that the NLuc-based microneutralization assay resulted in slightly higher neutralization values compared to the classic microneutralization assay, except for BTV-8 (BTV serotype 1 bias = 0.1313, upper limit of agreement (LoA) = 0.7301, lower LoA = − 0.4674; BTV serotype 4 bias = 0.3831, upper LoA = 0.9444, lower LoA = − 0.1783; BTV serotype 8 bias = −0.004352, upper LoA = 0.06106, lower LoA = − 0.06797). Nonetheless, results involving BTV serotype 8 need to be confirmed more robustly by using a wider set of serum samples reactive to BTV8 in further experiments. Overall, these data indicate the potential feasibility of luminescent recombinant viruses for the detection and quantification of serotype-specific NAbs. Vaccines 2025,13, 1102 16 of 18 12. 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