Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Southwest Saudi Arabia

Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Southwest Saudi Arabia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Southwest Saudi Arabia Ommer M. Dafalla, Abdulah A. Alashor, Mohammed O. Hussien, Elsiddig M. Noureldin, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7429013/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic virus with high public health and veterinary importance in Africa and the Middle East. Serological surveillance, functional neutralization testing, and virus titration via tissue culture are important for monitoring the effectiveness of vaccines and outbreaks. Objective This study aimed to increase the precision of immunological assessments after RVFV vaccination and provide a methodological approach to combine viral quantification, serological detection and functional neutralization testing. Methods Twenty serum samples were tested using the ID.vet RVFV competitive ELISA to detect antibodies specific to the viral nucleocapsid protein. Viral titration was conducted in Vero cells, and TCID₅₀/ml was calculated using the Reed and Muench method. VNT was performed at 24, 48, 72, and 96 hours after infection with different viral doses (100 to 100,000 TCID₅₀/ml), and the neutralizing ability of serial serum dilutions (1:2 to 1:1024) was tested. Compared with the control, protection was determined by CPE inhibition. Results ELISA revealed robust antibody signals up to a 1:32 dilution, with S/N < 40%, whereas for higher dilutions, antibody detection became inconclusive or negative. Virus titration was performed to verify a stock concentration of 10⁶. ⁵ TCID₅₀/ml. The VNT was time and dose- dependent, with good protection obtained at low serum dilutions and viral titers, with up to 97 protective effects at 1:2–1:8 dilutions against 100–1000 TCID₅₀/ml; however, this protection decreased at higher doses and higher serum dilutions. The results of ELISA and VNT were strongly correlated in the determination at low dilutions, whereas ELISA had decreased sensitivity at high dilutions, at which VNT was still capable of detecting neutralizing activity. Conclusion This combined strategy validates that competitive ELISA is applicable for early and medium antibody detection and VNT can functionally validate immune protection. These results provide evidence for the added value of the combination of these two methods in assessing RVFV vaccine-induced immunity and contribute to the further interpretation of antibody–virus kinetics. Rift Valley fever virus Smithburn vaccine Virus neutralization test Competitive ELISA TCID₅₀ Antibody response Vero cells CPE Immunodiagnostic Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The Rift Valley fever virus (RVFV) is a zoonotic arthropod-borne virus of substantial public health and veterinary importance. It is classified under the genus Phlebovirus of the family Phenuiviridae . It was first isolated during an epidemic in the Rift Valley in Kenya in the early 1930s and subsequently caused recurrent epidemics throughout sub-Saharan Africa, the Arabian Peninsula and the Indian Ocean islands, usually accompanied by high mortality among young livestock and severe disease in humans [ 1 , 2 ]. Aedes and Culex mosquitoes are the primary vectors of the virus, although transmission can also occur among humans upon direct contact with the blood or organs of the infected animals [ 3 ]. In enzootic areas, RVFV is mainly controlled through vector surveillance, early detection and vaccination of susceptible domestic animal species. Nevertheless, the inherent difficulty of evaluating immune response and viral virulence heterogeneity emphasizes the need for integrated diagnostics to assess vaccine effectiveness and antibody efficacy. A live attenuated vaccine (Smithburn strain) from the ancestral RVFV isolate is still in use because it induces strong immunogenic responses and long-lasting protection in animals [ 4 ]. Differences in viremia among vaccinated individuals may arise from variations in vaccine titers (during handling, transportation, or storage) and discrepancies in host antibody responses, which require the development of sensitive in vitro assays to assess the vaccine quality and immunological responses. Three important tools for assessing vaccine-induced immunity have become prominent: virus titration with TCID₅₀, competitive enzyme-linked immunosorbent assays, and virus neutralization tests [ 5 , 6 ]. Virus titration via tissue culture infectious dose 50% TCID₅₀ methodology allows for the quantification of infectious particles in batches of the vaccine. The method of Reed and Muench [ 7 ], a hallmark virological technique, allows for the determination of the dilution at which 50% of the inoculated wells show the effects of virus-induced CPE via this approach in Vero cells. The importance of such validation lies not only in ensuring vaccine consistency but also in determining the accurate concentration for downstream VNT. Accurate virus titration is important because VNT results evaluated via CPE inhibition are very sensitive to viral dose and Ab concentration [ 8 ]. When analyzing humoral immunity after vaccination to monitor antibody responses via ELISA, this is a scalable tool for high-throughput screening based on the criteria of positive, suspect, and negative responses. The competitive ELISA format used in this study was the ID.vet Rift Valley Fever Multispecies kit determines the competition between serum antibodies and a labelled antibody for viral antigen binding. The S/N value increased with increasing antibody concentration and decreased with increasing serial dilution. Competitive ELISAs have demonstrated high-value veterinary diagnostics because they are species-independent and field-friendly [ 9 , 10 ]. Nevertheless, although ELISAs are sensitive to antibody binding, they do not directly measure the neutralizing activity. Virus neutralization test (VNT) is the gold standard for testing protective antibodies. This finding highlights the ability of antisera to neutralize the infectivity of viruses in vitro by inhibiting cytopathic effects (CPE) in cell culture. Here, we determined neutralization over a range of viral concentrations (10 to 100,000 TCID₅₀/ml) and serum dilutions (1:2 to 1:1024) to generate a dose‒effect curve of protection. Vero cell monolayers have been included as a method of infection owing to their high susceptibility to RVFV and their widespread use in virology laboratories [ 11 ]. The sera tested were obtained from vaccinated animals, and those with the highest initial positivity for ELISA were mixed to obtain an immune source with a strong humoral immune response. The compound/virus mixture was serially diluted two-fold and incubated with various virus doses before inoculation into Vero cell monolayers. The cytopathic effects were noted for 96 hours and quantified to determine the percentage of protection using the normal formula, considering the reduction in the CPE compared to the control wells. One such threshold in the VNT was the neutralization titer (NT₅₀) or the dilution where ≥ 50% protection was observed. This is important for benchmarking vaccines and relating ELISA results to functional immunity [ 12 , 13 ]. The combination of these three diagnostic methods (virus titration, ELISA and VNT) provided a complete enumeration of the immune response. In the present study, competitive ELISA revealed high concentrations of antibodies at low dilutions, which decreased at high dilutions, a pattern similar to that normally observed in serum. Virus titration confirmed the potency of the vaccine stock, which was confirmed via VNT, with functional neutralization levels consistent with ELISA results, although extending further in some cases. Crucially, the kinetics of CPE under various antibody-virus combinations reflected the evolving nature of immune-mediated protection, where the greatest inactivation occurred at early times and with low viral titers. This study not only verifies the approach of assessing the RVFV VLP vaccine but also has implications for virological diagnostics in general by illustrating the importance of combining assays based on antigen-binding and function. In the context of newly emerging zoonoses and increased focus on pandemic preparedness, sound testing principles for serological evaluation are becoming increasingly important. Furthermore, knowledge gained from RVFV studies could be applied to other arboviruses transmitted in a similar manner and provoke similar immune responses, such as the West Nile, Zika, and dengue viruses [ 3 , 14 ]. Ultimately, our study aimed to increase the precision of immunological assessments after RVFV vaccination and provide a methodological approach to combine viral quantification, serological detection and functional neutralization testing. This type of analysis is necessary to confirm vaccine effectiveness and to understand the kinetics of antibodies and correlates of protection, especially in low-resource settings where RVFV is endemic. Material and Methods Virus dilution preparation for virus titration: The Rift Valley fever live attenuated vaccine Smithburn strain was obtained from the Rift Valley Fever Control Program, Weqaa Center, in the Jazan region (Batch No 136. Onderstepoort, South Africa). The vaccine was ten-fold serially diluted in RPMI 1640 medium. Serial tenfold dilutions were prepared in nine 1.5 ml tubes (each tube contained 1.8 ml of RPMI media supplemented with 1% antibiotic antimycotic and without serum) to obtain serial tenfold dilutions by adding 0.2 ml of the resolved vaccine to the first tube, and after vortexing, 0.2 ml was transferred to the next tube. Cells and medium: Vero cells (ATCC® CCL-81™) were grown in RPMI 1640 medium in 75 cm2 cell culture flasks (Nest Biotech) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin‒streptomycin (Thermo Scientific) and incubated at 37°C in a 5% CO₂ incubator. RVF virus titration: To ensure titration of the vaccine provided by the manufacturer, local titration was conducted to verify that the titer had not changed due to transportation or storage before it was used in the virus neutralization test (VNT). The viral titer in the vaccine batch was determined by calculating the tissue culture infective dose 50 (TCID50) using the Reed and Muench method (7). Vero cells were trypsinized for the test. Viable cells were counted using a hemocytometer [ 15 ] with 100 µl trypan blue dye (to separate the viable and dead cells) in 100 µl trypsinised cells. They were then suspended at 2 × 10⁴ cells/ml in RPMI + 10% FBS + 2% antibiotic/antimycotic (7 ml/plate; just one plate). Vero cells were added in at 2 × 10⁴ cells in 100 µl of cell suspension per well in a 96-well plate and incubated to 90% confluence, period to be around 24 hours. After aspiration of the medium from the vero cell monolayer and PBS wash, 100 µl of each dilution of virus was applied to six replicate wells (on rows B–G; columns 2–10) in both wells of 96-well plates; the negative control was column 11 (Table 1 ). After 37°C and 5% CO₂ incubation for one hour to allow viral adsorption, a hundred microliters of maintenance medium (RPMI + 2% FBS + 2% antibiotic/antimycotic) was added to each well. Wells containing 200 µl of PBS were used to protect the virus-inoculated wells from oxidation. The plate was subsequently incubated and observed daily under an inverted microscope for cytopathic effects (CPE) characterized by cell rounding, detachment, and clumping. The incubation period of the plate depended on the extent of CPE observed in the cells, with the endpoint defined as > 90% CPE at the highest virus concentration. In our experiment, the incubation period was four days long. The TCID₅₀ was calculated using the method suggested by Reed and Muench [ 7 ] according to the formula TCID50/0.1 ml =10 log total dilution above 50% − (I×log h) . The infection rate was calculated as follows: Infection rate = number of cumulative positive units/(number of cumulative positive units + number of cumulative negative units). The interpolated value of the 50% endpoint (I), also known as the proportional distance (PD) = I = 50% above positive-50/50% above positive-50% below positive. Where, h is the dilution factor. The TCID50 was expressed as TCID50/0.1 ml because each well was inoculated with 0.1 ml of each virus dilution. The reciprocal of the dilution factor is the virus infection titer; if we want to calculate it in milliliters, then TCID50/mL = 10 × 10 log total dilution above 50% - (I×log h). Table 1 Layout of the microtiter plate, indicating distribution of serial dilutions of the test virus (10⁻¹ to 10⁻⁹), PBS, and negative controls (-Ve). 1 2 3 4 5 6 7 8 9 10 11 12 A PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS B PBS 10 − 1 10 − 2 10 − 3 10 − 4 10 − 5 10 − 6 10 − 7 10 − 8 10 − 9 -Ve PBS C PBS 10 − 1 10 − 2 10 − 3 10 − 4 10 − 5 10 − 6 10 − 7 10 − 8 10 − 9 -Ve PBS D PBS 10 − 1 10 − 2 10 − 3 10 − 4 10 − 5 10 − 6 10 − 7 10 − 8 10 − 9 -Ve PBS E PBS 10 − 1 10 − 2 10 − 3 10 − 4 10 − 5 10 − 6 10 − 7 10 − 8 10 − 9 -Ve PBS F PBS 10 − 1 10 − 2 10 − 3 10 − 4 10 − 5 10 − 6 10 − 7 10 − 8 10 − 9 -Ve PBS G PBS 10 − 1 10 − 2 10 − 3 10 − 4 10 − 5 10 − 6 10 − 7 10 − 8 10 − 9 -Ve PBS H PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS Preparation of RVF antibody dilutions for competitive ELISA and VNT: Twenty sheep serum samples were collected one month post-vaccination (with the RVF Smithburn live attenuated vaccine, titer 10 6.5 ), the animals were vaccinated with a final concentration of 10 4.5 after resolving and diluting the vaccine, and screened for the presence of RVF antibodies using the ID Screen Rift Valley Fever Competition Multispecies Kit (ID.vet). Ten strongly positive samples (O.D. 0.05–0.08) in the test were mixed and filtered through a syringe filter (pore size 0.22 µm). The serum was inactivated by heating at 56°C for 1 hour, and 10 serial double-fold dilutions were prepared in RPMI 1640 (with 1% antibiotic antimycotic, without serum) as follows: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024. Fetal bovine serum (FBS) was diluted as a positive control and used as a negative control. Competitive ELISA: To detect antibodies, a Rift Valley Fever Competition Multispecies Kit (Innovative Diagnostic, ID.vet, France) was used, according to the manufacturer’s instructions. Serum dilutions (1:2 to 1:1024) were tested for the presence of antibodies in triplicate to ensure that the results were consistent. Two negative controls, two positive controls, two negative control samples (FBS), and two concentrated positive control samples were added to the plates. For each sample, the competition percentage signal-to-noise ratio (S/N%) was calculated as follows: S/N% = (OD sample/OD negative) × 100. Sample with S/N (%): A value less than or equal to 40% was considered positive. Values greater than 40% and less than or equal to 50% are considered doubtful. A value greater than 50% was considered negative. Preparation of virus dilutions for VNT: To detect serum-specific antibodies against RVFV and measure the ability of antibodies to neutralize viral infectivity, the VNT assay was performed as previously described with slight modifications [ 5 , 16 , 17 ]. After confirming the titration results and the information provided by the manufacturer indicating that the vaccine concentration was 10 ‒6.5 , we applied the dilution formula ( C1 × V1 = C2 × V2 ) to obtain a solution with a concentration of 10 ‒6 . where C1 is the initial concentration (10 –6.5 ), C2 is the final concentration (10 –6 ), V1 is the volume of the stock solution (1 ml), and V2 is the final total volume after dilution. The results showed that to achieve a concentration of 10 ‒6 , the vaccine must be resolved to a final volume of 3.16 ml. To prepare tenfold serial dilutions, in four 1.5 ml tubes (each tube containing 1.8 ml of RPMI media with 1% antibiotic antimycotic and without serum), 0.2 ml of resolved vaccine was added to the first tube, and after vortexing, 0.2 ml was transferred to the next tube (Table 2 ). Table 2 Prepared virus dilutions used in the VNT. Tube No Dilution TCID50\ml Tube 1 10 − 1 100000 TCID50\ml Tube 2 10 − 2 10000 TCID50\ml Tube 3 10 − 3 1000 TCID50\ml Tube 4 10 − 4 100 TCID50\ml Virus neutralization test: We conducted the test using four concentrations of the virus: 10 − 1 , 10 − 2 , 10 − 3 , and 10 − 4 . Each concentration was tested in a 96-well microtiter plate. Each virus concentration was tested with 10 positive serum double-fold dilutions (1:2–1:1024), prepared as described for the preparation of RVF antibody dilutions, and 10 negative serum double-fold dilutions. For each virus concentration, 2500 µl was distributed across 10 positive serum dilutions (1:2–1:1024) and 2500 µl was distributed across 10 negative serum dilutions (1:2–1:1024). In 1.5 ml tubes, 250 µl of each viral concentration was added to 250 µl of each positive and negative serum dilution, resulting in a total volume of 500 µl per virus–serum mixture (400 µl was needed for inoculation). The mixtures were incubated at 37°C for one hour. After the incubation period, 100 µl of each virus-serum mixture was inoculated into a 4-well Vero cells monolayer. Table 3 (Plate 1) illustrates the distribution of the virus-serum mixtures. Each column was inoculated with a different virus-serum mixture. For example, in Plate 1, wells A2 to D2 were inoculated with the virus 10 ‒1 and 1:2 antibody mixture, whereas wells E2 to H2 were inoculated with the virus 10 ‒1 and 1:2 negative antibody serum mixtures. To assess and calculate the difference in the cytopathic effect (CPE) between virus dilutions incubated with antibodies and those mixed with negative control serum, each plate (A2 to D2) and columns 2–11 were inoculated with a virus-positive antibody mixture, whereas E2–H2 and columns 2–11 were inoculated with virus-negative antibody serum mixtures. Each plate included a positive control (virus concentration without serum) in column 1 and a control (cells with medium) in column 12 to assess cell quality. The plates were incubated and observed daily for 4 days under an inverted microscope for CPE. Using the same method as for plate 1, plates 2, 3, 4, and 5 were prepared with the other virus dilution–serum mixtures. Table 3 Plate 1, distribution of virus 10 − 1 -antibody mixtures in rows A (2–11) to D (2–11) and virus 10 − 1 -FBS dilution mixtures in rows E (2‒11) to H (2‒11). Columns 1 and 12: Positive control and negative control Virus dilution RVF antibodies dilutions 1 2 3 4 5 6 7 8 9 10 11 12 + C 1 : 2 1 : 4 1 : 8 1 : 16 1:32 1:64 1:128 1:256 1:512 1:1024 -C A 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 -C B 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 -C C 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 -C D 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 -C Negative FBS + C 1 : 2 1 : 4 1 : 8 1 : 16 1:32 1:64 1:128 1:256 1:512 1:1024 -C E 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 -C F 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 -C G 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 -C H 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 10 − 1 -C Calculation of the Protection Rate: The protection rate (%) was used to quantify how effectively the antibody serum prevented viral CPE in cell culture using the following formula: protection rate % = (1‒ CPE mean of treated/CPE mean of virus control) × 100, where CPE mean of virus control = 100. [ 7 , 11 , 18 ]. If the mean CPE of the control was not 100, we used the actual value in the following formula: protection rate = ((control CPE mean – treated CPE mean)/control CPE mean)) × 100. The neutralization titer (NT) was calculated as follows: A 50% neutralization titer is defined as the reciprocal of the highest dilution of the antibody that induces a 50% reduction in CPE by the virus in vitro [ 12 , 19 – 21 ]. It is often employed in plaque reduction neutralization tests (PRNT) or microneutralization tests to quantify neutralizing antibody titers. Assay Validation and Interpretation: The positive control (virus with no serum) produced 100% CPE and the negative control (cells with no virus) produced 0% CPE at the end of each test; thus, testing the assay negativity could proceed to calculate the endpoint of the titration test. The neutralizing titer can be defined as the reciprocal of the highest dilution that results in ≥ 50% protection (e.g., 1:8 at 100,000 TCID₅₀/ml) [ 22 ] The variation in the number of positive wells and CPE range indicates some experimental variability, which is common in VNTs and supports the need for replicates and statistical confidence in reported titers [ 23 ]. Statistical analysis: Statistical packages for social sciences (SPSS ver. 25) and Microsoft Excel were used for data manipulation, analysis and visualization. The CBE rate (%) and protection rate (%) were calculated using Microsoft Excel. Spearman's correlation was used to examine the correlation between antibody dilution and CPE, and the test was considered significant when the P-value was less than 0.05. Study Results RVF competitive ELISA results: A positive sample was defined as S/N ≤ 40%, doubtful as 41–50%, and negative as > 50 according to the manufacturer’s interpretation criteria. Antibody detection was heavily identified at serum dilutions of 1:2 − 1:32 as represented in table (4), with S/N (3%-23%) representing positivity. The 1:64 dilution resulted in S/N values ranging between 41% and 47% and was categorized as doubtful. From dilutions 1:128 and above, the S/N % values were greater than 50%; hence, they were considered negative. Table 4 Signal-to-noise percentages (S/N%) obtained from three replicate wells at various serum dilutions. Serum Antibodies Dilutions Kit –Ve C Kit + Ve C FBS +Ve C 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 Well 1 OD 1.565 0.049 1.593 0.044 0.054 0.053 0.092 0.145 0.365 0.756 1.204 1.277 1.283 1.177 Well 1S/N 98% 3% 100% 3% 3% 3% 6% 9% 23% 47% 76% 80% 81% 74% Well 2 OD 1.62 0.055 1.594 0.047 0.051 0.054 0.087 0.158 0.362 0.726 1.063 1.015 1.248 1.216 Well 2 S/N 102% 3% 100% 3% 3% 3% 5% 10% 23% 46% 67% 64% 78% 76% Well 3 OD 0.042 0.053 0.101 0.128 0.352 0.652 1.097 1.060 1.241 1.267 Well 3 S/N 3% 3% 6% 8% 22% 41% 69% 67% 78% 80% Mean S/N 100% 3% 100% 3% 3% 3% 6% 9% 23% 45% 71% 70% 79% 77% Results Negative Positive Negative Positive Positive Positive Positive Positive Positive Doubtful Negative Negative Negative Negative The graphical plot (Fig. 1 ) shows the OD values and corresponding S/N ratios, which reflect the level of antibodies that bind to the viral antigen. A gradual increase in both parameters with increasing dilutions indicated a strong antibody response. Observations of CPE On virus titration and VNT plates, cytopathic effects (CPE) were examined at 100× magnification using an inverted microscope at 96 h post incubation. Figure 2 shows the dynamics of CPE development in RVFV-infected Vero cells, from a normal appearance (control) to total monolayer damage. The main findings of each stage are as follows: (A) Normal Vero cells, non-infected culture with a healthy monolayer, flat, elongated, and in close contact. (B) Low-level CPE (loose clusters of mildly rounded cells, sometimes detached) and later sporadic CPE (1–10%). (C) Moderate CPE with increased cell rounding, granulation, and focal monolayer disruption (30–50% CPE). Rounded or detached cells clustered into large foci, predominantly in darker areas (400× magnification). These may be early plaques or CPE foci. (D) Late CPE with scattered anchorage-dependent cells with cytoplasmic condensation and released interstitial-like cells (70% CPE). Note the well-defined empty spaces where cells have been released, and the adherent cells appear disorganized, and frequently clustered. (E) and (F) Destruction of the monolayer with massive cell lysis (90% CPE) or Full CPE 100%, desorption with most cells detached or lysed severe CPE with complete monolayer destruction, there was high virus activity and/or inadequate neutralization by antibody. Virus titration and TCID 50 calculation results A viral titration experiment was conducted to determine the infectious titer of the RVF vaccine. Ten-fold serial dilutions ranging from 10⁻¹ to 10⁻⁹ were applied to Vero cell cultures, with six replicate wells for each dilution. The cytopathic effects (CPE) were monitored daily. After 96 hours of incubation, 100% CPE was observed in the wells from dilutions of 10⁻¹ to 10⁻⁴. Partial CPE appeared at a dilution of 10⁻⁵, and the infection rate decreased markedly at dilutions of 10⁻⁷ or higher. Table (5) presents the percentage of observed CPE, cumulative positive and negative wells, and calculated infection rates across serial dilutions. Table 5 Infection rate (%) across 10-fold dilutions of virus. Dilution Observed CPE Cumulative Infection rate A/(A + B) Total well positive negative Positive (A) Negative (B) Total A + B 10 − 1 6 6 0 36 ↑ 0 ↓ 36 100 10 − 2 6 6 0 30 ↑ 0 ↓ 30 100 10 − 3 6 6 0 24 ↑ 0 ↓ 24 100 10 − 4 6 6 0 18 ↑ 0 ↓ 18 100 10 − 5 6 5 1 12 ↑ 1 ↓ 13 92 10 − 6 6 2 2 7 ↑ 3 ↓ 10 70 10 − 7 6 2 4 3 ↑ 7 ↓ 10 30 10 − 8 6 1 5 1 ↑ 12 ↓ 13 7.7 10 − 9 6 0 6 0 ↑ 18 ↓ 18 00 To estimate the TCID 50 , the Reed and Muench method was used according to the following formula: TCID 50 /ml =10 log total dilution above 50% − (I×log h) . For calculation, the interpolated value of the 50% endpoint (I) and dilution level were as follows: % infection rate above 50% = 70% at 10 − 6 and below 50% =30% at 10 − 7 . Apply the formula: – 6 + (50–70/70–50) × 1= – 6 + (– 20/40) = – 6 + (– 0.5) = – 6.5 Therefore, TCID 50 /ml = 10 –6.5 . Notably, our results were consistent with the titer provided by the vaccine producer, which was 10 –6.5 . Viral neutralization results: A viral neutralization test (VNT) was conducted to evaluate the presence and effectiveness of serum antibodies in preventing virus-induced CPE. The assay used serial viral dilutions (100–100000 TCID50/ml) and serum antibody dilutions (1:2 − 1:1024), which were evaluated after ninety-six hours of incubation in Vero cells. Figures (3) showed the effects of different dilutions of antibodies on CPE caused by virus concentrations over time (24–96 hours post-infection). In general, observations across virus loads : Rapid and widespread CPE development across all antibody dilutions was observed at the highest virus concentration ( 100 , 000 TCID50 /ml), and the CPE was already high, at approximately 45% at a 1:8 dilution, and reached 100% from 1:16 onwards. This finding indicates that only low dilutions and highly concentrated antibodies (1:2 and 1:4, respectively) exhibit high protection rates (90% and 75%, respectively) and can confer protection. At an intermediate viral load ( 10,000 TCID50 /ml) and at 96 h, the CPE ranged from 3% (1:2) to 100% (≥ 1:128), where the antibody effectiveness was better preserved at lower dilutions and CPE progression was slower. Up to a dilution of 1:16, the protection rate was 85%, and because the antibody concentration was insufficient for protection, higher dilutions resulted in substantial CPE. Effective neutralization was demonstrated at low virus concentrations (1000 and 100 TCID 50 /ml), even at dilutions up to 1:256 for longer durations. For example, protection remained above 50% up to a 1:512 dilution at a viral load of 100 TCID 50 /ml for 96 hours. These findings support the idea that a low viral load is more effectively controlled by pre-existing antibodies and aligns with known correlates of protection in an in vivo model. Correlation between antibody titer and protection rate Spearman's correlation test indicated that there was a significant, strong and inverse correlation between antibody dilution and CPE (P < 0.05). The rho coefficients were 0.813, 0.969, 0.994 and 0,079 for 100000 TCID50/ml, 10000 TCID50/ml, 1000 TCID50/ml and 100 TCID50/ml respectively. This result confirms that higher antibody concentrations effectively neutralize the virus. The calculated protection rate at each dilution also decreased significantly with increasing dilution, especially at 96 hours. This reflects the declining efficacy of the antibodies over time or under increased viral replication pressure. Time-dependent effect on CPE : The development of CPE is time-dependent: the median CPE increases from 24 hours to 96 hours under all conditions. The earliest measurable CPE begins at 48 hours in cases of a high viral load and low serum concentration, underscoring the importance of early immunological intervention. Table 6 and the graphical plot (Fig. 4 ) illustrate a decline in protection with increasing dilution and higher viral loads, emphasizing stronger neutralization at lower dilutions and lower viral concentrations. Table 6 Protection rate (%) at 96 h and ELISA results Dilution 100000 TCID 50 /ml 10000 TCID 50 /ml 1000 TCID 50 /ml 100 TCID 50 /ml Elisa results 1:2 90 97 97 96 Positive 1:4 75 94.5 94 96 Positive 1:8 55 90 92 87 Positive 1:16 0 85 90 87 Positive 1:32 0 40 87 87 Positive 1:64 0 15 82 80 Negative 1:128 0 0 69 80 Negative 1:256 0 0 69 80 Negative 1:512 0 0 11 60 Negative 1:1024 0 0 11 27 Negative To support and contextualize our VNT findings according to the references for CPE and neutralization assay standards [ 4 , 8 , 12 , 13 , 24 ], Table 7 showed a comparison of the results with standard or expected outcomes in viral neutralization assays, especially those measuring CPE as an endpoint. Table 8 presents the observations, interpretations, and potential explanations. The comparison results revealed that our VNT findings align very well with standard viral neutralization patterns, the serum contains potent antibodies, especially those against lower viral loads, and the assay functions as expected, showing clear dose-dependent antibody-virus interactions. The expected results of a strong neutralization response were a low CPE at a low antibody dilution (high concentration) and a gradual increase in CPE as the antibody concentration decreased. At high dilutions (1:128, 1:256, etc.), CPE approached 100%, indicating a loss of neutralization. Table 7 Comparison of VNT findings with standard or expected outcomes in viral neutralization assays Criterion Study results Standard Expectation Match? Low CPE at 1:2, 1:4 for low virus titer CPE ~ 3% for 100 & 1000 TCID 50 /ml Yes – strong neutralization yes Rising CPE with dilution CPE rises from 1:2 to 1:512 Expected as antibodies dilute yes High virus titers show 100% CPE even at 1:8–1:16 True for 100,000 TCID 50 /ml Suggests overwhelming infection yes Positive control (no serum) shows 100% CPE All virus doses show 100% except 100TCID 50 ml show 80% Confirms assay integrity yes Table 8 VNT study observations, interpretation, and potential explanations. Observation Interpretation Potential Explanation 10000 TCID 50 /ml still shows neutralization at 1:32 (CPE ~ 60%) Slightly stronger than typical Suggests serum has potent antibodies 1000 TCID 50 /ml shows CPE only ~ 30% up to 1:128 dilution Mild CPE at moderate dilution Indicates good neutralizing titer 100 TCID 50 /ml shows CPE ~ 65% even at 1:1024 Slight CPE at high dilutions is normal Low virus amounts allow longer antibody protection Discussion and Conclusion The antibody response to Rift Valley fever virus (RVFV) was evaluated via viral neutralization tests (VNTs), virus titration and IDvet competitive ELISA. The results also yielded a comprehensive picture of in vitro antibody effectiveness by offering a detailed profile of immunological performance across serum dilutions. In contrast to previous studies [ 4 ], we now focus on the implications of the ELISA values, TCID 50 estimates obtained from virus titration, and neutralization capacity detected by the VNT. ELISA results and trends in S/N ratios ranging from 3–23% were reported as positive, and competitive ELISA results revealed good antibody detection at dilutions ranging from 1:2 to 1:32. At a 1:64 dilution, the S/N ratio was near the questionable range (41%-47%), and a 1:128 dilution or greater was considered negative. These findings are in line with the established threshold guidelines for competitive ELISAs [ 25 ] and are consistent with previous studies that demonstrated the detection of antibodies as early as a 1:32 dilution after RVFV exposure [ 26 , 27 ]. Moreover, the gradual increase in S/N% and OD with further dilution is representative of well-established antibody-binding kinetics: strong binding at high concentrations followed by a decrease in the signal upon further dilution [ 10 ]. Paweska et al . [ 9 ] reported comparable results, supporting the fitness of competitive ELISA for field surveillance and vaccine response assessment. Virus Titration and TCID 50 estimation were performed in Vero cells as previously reported (53). A high-dose vaccine virus stock was denoted by a TCID 50 /ml of 10 − 6.5 , as measured by virus titration by the Reed method, and Muench stands for the percentage cumulative technique (PCT) method and 10-fold serial dilution. Consistent with the expected outcomes for live-attenuated vaccines, the precision of the calculated infectious dose setting was supported by the fact that 100% CPE was apparent at up to 10 –4 dilutions and 50% at a final dilution of approximately 10 –6.5 [ 7 , 28 ]. This is in line with the manufacturer's data and signals the presence of a high-titer reproducible inoculum required for neutralization studies [ 14 ]. VNT Results and Kinetics: The VNT results revealed a clear negative correlation between serum dilution and viral CPE, with substantial protection (> 90%) at dilutions of 1:2 and 1:4 (all tested virus concentrations). As the virus load and dilution increased, protection gradually decreased. This dose-dependent neutralization pattern has also been observed in CPE-based neutralization assays [ 8 , 24 ]. At high viral loads (100,000 TCID50/ml), protection was limited to low dilutions (1:2 − 1:4), whereas at intermediate viral loads (10,000 TCID50/ml), protection was extended to 1:16. Neutralization occurred up to a 1:256 dilution, with protection being the longest-lasting at low virus titers (1000 and 100 TCID50/ml). These results are in agreement with those of Manenti et al . [ 6 ] and Muruato et al . [ 13 ], who reported more durable protection at lower amounts of viral challenge. Cellular changes that occurred during CPE development were observed in a time-dependent manner between 24 and 96 hours. Under high-virus/low-antibody conditions, early signs of infection were evident at 48 h, and by 96 h, most of the high dilution groups presented full CPE. This finding is consistent with previous reports that the level of viral replication is associated with the extent of CPE development over time [ 11 , 29 ] and confirms the replication kinetics of RVFV in Vero cells. Correlation between VNT and ELISA: The strong correlation between VNT neutralization at low dilutions and ELISA positivity indicates that both assays are suitable for the detection of RVFV antibodies in the early stages of the immune response. The sensitivity of ELISA , even at dilutions ≥ 1:64, was inferior to that of VNT, whereas VNT showed detectable neutralizing activity. This difference might be attributable to the principles of the two tests, which, especially at 72- and 96-hours post-infection, might be attributed to the principles of the two tests. While ELISA measures the binding of an antibody, VNT quantifies functional neutralization of the virus [ 30 ]. Suthar et al . [ 12 ] reported that there were discrepancies and revealed that binding assays often underestimate protective immunity. Comparison with Other Studies Our results are also consistent with the neutralization and ELISA benchmark values reported in previous studies [ 3 , 4 , 31 ]. The Reed–Muench method is still a reference technique for TCID 50 estimation and has been widely applied in flavivirus and bunyavirus research. Additionally, the time-resolved protective profile of the VNT and the good precision of the ELISA test at low dilutions indicate that both methods are potentially valuable tools for the evaluation of antibodies. Crucially, our findings are consistent with the concept of ‘dilutional loss of protection’, which has been previously observed in RVFV and related arbovirus challenge models [ 1 , 2 ]. The VNT results provide a reliable measure of functional immunity, especially for low viral loads, despite the decrease in ELISA reactivity at high dilutions. Study Conclusion A comprehensive picture of the immune response to RVFV was obtained by integrating virus titration with VNT assays and ID.vet competitive ELISA. As a reliable seroconversion screening assay, ELISA effectively detected the binding antibodies in early and moderate serum samples. Crucial information on the ability of these antibodies to effectively block viral replication, depending on different virus titers and exposure times, was provided by a virus neutralization test (VNT). Our findings revealed significant intercorrelations between ELISA and VNT results. Although ELISA offers speed and pellucidity, especially for field surveillance, VNT remains the gold standard test for confirming protective immunity. The strong correlation between VNT neutralization titers and ELISA positivity at an early time point after challenge was in line with prior studies of arbovirus exposure, supporting the utility of combined testing strategies for epidemiological immunoprofiles [ 1 , 3 , 12 ]. The implication of this study is that protective antibodies are time-dependent. Neutralization ability and cytopathic effect (CPE) are also very different at 24–96 hours of infection, emphasizing the importance of studying antibodies in a kinetic context. Our results further indicate that the Reed-Muench method is convenient for the accurate determination of TCID 50 and can even be utilized for determining vaccine potency. Overall, this study extends the understanding of the time-dependent association between antibody binding (ELISA) and virus neutralization (VNT) and confirms previous reports on the kinetics of antibody responses to RVFV infection. These insights are relevant for outbreak preparedness, diagnostic algorithms for RVFV and other zoonotic viral pathogens, and vaccine evaluation. The combined testing routinely employed is a good model of how functional testing, within the context of serological antibody testing, can be achieved, and is ultimately required to increase surveillance for human as well as animal zoonotic viruses. Abbreviations RVF: Rift Valley fever virus.; RPMI: Roswell Park Memorial Institute O.D: Optical density; S/N: Signal-to-noise ratio; RNA: Ribonucleic acid RT‒qPCR: reverse transcriptase quantification polymerase chain reaction CPE: Cytopathic effect; TCID₅₀: 50% tissue culture infective dose I: Interpolated value of the 50% endpoint; PD: proportional distance; VNT: virus neutralization test C1: initial concentration; C2: final concentration; V1: volume taken from the stock (initial concentration); V2: final volume; Vero cell: African Green Monkey Kidney Cell Line FBS: fetal bovine serum; PBS: phosphate-buffered saline; PCT: percentage cumulative technique Declarations Acknowledgements This work was supported by the National Center for the Prevention and Control of Plants Pests and Animal Disease (Wegaa Center), Saudi Arabia. We thank the animal herd's owners and the other support staff at the study sites for their cooperation and assistance in vaccination and samples collection . Sincer thanks and appreciation to the Dr. Khalid Suleiman , Weqaa Central Laboratory in Riyadh, has revised and proof-read the manuscript. Special thanks also to Dr. Ahmed Al-Qadam , Wegaa Central Laboratory in Jazan for his technical support in documenting, photographing, and producing the images. Authors’ contributions OD, MH, MN and AH carried out the experimental work and data curation. OD, EN, and SA wrote the manuscript, Validation, and reviewing. TA, statistical analysis and reviewing OD, MM, NA, and AA participated in the design of the study, and editing SA, FA, and FB study supervision and reviewing All authors read and approved the final manuscript . Funding Not applicable . Availability of data and materials The data used to support the findings of this study are available from the Jazan laboratory, Wegaa Center, Saudi Arabia . 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We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7429013","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":526998109,"identity":"22e7fe63-f1da-4a3a-ba10-a766f930c834","order_by":0,"name":"Ommer M. 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06:26:54","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":156041,"visible":true,"origin":"","legend":"","description":"","filename":"7f247b872e2d41a8bacac2b08a9a421f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7429013/v1/c8162a2c4b4c4f9ba14d4ea4.xml"},{"id":93198483,"identity":"5fc67440-de82-492c-b663-f3e49666051d","added_by":"auto","created_at":"2025-10-10 06:26:54","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161429,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7429013/v1/c23dbac64cb908bad3e4049c.html"},{"id":93198468,"identity":"ea39c154-a9a8-43fa-840e-cf7ca7e94a43","added_by":"auto","created_at":"2025-10-10 06:26:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":156352,"visible":true,"origin":"","legend":"\u003cp\u003eELISA optical density values and signal-to-noise ratios at serial serum dilutions (1:2 -1:1024).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7429013/v1/88517e43ea6836f9e0841be6.png"},{"id":93198789,"identity":"68b75f7a-f40f-4db5-9ffb-1b8df382b23c","added_by":"auto","created_at":"2025-10-10 06:34:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1195858,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal stages of CPE development in RVFV-infected Vero cells. \u003cstrong\u003e(A)\u003c/strong\u003e Normal uninfected Vero cells. \u003cstrong\u003e(B)\u003c/strong\u003e Initial low-level CPE: 1–10% CPE. \u003cstrong\u003e(C)\u003c/strong\u003eModerate CPE with enhanced cell rounding of 30–50% CPE. \u003cstrong\u003e(D)\u003c/strong\u003e Advanced CPE with 60–70% CPE. \u003cstrong\u003e(E)\u003c/strong\u003e Destruction of the monolayer with massive cell lysis and 90% CPE. \u003cstrong\u003e(F)\u003c/strong\u003e Full CPE, with most cells detached or lysed (approx. 100% CPE)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7429013/v1/fb23d8c9a20f21cb29a586ac.png"},{"id":93198469,"identity":"14037c9c-c5fd-4b4e-9c24-28f7e8c34bcb","added_by":"auto","created_at":"2025-10-10 06:26:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180370,"visible":true,"origin":"","legend":"\u003cp\u003eVNT results showing CPE inhibition across varying antibody dilutions and virus concentrations over four incubation periods. The figure shows the CPE range, mean percentage, number of positive wells, and calculated protection rates for each condition.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7429013/v1/889f3f2c70e0ec4ca836b3de.png"},{"id":93199759,"identity":"464e3047-3962-4a2e-9c54-614d9f4fc8d0","added_by":"auto","created_at":"2025-10-10 06:42:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80007,"visible":true,"origin":"","legend":"\u003cp\u003eProtection rate (%) against different serum antibody dilutions at 96 hours post-infection for various virus concentrations.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7429013/v1/55b2c727b050b7b4c2938779.png"},{"id":93660226,"identity":"b9ea9c77-8dcb-4cb9-842d-ee6b8fc60414","added_by":"auto","created_at":"2025-10-16 08:02:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3346728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7429013/v1/b3d30e7b-34e8-46e5-967e-9b5fcf14e0ce.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Southwest Saudi Arabia","fulltext":[{"header":"Background","content":"\u003cp\u003eThe Rift Valley fever virus (RVFV) is a zoonotic arthropod-borne virus of substantial public health and veterinary importance. It is classified under the genus \u003cem\u003ePhlebovirus\u003c/em\u003e of the family \u003cem\u003ePhenuiviridae\u003c/em\u003e. It was first isolated during an epidemic in the Rift Valley in Kenya in the early 1930s and subsequently caused recurrent epidemics throughout sub-Saharan Africa, the Arabian Peninsula and the\u0026ensp;Indian Ocean islands, usually accompanied by high mortality among young livestock and severe disease in humans [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Aedes and Culex mosquitoes are the primary vectors of\u0026ensp;the virus, although transmission can also occur among humans upon direct contact with the blood or organs of the infected animals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In enzootic areas,\u0026ensp;RVFV is mainly controlled through vector surveillance, early detection and vaccination of susceptible domestic animal species. Nevertheless, the inherent difficulty of evaluating immune response and viral virulence heterogeneity emphasizes the need for integrated diagnostics to assess vaccine\u0026ensp;effectiveness and antibody efficacy.\u003c/p\u003e\u003cp\u003eA live attenuated vaccine (Smithburn strain) from the ancestral RVFV isolate is still in use because it induces strong immunogenic responses\u0026ensp;and long-lasting protection in animals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Differences in viremia among vaccinated individuals may arise from variations in\u0026ensp;vaccine titers (during handling, transportation, or storage) and discrepancies in host antibody responses, which require the development of sensitive in vitro assays to assess the vaccine quality and immunological responses. Three important tools for assessing vaccine-induced immunity have become prominent: virus titration with TCID₅₀, competitive enzyme-linked immunosorbent assays, and virus neutralization tests [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Virus titration via tissue culture infectious dose 50% TCID₅₀ methodology allows for the quantification of infectious particles in batches of the vaccine. The method of Reed and Muench [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], a hallmark virological technique, allows for the determination of the dilution at which 50% of the inoculated wells show the effects of virus-induced CPE via this approach in Vero cells. The importance of such validation lies not only in ensuring vaccine consistency but also in determining the accurate concentration for downstream VNT. Accurate virus titration is important because VNT results evaluated via CPE inhibition are very sensitive\u0026ensp;to viral dose and Ab concentration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhen analyzing humoral immunity after vaccination to monitor antibody responses via ELISA, this is a scalable tool for high-throughput screening based on the criteria of positive, suspect, and negative responses. The competitive ELISA format used in this study\u0026ensp;was the ID.vet Rift Valley Fever Multispecies kit determines the competition between serum antibodies and a labelled antibody for viral antigen binding. The\u0026ensp;S/N value increased with increasing antibody concentration and decreased with increasing serial dilution. Competitive ELISAs have demonstrated high-value veterinary diagnostics because they are species-independent and field-friendly [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNevertheless, although ELISAs are sensitive to antibody binding, they do not directly measure the neutralizing activity. Virus neutralization test (VNT)\u0026ensp;is the gold standard for testing protective antibodies. This finding highlights\u0026ensp;the ability of antisera to neutralize the infectivity of viruses in vitro by inhibiting cytopathic effects (CPE) in cell culture. Here, we determined neutralization over a range of viral concentrations (10 to 100,000 TCID₅₀/ml) and\u0026ensp;serum dilutions (1:2 to 1:1024) to generate a dose‒effect curve of protection. Vero cell monolayers have been included as a method of infection owing to their high susceptibility to RVFV and their widespread use in virology laboratories [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe sera tested were obtained from vaccinated animals, and those with the highest initial positivity for ELISA were\u0026ensp;mixed to obtain an immune source with a strong humoral immune response. The compound/virus mixture was serially\u0026ensp;diluted two-fold and incubated with various virus doses before inoculation into Vero cell monolayers. The cytopathic effects were\u0026ensp;noted for 96 hours and quantified to determine the percentage of protection using the normal formula, considering the reduction in the CPE compared to the control wells. One such threshold in the VNT was\u0026ensp;the neutralization titer (NT₅₀) or the dilution where \u0026ge;\u0026thinsp;50% protection was observed. This is\u0026ensp;important for benchmarking vaccines and relating ELISA results to functional immunity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe combination of these three diagnostic methods\u0026ensp;(virus titration, ELISA and VNT) provided a complete enumeration of the immune response. In the present study, competitive ELISA revealed high concentrations\u0026ensp;of antibodies at low dilutions, which decreased at high dilutions, a pattern similar to that normally observed in serum. Virus titration confirmed the potency of\u0026ensp;the vaccine stock, which was confirmed via VNT, with functional neutralization levels consistent with ELISA results, although extending further in some cases. Crucially, the kinetics of CPE under various antibody-virus combinations reflected the evolving nature of immune-mediated protection, where the greatest inactivation occurred at early times\u0026ensp;and with low viral titers.\u003c/p\u003e\u003cp\u003eThis study not only verifies the approach of assessing the RVFV VLP vaccine but also has implications for virological diagnostics in general by illustrating the importance of\u0026ensp;combining assays based on antigen-binding and function. In the context\u0026ensp;of newly emerging zoonoses and increased focus on pandemic preparedness, sound testing principles for serological evaluation are becoming increasingly important. Furthermore, knowledge gained from RVFV studies could be applied to other arboviruses transmitted in a similar manner and provoke similar immune responses, such as the West Nile, Zika, and dengue viruses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUltimately, our study aimed to increase the precision of\u0026ensp;immunological assessments after RVFV vaccination and provide a methodological approach to combine viral quantification, serological detection and functional neutralization testing. This type of analysis is necessary to confirm vaccine effectiveness and\u0026ensp;to understand the kinetics of antibodies and correlates of protection, especially in low-resource settings where RVFV is endemic.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eVirus dilution preparation for virus titration:\u003c/h2\u003e\u003cp\u003eThe Rift Valley fever live attenuated vaccine Smithburn strain was obtained from the Rift Valley Fever Control Program, Weqaa Center, in the Jazan region (Batch No 136. Onderstepoort, South Africa). The vaccine was ten-fold serially diluted in RPMI 1640 medium. Serial tenfold dilutions were prepared in nine 1.5 ml tubes (each tube contained 1.8 ml of RPMI media supplemented with 1% antibiotic antimycotic and without serum) to obtain serial tenfold dilutions by adding 0.2 ml of the resolved vaccine to the first tube, and after vortexing, 0.2 ml was transferred to the next tube.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCells and medium:\u003c/h3\u003e\n\u003cp\u003eVero cells (ATCC\u0026reg; CCL-81\u0026trade;) were grown in RPMI 1640 medium in 75 cm2 cell culture flasks (Nest Biotech) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin‒streptomycin (Thermo Scientific) and incubated at 37\u0026deg;C in a 5% CO₂ incubator.\u003c/p\u003e\n\u003ch3\u003eRVF virus titration:\u003c/h3\u003e\n\u003cp\u003eTo ensure titration of the vaccine provided by the manufacturer, local titration was conducted to verify that the titer had not changed due to transportation or storage before it was used in the virus neutralization test (VNT).\u003c/p\u003e\u003cp\u003eThe viral titer in the vaccine batch was determined by calculating the tissue culture infective dose 50 (TCID50) using the Reed and Muench method (7).\u003c/p\u003e\u003cp\u003eVero cells were trypsinized for the test. Viable cells were counted using a hemocytometer [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] with 100 \u0026micro;l trypan blue dye (to separate the viable and dead cells) in 100 \u0026micro;l trypsinised cells. They were then suspended at 2 \u0026times; 10⁴ cells/ml in RPMI\u0026thinsp;+\u0026thinsp;10% FBS\u0026thinsp;+\u0026thinsp;2% antibiotic/antimycotic (7 ml/plate; just one plate). Vero cells were added in at 2 \u0026times; 10⁴ cells in 100 \u0026micro;l of cell suspension per well in a 96-well plate and incubated to 90% confluence, period to be around 24 hours. After aspiration of the medium from the vero cell monolayer and PBS wash, 100 \u0026micro;l of each dilution of virus was applied to six replicate wells (on rows B\u0026ndash;G; columns 2\u0026ndash;10) in both wells of 96-well plates; the negative control was column 11 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After 37\u0026deg;C and 5% CO₂ incubation for one hour to allow viral adsorption, a hundred microliters of maintenance medium (RPMI\u0026thinsp;+\u0026thinsp;2% FBS\u0026thinsp;+\u0026thinsp;2% antibiotic/antimycotic) was added to each well. Wells containing 200 \u0026micro;l of PBS were used to protect the virus-inoculated wells from oxidation. The plate was subsequently incubated and observed daily under an inverted microscope for cytopathic effects (CPE) characterized by cell rounding, detachment, and clumping. The incubation period of the plate depended on the extent of CPE observed in the cells, with the endpoint defined as \u0026gt;\u0026thinsp;90% CPE at the highest virus concentration. In our experiment, the incubation period was four days long. The TCID₅₀ was calculated using the method suggested by Reed and Muench [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] according to the formula TCID50/0.1 ml =10 \u003csup\u003elog total dilution above 50% \u0026minus; (I\u0026times;log h)\u003c/sup\u003e. The infection rate was calculated as follows:\u003c/p\u003e\u003cp\u003eInfection rate\u0026thinsp;=\u0026thinsp;number of cumulative positive units/(number of cumulative positive units\u0026thinsp;+\u0026thinsp;number of cumulative negative units). The interpolated value of the 50% endpoint (I), also known as the proportional distance (PD)\u0026thinsp;=\u0026thinsp;I\u0026thinsp;=\u0026thinsp;50% above positive-50/50% above positive-50% below positive. Where, h is the dilution factor. The TCID50 was expressed as TCID50/0.1 ml because each well was inoculated with 0.1 ml of each virus dilution. The reciprocal of the dilution factor is the virus infection titer; if we want to calculate it in milliliters, then TCID50/mL\u0026thinsp;=\u0026thinsp;10 \u0026times; 10 log total dilution above 50% - (I\u0026times;log h).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLayout of the microtiter plate, indicating distribution of serial dilutions of the test virus (10⁻\u0026sup1; to 10⁻⁹), PBS, and negative controls (-Ve).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"13\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" 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colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u0026thinsp;\u0026minus;\u0026thinsp;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c6\"\u003e\u003cp\u003e10\u0026thinsp;\u0026minus;\u0026thinsp;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-Ve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u0026thinsp;\u0026minus;\u0026thinsp;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-Ve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u0026thinsp;\u0026minus;\u0026thinsp;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-Ve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u0026thinsp;\u0026minus;\u0026thinsp;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-Ve\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003ePBS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003ePreparation of RVF antibody dilutions for competitive ELISA and VNT:\u003c/h3\u003e\n\u003cp\u003eTwenty sheep serum samples were collected one month post-vaccination (with the RVF Smithburn live attenuated vaccine, titer 10\u003csup\u003e6.5\u003c/sup\u003e), the animals were vaccinated with a final concentration of 10\u003csup\u003e4.5\u003c/sup\u003e after resolving and diluting the vaccine, and screened for the presence of RVF antibodies using the ID Screen Rift Valley Fever Competition Multispecies Kit (ID.vet). Ten strongly positive samples (O.D. 0.05\u0026ndash;0.08) in the test were mixed and filtered through a syringe filter (pore size 0.22 \u0026micro;m). The serum was inactivated by heating at 56\u0026deg;C for 1 hour, and 10 serial double-fold dilutions were prepared in RPMI 1640 (with 1% antibiotic antimycotic, without serum) as follows: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024.\u003c/p\u003e\u003cp\u003eFetal bovine serum (FBS) was diluted as a positive control and used as a negative control.\u003c/p\u003e\n\u003ch3\u003eCompetitive ELISA:\u003c/h3\u003e\n\u003cp\u003eTo detect antibodies, a Rift Valley Fever Competition Multispecies Kit (Innovative Diagnostic, ID.vet, France) was used, according to the manufacturer\u0026rsquo;s instructions. Serum dilutions\u003c/p\u003e\u003cp\u003e(1:2 to 1:1024) were tested for the presence of antibodies in triplicate to ensure that the results were consistent. Two negative controls, two positive controls, two negative control samples (FBS), and two concentrated positive control samples were added to the plates. For each sample, the competition percentage signal-to-noise ratio (S/N%) was calculated as follows: S/N% = (OD sample/OD negative) \u0026times; 100.\u003c/p\u003e\u003cp\u003eSample with S/N (%):\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eA value less than or equal to 40% was considered positive.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eValues greater than 40% and less than or equal to 50% are considered doubtful.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eA value greater than 50% was considered negative.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of virus dilutions for VNT:\u003c/h2\u003e\u003cp\u003eTo detect serum-specific antibodies against RVFV and measure the ability of antibodies to neutralize viral infectivity, the VNT assay was performed as previously described with slight modifications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAfter confirming the titration results and the information provided by the manufacturer indicating that the vaccine concentration was 10\u003csup\u003e‒6.5\u003c/sup\u003e, we applied the dilution formula (\u003cem\u003eC1 \u0026times; V1\u0026thinsp;=\u0026thinsp;C2 \u0026times; V2\u003c/em\u003e) to obtain a solution with a concentration of 10\u003csup\u003e‒6\u003c/sup\u003e. where C1 is the initial concentration (10\u003csup\u003e\u0026ndash;6.5\u003c/sup\u003e), C2 is the final concentration (10\u003csup\u003e\u0026ndash;6\u003c/sup\u003e), V1 is the volume of the stock solution (1 ml), and V2 is the final total volume after dilution. The results showed that to achieve a concentration of 10\u003csup\u003e‒6\u003c/sup\u003e, the vaccine must be resolved to a final volume of 3.16 ml. To prepare tenfold serial dilutions, in four 1.5 ml tubes (each tube containing 1.8 ml of RPMI media with 1% antibiotic antimycotic and without serum), 0.2 ml of resolved vaccine was added to the first tube, and after vortexing, 0.2 ml was transferred to the next tube (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrepared virus dilutions used in the VNT.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTube No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDilution\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTCID50\\ml\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTube 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100000 TCID50\\ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTube 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10000 TCID50\\ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTube 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1000 TCID50\\ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTube 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u0026thinsp;\u0026minus;\u0026thinsp;\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100 TCID50\\ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eVirus neutralization test:\u003c/h3\u003e\n\u003cp\u003eWe conducted the test using four concentrations of the virus: 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e. Each concentration was tested in a 96-well microtiter plate.\u003c/p\u003e\u003cp\u003eEach virus concentration was tested with 10 positive serum double-fold dilutions (1:2\u0026ndash;1:1024), prepared as described for the preparation of RVF antibody dilutions, and 10 negative serum double-fold dilutions.\u003c/p\u003e\u003cp\u003eFor each virus concentration, 2500 \u0026micro;l was distributed across 10 positive serum dilutions (1:2\u0026ndash;1:1024) and 2500 \u0026micro;l was distributed across 10 negative serum dilutions (1:2\u0026ndash;1:1024). In 1.5 ml tubes, 250 \u0026micro;l of each viral concentration was added to 250 \u0026micro;l of each positive and negative serum dilution, resulting in a total volume of 500 \u0026micro;l per virus\u0026ndash;serum mixture (400 \u0026micro;l was needed for inoculation). The mixtures were incubated at 37\u0026deg;C for one hour. After the incubation period, 100 \u0026micro;l of each virus-serum mixture was inoculated into a 4-well Vero cells monolayer. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (Plate 1) illustrates the distribution of the virus-serum mixtures. Each column was inoculated with a different virus-serum mixture. For example, in Plate 1, wells A2 to D2 were inoculated with the virus 10\u003csup\u003e‒1\u003c/sup\u003e and 1:2 antibody mixture, whereas wells E2 to H2 were inoculated with the virus 10\u003csup\u003e‒1\u003c/sup\u003e and 1:2 negative antibody serum mixtures. To assess and calculate the difference in the cytopathic effect (CPE) between virus dilutions incubated with antibodies and those mixed with negative control serum, each plate (A2 to D2) and columns 2\u0026ndash;11 were inoculated with a virus-positive antibody mixture, whereas E2\u0026ndash;H2 and columns 2\u0026ndash;11 were inoculated with virus-negative antibody serum mixtures. Each plate included a positive control (virus concentration without serum) in column 1 and a control (cells with medium) in column 12 to assess cell quality.\u003c/p\u003e\u003cp\u003eThe plates were incubated and observed daily for 4 days under an inverted microscope for CPE.\u003c/p\u003e\u003cp\u003eUsing the same method as for plate 1, plates 2, 3, 4, and 5 were prepared with the other virus dilution\u0026ndash;serum mixtures.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePlate 1, distribution of virus 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e-antibody mixtures in rows A (2\u0026ndash;11) to D (2\u0026ndash;11) and virus 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e-FBS dilution mixtures in rows E (2‒11) to H (2‒11). Columns 1 and 12: Positive control and negative control\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eVirus dilution\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"12\" nameend=\"c14\" namest=\"c3\"\u003e\u003cp\u003eRVF antibodies dilutions\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+ C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 : 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1 : 4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1 : 8\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1 : 16\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1:32\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1:64\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1:128\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1:256\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1:512\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1:1024\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eNegative FBS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+ C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 : 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1 : 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1 : 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1 : 16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1:32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1:64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1:128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1:256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1:512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1:1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eCalculation of the Protection Rate:\u003c/h3\u003e\n\u003cp\u003eThe protection rate (%) was used to quantify how effectively the antibody serum prevented viral CPE in cell culture using the following formula: protection rate % = (1‒ CPE mean of treated/CPE mean of virus control) \u0026times; 100, where CPE mean of virus control\u0026thinsp;=\u0026thinsp;100. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. If the mean CPE of the control was not 100, we used the actual value in the following formula: protection rate = ((control CPE mean \u0026ndash; treated CPE mean)/control CPE mean)) \u0026times; 100.\u003c/p\u003e\u003cp\u003eThe neutralization titer (NT) was calculated as follows:\u003c/p\u003e\u003cp\u003eA 50% neutralization titer is defined as the reciprocal of the\u0026ensp;highest dilution of the antibody that induces a 50% reduction in CPE by the virus in vitro [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It is often employed in \u0026ensp;plaque reduction neutralization tests (PRNT) or microneutralization tests to quantify neutralizing antibody titers.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAssay Validation and Interpretation:\u003c/h2\u003e\u003cp\u003eThe positive control (virus with no serum) produced 100% CPE and the negative control (cells with no virus) produced 0% CPE at the end of each test; thus, testing the assay negativity could proceed to calculate the endpoint of\u0026ensp;the titration test.\u003c/p\u003e\u003cp\u003eThe neutralizing titer can be defined as the reciprocal of the highest dilution that results in \u0026ge;\u0026thinsp;50% protection (e.g., 1:8 at 100,000 TCID₅₀/ml) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe variation in the number of positive wells and CPE range indicates some experimental variability, which is common in VNTs and supports the need for replicates and statistical confidence in reported titers [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis:\u003c/h2\u003e\u003cp\u003eStatistical packages for social sciences (SPSS ver. 25) and Microsoft Excel were used for data manipulation, analysis and visualization.\u003c/p\u003e\u003cp\u003eThe CBE rate (%) and protection rate (%) were calculated using Microsoft Excel. Spearman's correlation was used to examine the correlation between antibody dilution and CPE, and the test was considered significant when the \u003cem\u003eP-value\u003c/em\u003e was less than 0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Study Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003cp\u003e\u003cb\u003eRVF competitive ELISA results: A\u003c/b\u003e positive sample was defined as S/N\u0026thinsp;\u0026le;\u0026thinsp;40%, doubtful as 41\u0026ndash;50%, and\u0026ensp;negative as \u0026gt;\u0026thinsp;50 according to the manufacturer\u0026rsquo;s interpretation criteria. Antibody detection was heavily identified at serum dilutions of 1:2\u0026thinsp;\u0026minus;\u0026thinsp;1:32 as represented in table (4), with S/N\u0026ensp;(3%-23%) representing positivity. The 1:64 dilution resulted in\u0026ensp;S/N values ranging between 41% and 47% and was categorized as doubtful. From dilutions 1:128 and above, the S/N % values were\u0026ensp;greater than 50%; hence, they were considered negative.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSignal-to-noise percentages (S/N%) obtained from three replicate wells at various serum dilutions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"15\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"15\" nameend=\"c15\" namest=\"c1\"\u003e\u003cp\u003eSerum Antibodies Dilutions\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eKit \u0026ndash;Ve C\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eKit\u0026thinsp;+\u0026thinsp;Ve C\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eFBS\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+Ve C\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e1:2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e1:4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e1:8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e1:16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e1:32\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cb\u003e1:64\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e1:128\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cb\u003e1:256\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u003cb\u003e1:512\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e\u003cb\u003e1:1024\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWell 1 OD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.565\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.049\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.593\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.044\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.053\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.092\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.365\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.756\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.204\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.277\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e1.283\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e1.177\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWell 1S/N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e98%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e23%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e47%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e76%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e80%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e81%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e74%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWell 2 OD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.594\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.051\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.087\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.158\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.362\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.726\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.063\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e1.248\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e1.216\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWell 2 S/N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e102%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e23%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e46%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e64%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e78%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e76%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWell 3 OD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.042\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.053\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.352\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.652\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.060\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e1.241\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e1.267\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWell 3 S/N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e8%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e22%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e41%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e69%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e78%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e80%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMean S/N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e23%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e45%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e71%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e70%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e79%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e77%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResults\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eDoubtful\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe graphical plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) shows the OD values and corresponding S/N ratios, which reflect the level of antibodies that bind to the viral antigen. A gradual increase in both parameters with increasing dilutions indicated a strong antibody response.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eObservations of CPE\u003c/strong\u003e\u003cp\u003eOn virus titration and VNT plates, cytopathic\u0026ensp;effects (CPE) were examined at 100\u0026times; magnification using an inverted microscope at 96 h post incubation.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the dynamics of\u0026ensp;CPE development in RVFV-infected Vero cells, from a normal appearance (control) to total monolayer damage. The main findings\u0026ensp;of each stage are as follows:\u003c/p\u003e\u003cp\u003e(A) Normal Vero\u0026ensp;cells, non-infected culture with a healthy monolayer, flat, elongated, and in close contact. (B) Low-level CPE (loose\u0026ensp;clusters of mildly rounded cells, sometimes detached) and later sporadic CPE (1\u0026ndash;10%). (C) Moderate CPE with increased cell rounding, granulation, and focal monolayer disruption (30\u0026ndash;50% CPE). Rounded or detached cells clustered into large foci,\u0026ensp;predominantly in darker areas (400\u0026times; magnification). These may be early plaques\u0026ensp;or CPE foci. (D) Late\u0026ensp;CPE with scattered anchorage-dependent cells with cytoplasmic condensation and released interstitial-like cells (70% CPE). Note the well-defined empty spaces where cells have been released, and the adherent cells appear disorganized, and frequently clustered. (E) and (F) Destruction of the monolayer with\u0026ensp;massive cell lysis (90% CPE) or Full CPE 100%, desorption with most cells detached or lysed severe CPE with complete monolayer destruction, there was high virus activity and/or inadequate neutralization by antibody.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eVirus titration and TCID\u003csub\u003e50\u003c/sub\u003e calculation results\u003c/strong\u003e\u003cp\u003eA viral titration experiment was conducted to determine the infectious titer of the RVF vaccine. Ten-fold serial dilutions ranging from 10⁻\u0026sup1; to 10⁻⁹ were applied to Vero cell cultures, with six replicate wells for each dilution. The cytopathic effects (CPE) were monitored daily. After 96 hours of incubation, 100% CPE was observed in the wells from dilutions of 10⁻\u0026sup1; to 10⁻⁴. Partial CPE appeared at a dilution of 10⁻⁵, and the infection rate decreased markedly at dilutions of 10⁻⁷ or higher. Table\u0026nbsp;(5) presents the percentage of observed CPE, cumulative positive and negative wells, and calculated infection rates across serial dilutions.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInfection rate (%) across 10-fold dilutions of virus.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDilution\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eObserved CPE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eCumulative\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eInfection rate A/(A\u0026thinsp;+\u0026thinsp;B)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal well\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003epositive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003enegative\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePositive (A)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNegative (B)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTotal A\u0026thinsp;+\u0026thinsp;B\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36 \u0026uarr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 \u0026darr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30 \u0026uarr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 \u0026darr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24 \u0026uarr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 \u0026darr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18 \u0026uarr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 \u0026darr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12 \u0026uarr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1 \u0026darr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e92\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7 \u0026uarr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3 \u0026darr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3 \u0026uarr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7 \u0026darr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1 \u0026uarr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12 \u0026darr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0 \u0026uarr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18 \u0026darr;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eTo estimate the TCID\u003csub\u003e50\u003c/sub\u003e, the Reed and Muench method was used according to the following formula:\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTCID\u003csub\u003e50\u003c/sub\u003e/ml =10 \u003csup\u003elog total dilution above 50% \u0026minus; (I\u0026times;log h)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor calculation, the interpolated value of the 50% endpoint (I) and dilution level were as follows: % infection rate above 50% = 70% at 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e and below 50% =30% at 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eApply the formula: \u0026ndash; 6 + (50\u0026ndash;70/70\u0026ndash;50) \u003csub\u003e\u0026times;\u003c/sub\u003e 1= \u0026ndash; 6 + (\u0026ndash; 20/40) = \u0026ndash; 6 + (\u0026ndash; 0.5) = \u0026ndash; 6.5\u003c/p\u003e\u003cp\u003eTherefore, TCID\u003csub\u003e50\u003c/sub\u003e/ml\u0026thinsp;=\u0026thinsp;10\u003csup\u003e\u0026ndash;6.5\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNotably, our results were consistent with the titer provided by the vaccine producer, which was 10\u003csup\u003e\u0026ndash;6.5\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eViral neutralization results:\u003c/h2\u003e\u003cp\u003eA viral neutralization test (VNT) was conducted to evaluate the presence and effectiveness of serum antibodies in preventing virus-induced CPE. The assay used serial viral dilutions (100\u0026ndash;100000 TCID50/ml) and serum antibody dilutions (1:2\u0026thinsp;\u0026minus;\u0026thinsp;1:1024), which were evaluated after ninety-six hours of incubation in Vero cells. Figures\u0026nbsp;(3) showed the effects of different dilutions of antibodies on CPE caused by virus concentrations over time (24\u0026ndash;96 hours post-infection).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn general, observations across virus loads\u003c/b\u003e: Rapid and widespread CPE development across all antibody dilutions was observed at the highest virus concentration (\u003csub\u003e100\u003c/sub\u003e,\u003csub\u003e000 TCID50\u003c/sub\u003e/ml), and the CPE was already high, at approximately 45% at a 1:8 dilution, and reached 100% from 1:16 onwards. This finding indicates that only low dilutions and highly concentrated antibodies (1:2 and 1:4, respectively) exhibit high protection rates (90% and 75%, respectively) and can confer protection.\u003c/p\u003e\u003cp\u003eAt an intermediate viral load (\u003csub\u003e10,000 TCID50\u003c/sub\u003e/ml) and at 96 h, the CPE ranged from 3% (1:2) to 100% (\u0026ge;\u0026thinsp;1:128), where the antibody effectiveness was better preserved at lower dilutions and CPE progression was slower. Up to a dilution of 1:16, the protection rate was 85%, and because the antibody concentration was insufficient for protection, higher dilutions resulted in substantial CPE.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEffective neutralization was demonstrated at low virus concentrations (1000 and 100 TCID\u003csub\u003e50\u003c/sub\u003e/ml), even at dilutions up to 1:256 for longer durations. For example, protection remained above 50% up to a 1:512 dilution at a viral load of 100 TCID\u003csub\u003e50\u003c/sub\u003e/ml for 96 hours. These findings support the idea that a low viral load is\u003c/p\u003e\u003cp\u003emore effectively controlled by pre-existing antibodies and aligns with known correlates of protection in an in vivo model.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCorrelation between antibody titer and protection rate\u003c/strong\u003e\u003cp\u003eSpearman's correlation test indicated that there was a significant, strong and inverse correlation between antibody dilution and CPE (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The rho coefficients were 0.813, 0.969, 0.994 and 0,079 for 100000 TCID50/ml, 10000 TCID50/ml, 1000 TCID50/ml and 100 TCID50/ml respectively. This result confirms that higher antibody concentrations effectively neutralize the virus. The calculated protection rate at each dilution also decreased significantly with increasing dilution, especially at 96 hours. This reflects the declining efficacy of the antibodies over time or under increased viral replication pressure.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTime-dependent effect on CPE\u003c/b\u003e: The development of CPE is time-dependent: the median CPE increases from 24 hours to 96 hours under all conditions. The earliest measurable CPE begins at 48 hours in cases of a high viral load and low serum concentration, underscoring the importance of early immunological intervention.\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and the graphical plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) illustrate a decline in protection with increasing dilution and higher viral loads, emphasizing stronger neutralization at lower dilutions and lower viral concentrations.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eProtection rate (%) at 96 h and ELISA results\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDilution\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100000 TCID\u003csub\u003e50\u003c/sub\u003e/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10000 TCID\u003csub\u003e50\u003c/sub\u003e/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1000 TCID\u003csub\u003e50\u003c/sub\u003e/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100 TCID\u003csub\u003e50\u003c/sub\u003e/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eElisa results\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e94.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo support and contextualize our VNT findings according to the references for CPE and neutralization assay standards [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e showed a comparison of the results with standard or expected outcomes in viral neutralization assays, especially those measuring CPE as an endpoint. Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e presents the observations, interpretations, and potential explanations. The comparison results revealed that our VNT findings align very well with standard viral neutralization patterns, the serum contains potent antibodies, especially those against lower viral loads, and the assay functions as expected, showing clear dose-dependent antibody-virus interactions.\u003c/p\u003e\u003cp\u003eThe expected results of a strong neutralization response were a low CPE at a low antibody dilution (high concentration) and a gradual increase in CPE as the antibody concentration decreased. At high dilutions (1:128, 1:256, etc.), CPE approached 100%, indicating a loss of neutralization.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of VNT findings with standard or expected outcomes in viral neutralization assays\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCriterion\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStudy results\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStandard Expectation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMatch?\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow CPE at 1:2, 1:4 for low virus titer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCPE\u0026thinsp;~\u0026thinsp;3% for 100 \u0026amp; 1000 TCID\u003csub\u003e50\u003c/sub\u003e/ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYes \u0026ndash; strong neutralization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eyes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRising CPE with dilution\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCPE rises from 1:2 to 1:512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eExpected as antibodies dilute\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eyes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh virus titers show 100% CPE even at 1:8\u0026ndash;1:16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTrue for 100,000 TCID\u003csub\u003e50\u003c/sub\u003e/ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSuggests overwhelming infection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eyes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive control (no serum) shows 100% CPE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAll virus doses show 100%\u003c/p\u003e\u003cp\u003eexcept 100TCID\u003csub\u003e50\u003c/sub\u003eml show 80%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConfirms assay integrity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eyes\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eVNT study observations, interpretation, and potential explanations.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eObservation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInterpretation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePotential Explanation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10000 TCID\u003csub\u003e50\u003c/sub\u003e/ml still shows neutralization at 1:32 (CPE\u0026thinsp;~\u0026thinsp;60%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSlightly stronger than typical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSuggests serum has potent antibodies\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1000 TCID\u003csub\u003e50\u003c/sub\u003e/ml shows CPE only\u0026thinsp;~\u0026thinsp;30% up to 1:128 dilution\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMild CPE at moderate dilution\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIndicates good neutralizing titer\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 TCID\u003csub\u003e50\u003c/sub\u003e/ml shows CPE\u0026thinsp;~\u0026thinsp;65% even at 1:1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSlight CPE at high dilutions is normal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLow virus amounts allow longer antibody protection\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion and Conclusion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003cp\u003eThe antibody response to Rift Valley fever virus (RVFV) was evaluated via viral neutralization tests (VNTs),\u0026ensp; virus titration and IDvet competitive ELISA. The results also\u0026ensp; yielded a comprehensive picture of in vitro antibody effectiveness by offering a detailed profile of immunological performance across serum dilutions. In contrast to previous studies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], we\u0026ensp; now focus on the implications of the ELISA values, TCID\u003csub\u003e50\u003c/sub\u003e estimates obtained from virus titration, and neutralization capacity detected by the VNT. ELISA results\u0026ensp; and trends in S/N ratios ranging from 3\u0026ndash;23% were reported as positive, and competitive ELISA results revealed good antibody detection at dilutions ranging from\u0026ensp; 1:2 to 1:32. At a 1:64 dilution, the S/N ratio was near\u0026ensp;the questionable range (41%-47%), and a 1:128 dilution or greater was considered negative. These\u0026ensp; findings are in line with the established threshold guidelines for competitive ELISAs [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and are consistent with previous studies that demonstrated the detection of antibodies as early as a 1:32 dilution after RVFV exposure [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, the gradual increase in S/N% and OD with further dilution is representative\u0026ensp;of well-established antibody-binding kinetics: strong binding at high concentrations followed by a decrease in the signal upon further dilution [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Paweska \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u0026ensp; reported comparable results, supporting the fitness of competitive ELISA for field surveillance and vaccine response assessment.\u003c/p\u003e\u003cp\u003eVirus Titration\u0026ensp; and TCID\u003csub\u003e50\u003c/sub\u003e estimation were performed in Vero cells as previously reported (53).\u003c/p\u003e\u003cp\u003eA high-dose vaccine virus stock was denoted by a TCID\u003csub\u003e50\u003c/sub\u003e/ml of 10\u003csup\u003e\u0026minus;\u0026thinsp;6.5 ,\u003c/sup\u003e as measured by virus titration by the Reed method, and Muench stands for the percentage cumulative technique (PCT) method and 10-fold serial dilution. Consistent with the expected outcomes for live-attenuated vaccines, the precision of the calculated infectious dose setting was supported by the fact that 100% CPE was apparent at up to\u0026ensp; 10\u003csup\u003e\u0026ndash;4\u003c/sup\u003e dilutions and 50% at a final dilution of approximately \u003csub\u003e10\u003c/sub\u003e\u003csup\u003e\u0026ndash;6.5\u003c/sup\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This is in line with the manufacturer's data and signals the presence of a high-titer reproducible inoculum\u0026ensp; required for neutralization studies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eVNT Results and\u0026ensp;Kinetics:\u003c/h2\u003e\u003cp\u003eThe VNT results revealed a clear negative correlation between serum dilution and viral CPE,\u0026ensp; with substantial protection (\u0026gt;\u0026thinsp;90%) at dilutions of 1:2 and 1:4 (all tested virus concentrations). As\u0026ensp; the virus load and dilution increased, protection gradually decreased. This dose-dependent neutralization pattern\u0026ensp; has also been observed in CPE-based neutralization assays [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt\u0026ensp; high viral loads (100,000 TCID50/ml), protection was limited to low dilutions (1:2\u0026thinsp;\u0026minus;\u0026thinsp;1:4), whereas at intermediate viral loads (10,000 TCID50/ml), protection was extended to 1:16. Neutralization occurred up to a 1:256 dilution, with protection being the \u0026ensp;longest-lasting at low virus titers (1000 and 100 TCID50/ml). These results are in agreement\u0026ensp; with those of Manenti \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and Muruato \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], who reported more\u0026ensp;durable protection at lower amounts of viral challenge.\u003c/p\u003e\u003cp\u003eCellular changes that occurred during CPE development were observed in a time-dependent\u0026ensp;manner between 24 and 96 hours. Under high-virus/low-antibody conditions, early signs of infection were evident at 48 h,\u0026ensp; and by 96 h, most of the high dilution groups presented full CPE. This finding is consistent with previous reports that the level of viral replication is associated with the\u0026ensp; extent of CPE development over time [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and confirms the replication kinetics of RVFV in Vero cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCorrelation between VNT and\u0026ensp;ELISA:\u003c/h2\u003e\u003cp\u003eThe strong correlation between VNT neutralization\u0026ensp; at low dilutions and ELISA positivity indicates that both assays are suitable for the detection of RVFV antibodies in the early stages of the immune response. The sensitivity of ELISA\u0026ensp;, even at dilutions\u0026thinsp;\u0026ge;\u0026thinsp;1:64, was inferior to that of VNT, whereas VNT showed detectable neutralizing activity. This difference might be attributable to the principles of the two tests, which, especially at 72- and 96-hours post-infection, might be attributed to the principles of the two tests. While ELISA measures the binding of an antibody, VNT\u0026ensp;quantifies functional neutralization of the virus [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Suthar \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] reported that there were discrepancies and revealed that binding assays often\u0026ensp; underestimate protective immunity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eComparison\u0026ensp;with Other Studies\u003c/h2\u003e\u003cp\u003eOur results are also consistent with the neutralization and ELISA\u0026ensp; benchmark values reported in previous studies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The Reed\u0026ndash;Muench method is still a reference technique for TCID\u003csub\u003e50\u003c/sub\u003e estimation and has been widely\u0026ensp; applied in flavivirus and bunyavirus research. Additionally, the time-resolved protective profile of the VNT and the good precision of the ELISA test at low dilutions indicate that both methods are \u0026ensp;potentially valuable tools for the evaluation of antibodies.\u003c/p\u003e\u003cp\u003eCrucially, our findings are consistent with the concept of \u0026lsquo;dilutional loss of protection\u0026rsquo;, which has been previously observed in RVFV and related\u0026ensp;arbovirus challenge models [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The VNT results provide a reliable measure of functional immunity, especially for low viral loads, despite the decrease in ELISA reactivity at high\u0026ensp;dilutions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStudy Conclusion\u003c/h2\u003e\u003cp\u003eA comprehensive picture of the immune response to RVFV was obtained by integrating virus titration with VNT assays and ID.vet\u0026ensp; competitive ELISA. As a reliable seroconversion screening assay, ELISA effectively detected the binding\u0026ensp;antibodies in early and moderate serum samples. Crucial information on the ability of these antibodies to effectively block viral replication,\u0026ensp; depending on different virus titers and exposure times, was provided by a virus neutralization test (VNT).\u003c/p\u003e\u003cp\u003eOur findings revealed \u0026ensp;significant intercorrelations between ELISA and VNT results. Although ELISA offers speed\u0026ensp; and pellucidity, especially for field surveillance, VNT remains the gold standard test for confirming protective immunity. The strong correlation between VNT neutralization titers and ELISA positivity at an early time point after challenge was in line with prior studies of arbovirus\u0026ensp; exposure, supporting the utility of combined testing strategies for epidemiological immunoprofiles [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe implication of this study is that protective\u0026ensp;antibodies are time-dependent. Neutralization ability and cytopathic effect (CPE) are also very different at 24\u0026ndash;96 hours of infection, emphasizing the importance of studying antibodies in a kinetic context. Our results further indicate that the Reed-Muench\u0026ensp; method is convenient for the accurate determination of TCID\u003csub\u003e50\u003c/sub\u003e and can even be utilized for determining vaccine potency.\u003c/p\u003e\u003cp\u003eOverall, this study extends the understanding of the time-dependent association between antibody binding (ELISA) and virus neutralization (VNT)\u0026ensp; and confirms previous reports on the kinetics of antibody responses to RVFV infection. These insights are relevant for outbreak preparedness, diagnostic algorithms for RVFV and other zoonotic\u0026ensp;viral pathogens, and vaccine evaluation. The\u0026ensp;combined testing routinely employed is a good model of how functional testing, within the context of serological antibody testing, can be achieved, and is ultimately required to increase surveillance for human as well as animal zoonotic viruses.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRVF: Rift Valley fever virus.; RPMI: Roswell Park Memorial Institute\u003c/p\u003e\n\u003cp\u003eO.D: Optical density; S/N: Signal-to-noise ratio; RNA: Ribonucleic acid\u003c/p\u003e\n\u003cp\u003eRT‒qPCR: reverse transcriptase quantification polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eCPE: Cytopathic effect; TCID₅₀: 50% tissue culture infective dose\u003c/p\u003e\n\u003cp\u003eI: Interpolated value of the 50% endpoint; PD: proportional distance; VNT: virus neutralization test\u003c/p\u003e\n\u003cp\u003eC1: initial concentration; C2: final concentration; V1: volume taken from the stock (initial concentration); V2: final volume; Vero cell: African Green Monkey Kidney Cell Line\u003c/p\u003e\n\u003cp\u003eFBS: fetal bovine serum; PBS: phosphate-buffered saline; PCT: percentage cumulative technique\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;This work was supported by the National Center for the Prevention and\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eControl of Plants Pests and Animal Disease (Wegaa Center), Saudi Arabia. We thank the animal herd\u0026apos;s owners and the other\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003esupport staff at the study sites for their cooperation and assistance in vaccination and samples collection\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eSincer thanks and appreciation to the \u003cem\u003eDr. Khalid Suleiman\u003c/em\u003e, Weqaa Central Laboratory in Riyadh, has revised and proof-read the manuscript.\u003c/p\u003e\n\u003cp\u003eSpecial thanks also to \u003cem\u003eDr. Ahmed Al-Qadam\u003c/em\u003e, Wegaa Central Laboratory in Jazan for his technical support in documenting, photographing, and producing the images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003eOD, MH, MN and AH carried out the experimental work and data curation.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eOD, EN, and SA wrote the\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003emanuscript, Validation, and reviewing.\u003c/p\u003e\n\u003cp\u003eTA, statistical analysis and reviewing\u003c/p\u003e\n\u003cp\u003eOD, MM, NA, and AA participated in the design of the study, and editing\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eSA, FA, and FB study supervision and reviewing \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;Not applicable\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;The data used to support the findings of this study are available from the\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eJazan laboratory, Wegaa Center, Saudi Arabia\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;The use of animal subjects for this study was approved by National Center for the Prevention and Control of Plants Pests and Animal Disease (Wegaa Center), Ethical\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eCommittee\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;Not applicable\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;The authors declare that they have no competing interests\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBouloy, M. \u003cem\u003eet al.\u003c/em\u003e (2001) \u0026apos;Genetic evidence for an Interferon-Antagonistic function of Rift Valley Fever Virus Nonstructural Protein NSS,\u0026apos; \u003cem\u003eJournal of Virology\u003c/em\u003e, 75(3), pp. 1371\u0026ndash;1377. https://doi.org/10.1128/jvi.75.3.1371-1377.2001.\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;pin, M. 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(2025). \u003cem\u003evirus titratioin in cell line - Search Videos\u003c/em\u003e. [online] Available at: https://www.bing.com/videos/riverview/relatedvideo?q=virus%20titratioin%20in%20 cell%20line\u0026amp;mid=B7B396F5EDD3CDD17379B7B396F5EDD3CDD17379\u0026amp;ajaxhist=06 [Accessed 10 Jun. 2025].\u003c/li\u003e\n\u003cli\u003e\u003cem\u003ePpr-labs-oie-network.org. (2021). Standard Operating Procedures/Materials and protocols - OIE reference laboratory network for peste des petits ruminants (PPR)\u003c/em\u003e. [online] Available at: https://www.ppr-labs-oie-network.org/en/materials-and-protocols/standard-operating-procedures [Accessed 10 Jun. 2025].\u003c/li\u003e\n\u003cli\u003eEbrahim, G.J. (2007) \u0026apos;Virology: principles and applications J. Carter, V. Saunders (eds),\u0026apos; \u003cem\u003eJournal of Tropical Pediatrics\u003c/em\u003e, 55(1), p. 66. https://doi.org/10.1093/tropej/fmn001.\u003c/li\u003e\n\u003cli\u003eWho.int. (2024). \u003cem\u003eManual for the laboratory diagnosis and virological surveillance of influenza. \u003c/em\u003e[online] Available at: https://www.who.int/publications/i/item/manual-for-the-laboratory-diagnosis-and-virological-surveillance-of-influenza.\u003c/li\u003e\n\u003cli\u003eBeaty, B.J., Calisher, C.H., \u0026amp; Shope, R.E. (1989). Arboviruses. \u003cem\u003eIn Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections\u003c/em\u003e (6th ed., pp. 797-855). American Public Health Association.In: Schmidt, N.J., Emmons, R.W. (Eds.), Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 7th ed., 1989.\u003c/li\u003e\n\u003cli\u003ePadoan, A. \u003cem\u003eet al.\u003c/em\u003e (2020b) \u0026apos;Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics,\u0026apos; \u003cem\u003eClinical Chemistry and Laboratory Medicine (CCLM)\u003c/em\u003e, 58(7), pp. 1081\u0026ndash;1088. https://doi.org/10.1515/cclm-2020-0443.\u003c/li\u003e\n\u003cli\u003eKrause, P.R. \u003cem\u003eet al.\u003c/em\u003e (2021) \u0026apos;SARS-CoV-2 variants and vaccines,\u0026apos; \u003cem\u003eNew England Journal of Medicine\u003c/em\u003e, 385(2), pp. 179\u0026ndash;186. https://doi.org/10.1056/nejmsr2105280.\u003c/li\u003e\n\u003cli\u003eAmanat, F. \u003cem\u003eet al.\u003c/em\u003e (2020) \u0026apos;A serological assay to detect SARS-CoV-2 seroconversion in humans,\u0026apos; \u003cem\u003eNature Medicine\u003c/em\u003e, 26(7), pp. 1033\u0026ndash;1036. https://doi.org/10.1038/s41591-020-0913-5.\u003c/li\u003e\n\u003cli\u003eWang, P. \u003cem\u003eet al.\u003c/em\u003e (2021) \u0026apos;Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7,\u0026apos; \u003cem\u003eNature\u003c/em\u003e, 593(7857), pp. 130\u0026ndash;135. https://doi.org/10.1038/s41586-021-03398-2.\u003c/li\u003e\n\u003cli\u003eCase, J.B., Rothlauf, P.W., \u003cem\u003eet al.\u003c/em\u003e (2020) \u0026apos;Neutralizing antibody and soluble ACE2 inhibition of a Replication-Competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2,\u0026apos; \u003cem\u003eCell Host \u0026amp; Microbe\u003c/em\u003e, 28(3), pp. 475-485.e5. https://doi.org/10.1016/j.chom.2020.06.021.\u003c/li\u003e\n\u003cli\u003eInnovative Diagnostics. (n.d.). \u003cem\u003eID Screen\u0026reg; Rift Valley Fever Competition Multispecies\u003c/em\u003e. [online] Available at: https://www.innovative-diagnostics.com/produit/id-screen-rift-valley-fever-competition-multispecies/.\u003c/li\u003e\n\u003cli\u003eKortekaas, J. \u003cem\u003eet al.\u003c/em\u003e (2015) \u0026apos;Crimean-Congo Hemorrhagic fever virus subunit vaccines induce high levels of neutralizing antibodies but no protection in STAT1 knockout mice,\u0026apos; \u003cem\u003eVector-Borne and Zoonotic Diseases\u003c/em\u003e, 15(12), pp. 759\u0026ndash;764. https://doi.org/10.1089/vbz.2015.1855.\u003c/li\u003e\n\u003cli\u003ePaweska, J.T., Van Vuren, P.J. and Swanepoel, R. (2007) \u0026apos;Validation of an indirect ELISA based on a recombinant nucleocapsid protein of Rift Valley fever virus for the detection of IgG antibody in humans,\u0026apos; \u003cem\u003eJournal of Virological Methods\u003c/em\u003e, 146(1\u0026ndash;2), pp. 119\u0026ndash;124. https://doi.org/10.1016/j.jviromet.2007.06.006.\u003c/li\u003e\n\u003cli\u003eWHO ANIMAL INFLUENZA MANUAL 1 WHO Manual on Animal Influenza Diagnosis and Surveillance World Health Organization Department of Communicable Disease Surveillance and Response WHO Global Influenza Programme. (n.d.). Available at: https://www.chinacdc.cn/jkyj/crb2/yl/rgrgzbxqlg/jswj_qlg/202409/W02024090934012806662pdf [Accessed 10 Jun. 2025].\u003c/li\u003e\n\u003cli\u003eRiss, T.L. \u003cem\u003eet al.\u003c/em\u003e (2021) \u0026apos;Treating cells as reagents to design reproducible assays,\u0026apos; \u003cem\u003eSLAS DISCOVERY\u003c/em\u003e, 26(10), pp. 1256\u0026ndash;1267. https://doi.org/10.1177/24725552211039754.\u003c/li\u003e\n\u003cli\u003eWallace, R.P. \u003cem\u003eet al.\u003c/em\u003e (2023) \u0026apos;Synthetically mannosylated antigens induce antigen-specific humoral tolerance and reduce anti-drug antibody responses to immunogenic biologics,\u0026apos; \u003cem\u003eCell Reports Medicine\u003c/em\u003e, 5(1), p. 101345. https://doi.org/10.1016/j.xcrm.2023.101345.\u003c/li\u003e\n\u003cli\u003eWeingartl HM, Drebot MA, Hub\u0026aacute;lek Z, Halouzka J, Andonova M, Dibernardo A, Cottam-Birt C, Larence J, Marszal P. Comparison of assays for the detection of West Nile virus antibodies in chicken serum. \u003cem\u003eCan J Vet Res\u003c/em\u003e. 2003 May;67(2):128-32. PMID: 12760478; PMCID: PMC227040.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rift Valley fever virus, Smithburn vaccine, Virus neutralization test, Competitive ELISA, TCID₅₀, Antibody response, Vero cells, CPE, Immunodiagnostic","lastPublishedDoi":"10.21203/rs.3.rs-7429013/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7429013/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eRift Valley fever virus (RVFV) is a mosquito-borne zoonotic virus\u0026ensp;with high public health and veterinary importance in Africa and the Middle East. Serological surveillance, functional neutralization testing, and virus titration via tissue culture are important for monitoring the effectiveness of vaccines and outbreaks.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eThis study aimed to increase the precision of\u0026ensp;immunological assessments after RVFV vaccination and provide a methodological approach to combine viral quantification, serological detection and functional neutralization testing.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTwenty serum samples were tested using the ID.vet RVFV competitive ELISA to detect antibodies specific to the viral nucleocapsid protein. Viral titration was conducted in Vero cells, and TCID₅₀/ml was calculated using the Reed and Muench\u0026ensp; method. VNT was performed at 24, 48, 72, and 96 hours after infection with different viral doses (100 to 100,000\u0026ensp;TCID₅₀/ml), and the neutralizing ability of serial serum dilutions (1:2 to 1:1024) was tested. Compared with the \u0026ensp;control, protection was determined by CPE inhibition.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eELISA revealed robust antibody signals up to a 1:32 dilution, with S/N\u0026thinsp;\u0026lt;\u0026thinsp;40%, whereas for higher dilutions, antibody detection became inconclusive or negative. Virus titration was performed to verify a stock concentration\u0026ensp; of 10⁶. ⁵ TCID₅₀/ml. The VNT was time and dose- dependent, with good\u0026ensp; protection obtained at low serum dilutions and viral titers, with up to 97 protective effects at 1:2\u0026ndash;1:8 dilutions against 100\u0026ndash;1000 TCID₅₀/ml; however, this protection decreased at higher doses and higher serum dilutions. The results of ELISA and VNT were strongly correlated in the determination at low dilutions, whereas ELISA had decreased sensitivity at high dilutions, at which VNT was still capable\u0026ensp;of detecting neutralizing activity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis combined strategy validates that competitive ELISA\u0026ensp; is applicable for early and medium antibody detection and VNT can functionally validate immune protection. These results provide evidence for the added value of the combination of these two methods in assessing RVFV vaccine-induced immunity and contribute to the\u0026ensp; further interpretation of antibody\u0026ndash;virus kinetics.\u003c/p\u003e","manuscriptTitle":"Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Southwest Saudi Arabia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-10 06:26:49","doi":"10.21203/rs.3.rs-7429013/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"62fed124-a094-4530-be05-21cf6509c176","owner":[],"postedDate":"October 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-16T07:54:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-10 06:26:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7429013","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7429013","identity":"rs-7429013","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}