Search Results (155)

Background: Due to the wide host range of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), vaccination has been recommended for susceptible species in zoological collections, particularly to protect endangered species. The Zoetis^® Experimental Mink Coronavirus Vaccine (Subunit) was temporarily authorized in 2021–2024 for emergency use in North America for this purpose. However, there are limited data regarding its safety or efficacy in non-domestic mammals. The present study was conducted to assess the ability of this vaccine to elicit serum neutralizing titers against SARS-CoV-2 in selected animals from the Toronto Zoo (TZ) vaccinated during 2022. Methods: Serum samples were collected from 24 individuals across four families (Cervidae, Felidae, Ursidae, and Hyaenidae) and tested using a surrogate virus neutralization test (sVNT) and a plaque-reduction neutralization test (PRNT). Results: The results showed that all species developed some neutralizing titers after at least one vaccine dose, except for polar bears, which showed no seroconversion. Felids and hyenas had the highest neutralizing titers, which peaked at 3 and declined between 4 and 6 months after boost. These differences may stem from species-specific immune responses or lack of vaccination protocols tailored to individual species. Conclusions: While natural infection with SARS-CoV-2 could not be ruled out in the cohort of this study, insights from our results have the potential to inform future vaccine recommendations for non-domestic species. Furthermore, our study highlighted the value of competitive assays in assessing serological responses across a broad range of exotic species, for which reagents, such as anti-isotype antibodies, are often unavailable. Full article

Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) are major viral pathogens of horses that contribute to respiratory disease, abortion, and neurological disorders, leading to economic losses in the equine industry. Accurate serological diagnosis is critical for disease surveillance and control. This study aimed to develop and validate an in-house enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against EHV-1 and EHV-4 in horse sera. Serum samples previously confirmed by virus neutralization test (VNT) and a commercial ELISA were used to optimize the assay. Cut-off values were established using negative controls, and 155 serum samples were tested. The developed ELISA demonstrated 80.64% positivity for EHV-1 and 79.35% for EHV-4. When compared to VNT, the assay showed 85% specificity and 100% sensitivity. A high similarity (99%) was also observed when compared with the commercial ELISA. The overall seroprevalence was found to be 54.19% for EHV-1 and 75.48% for EHV-4, with an SN₅₀ value of 1/10 for both. The validation results confirmed high repeatability and reliability across plates. These findings suggest that the developed in-house ELISA is a practical, cost-effective, and accurate tool for the serological monitoring of EHV-1 and EHV-4 infections in horse populations. Full article

Schmallenberg virus (SBV) is a negative-sense RNA virus transmitted by insect vectors, causing arthrogryposis-hydranencephaly syndrome in newborn ruminants. Since its discovery in Germany and the Netherlands in 2011, SBV has rapidly spread across multiple European countries, resulting in significant economic losses in the livestock industry. With the increasing global animal trade and the expanded range of insect transmission, the risk of SBV introduction into non-endemic regions is also rising. As the gold standard for serological testing, the virus neutralization test (VNT) is crucial for tracking the spread of SBV and evaluating the efficacy of vaccines. However, in non-endemic regions, the lack of local viral strains and the biosafety risks associated with introducing foreign strains pose challenges to the implementation of VNT. In this study, we employed reverse genetics techniques using vesicular stomatitis virus (VSV) to substitute the VSV G protein with the envelope glycoproteins of SBV, thereby successfully generating and rescuing the recombinant virus rVSVΔG-eGFP-SBVGPC. The recombinant virus was then thoroughly characterized in terms of SBV Gc protein expression, viral morphology, and growth kinetics. Importantly, rVSVΔG-eGFP-SBVGPC exhibited SBV-specific cell tropism and was capable of reacting with SBV-positive serum, enabling the measurement of neutralizing antibody titers. The results suggest that this recombinant virus can serve as a feasible alternative for SBV neutralization tests, with promising potential for application in serological screening and vaccine evaluation. Full article

In the last decades, there has been huge interest in Per- and Polyfluoroalkyl Substances (PFAS) worldwide because of the toxic effects on humans. In 2013, a large-scale contamination of PFASs in the Veneto region was caused by a fluorochemical plant in Vicenza. About 130,000 inhabitants were exposed to PFAS in their drinking water. To date, relatively few studies have investigated the associations between blood serum PFAS concentrations and oxidative stress in semen. This study compared the antioxidant activity, lipoperoxide levels and protection or induction of oxidative DNA damage by sperm nuclear basic proteins (SNBP) in subjects living in Veneto exposed to PFAS (VNT) with those living in a non-PFAS contaminated area (VSL). Although the semen parameters were within the WHO range, the VNT semen samples showed higher levels of lipoperoxides and lower antioxidant activity compared to the VSL samples. These differences were statistically significant. We also examined DNA damage following SNBP addition under pro-oxidative conditions, finding a significantly different distribution of DNA damage types between the two groups, where 0 means no damage and 1 to 3 means increasing damage with 3 indicating maximum damage. SNBP of VNT subjects showed a reduced ability to protect DNA from oxidative damage. In the VSL group, damage 0 was found in 56% of subjects, 35% of the VNT group show damage 1, 36% damage 2 and 18% damage 3, while only 11% of VNT subjects show damage 0. Additionally, VNT with 0-grade DNA oxidative damage also exhibited reduced antioxidant activity and higher levels of lipoperoxides, in contrast to VSL. The results of this study indicate that exposure to PFAS produces oxidative stress in the semen of VNT subjects, who were also found to have blood serum perfluorooctanoic acid (PFOA) levels above the threshold. This suggests the possibility of infertility issues and emphasises the necessity for additional research into the long-term consequences of oxidative stress on male fertility and the health of offspring. Full article

Community-based serosurveillance for emerging zoonotic viruses can provide a powerful and cost-effective measurement of cryptic spillovers. Betacoronaviruses, including SARS-CoV, SARS-CoV-2 and MERS-CoV, are known to infect bats and can cause severe respiratory illness in humans, yet remain under-surveyed in high-risk populations. This study aimed to determine the seroprevalence of betacoronaviruses in an occupational cohort in contact with bats before and after the emergence of SARS-CoV-2. Serum samples from pre- and post-COVID-19 pandemic were screened using antigen-based multiplex microsphere immunoassays (MMIAs) and a multiplex surrogate virus neutralization test (sVNT). Pre-pandemic samples showed no SARS-CoV-2 antibodies, while post-pandemic samples from vaccinated participants displayed binding and neutralizing antibodies against SARS-CoV-2 and a related bat CoV. Furthermore, one participant (1/237, 0.43%) had persistent antibodies against MERS-CoV in 2017, 2018 and 2021 but was seronegative in 2023, despite reporting no history of traveling abroad or severe pneumonia. The observed sustained antibody levels indicate a possible exposure to MERS-CoV or a MERS-CoV-like virus, although the etiology and clinical relevance of this finding remains unclear. Ongoing surveillance in high-risk populations remains crucial for understanding virus epidemiology and mitigating zoonotic transmission risk. Full article

Foot-and-mouth disease virus (FMDV) continues to pose a significant threat to livestock health and the global agricultural economy, particularly in endemic regions of Asia, Africa, and the Middle East. Current vaccines based on chemically inactivated FMDV present several challenges, including biosafety risks, high production costs, and limited effectiveness against emerging viral variants. To overcome these limitations, we developed virus-like particle (VLP) vaccines targeting FMDV serotypes O, A, and Asia1 using a recombinant Escherichia coli expression system. The resulting VLPs self-assembled into 25–30 nm particles with native-like morphology and antigenic properties, as confirmed by transmission electron microscopy, SDS-PAGE, and Western blot analysis. Immunogenicity was evaluated in mice and pigs using ELISA and virus neutralization tests (VNT), and protective efficacy was assessed through viral challenge studies. All VLPs induced strong serotype-specific antibody responses, with ELISA PI values exceeding 50% and significantly increased VNT titers after booster immunization. In mice, PD₅₀ values were 73.5 (A-type), 32.0 (O-type), and 55.7 (Asia1-type); in pigs, PD₅₀ values reached 10.6 (O-type) and 22.6 (Asia1-type). Notably, the vaccines induced robust immune responses even at lower antigen doses, suggesting the feasibility of dose-sparing formulations. These findings demonstrate that FMDV VLPs produced in E. coli are highly immunogenic and capable of eliciting protective immunity, highlighting their promise as safe, scalable, and cost-effective alternatives to conventional inactivated FMD vaccines. Full article

Background/Objectives: Patients suffering from inflammatory bowel diseases (IBDs) undergoing treatment with anti-TNF antibodies mount a diminished humoral immune response to vaccination against SARS-CoV-2 compared to healthy controls. The characterization of variant-specific immune responses is particularly warranted among immunosuppressed patients, where reduced responses may necessitate further medical interventions. Methods: This pilot study investigated the humoral immune response of vaccinated IBD patients on anti-TNF medication and a comparable group of healthy individuals against the viral variants Alpha, Beta, Gamma, Delta, and Omicron BA.1 and BA.5. While total IgG antibodies targeting the receptor binding site of the spike protein of SARS-CoV-2 were quantified using a chemiluminescence microparticle immunoassay (CMIA), their potential neutralizing capacity was determined using commercial and variant-specific in-house surrogate virus neutralization tests (sVNTs) against a variant-specific in-house VSV-pseudotyped virus neutralization test (pVNT) as the gold standard. Results: Employing variant-specific assays recapitulated the immune escape functions of virus variants. Conspicuously, antibody reactivity against Alpha and Omicron BA.1 and BA.5 was strikingly poor in IBD patient sera post-initial vaccination compared to healthy individuals. A comparison of the diagnostic performance of assays with the pVNT revealed that identification of patients with inadequate humoral responses by CMIA and sVNT may require adjustments to cut-off values and end-point titration of sera. Following adaptation of cut-off values, patient sera exhibited reduced reactivity against all tested variants. The assay panel used substantiated the impact of anti-TNF therapy in IBD patients as to reduced strength, function, and breadth of the immune response to several SARS-CoV-2 variants. The immune response measured following the second vaccination was comparable to the antibody response observed in healthy individuals following the first vaccination. Conclusion: Variant-specific sVNTs and pVNTs have the potential to serve as valuable tools for evaluating the efficacy of adapted vaccines and to inform clinical interventions in the care of immunosuppressed patients. Anti-TNF-treated individuals with antibody levels below the optimized CMIA threshold should be considered for early booster vaccination and/or close immunological monitoring. Full article

Background/Objectives: Foot-and-mouth disease virus (FMDV) poses a continuous threat to livestock health and agricultural economies. Current vaccines require high biosafety standards and are costly to produce. While novel vaccine technologies have been explored, most fail to meet industrial scalability, cost-efficiency, or multiserotype flexibility required for effective FMD control. This study aimed to evaluate the feasibility of using a high-cell density transient gene expression (TGE) system in CHO cells for the production of FMDV virus-like particles (VLPs) as a recombinant vaccine platform. Methods: VLP expression was optimized by adjusting cDNA and polyethyleneimine (PEI) concentrations. Expression yields were compared at 24 and 48 h post-transfection to determine optimal harvest timing. We further tested the system’s capacity to express different serotypes and chimeric constructs, incorporating VP1 sequences from various FMDV strains. Immunogenicity was evaluated in swine using VLPs from the A2001 Argentina strain as a model. Results: Optimal VLP expression was achieved at 24 h post-transfection. Chimeric constructs incorporating heterologous VP1 regions were successfully expressed. Immunized pigs developed protective antibody titers as measured by a virus neutralization test (VNT, log₁₀ titer 1.43) and liquid-phase blocking ELISA (LPBE, titer 2.20) at 28 days post-vaccination (dpv). Titers remained above protective thresholds up to 60 dpv with a single dose. A booster at 28 dpv further elevated titers to levels comparable to those induced by the inactivated vaccine. Conclusions: Our results demonstrate the feasibility of using CHO cell-based TGE for producing immunogenic FMDV VLPs. This platform shows promise for scalable, cost-effective, and biosafe development of recombinant FMD vaccines. Full article

A wide experimental campaign was developed on an automotive turbocharged diesel engine, using two blends between diesel oil and waste cooking oil methyl esters (WCOME) and neat biodiesel. A conventional B7 diesel oil was considered as a reference fuel. The two blends, respectively, included 40 and 70% of WCOME, on a volumetric basis. The influence of biodiesel was analyzed by testing the engine in two part-load operating conditions, applying proper control strategies to the exhaust gas recirculation (EGR) circuit and rail pressure, to assess the interactions between the engine management and the tested fuels. The variable nozzle turbine (VNT) was controlled to obtain a constant level of intake pressure in the two experimental points. Referring to biodiesel effects at constant operating mode, higher WCOME content generally resulted in better efficiency and soot emission, while NO_X emission was negatively affected. EGR activation allowed for limited NO formation but with penalties in soot emission. Furthermore, interactions between the EGR circuit and turbocharger operations and control led to higher fuel consumption and lower efficiency. Finally, the increase in rail pressure corresponded to better soot emission and penalties in NO_X emission. Combining all these effects, the selection of EGR rate and rail pressure values higher than the standard levels resulted in better efficiency, NO_X, and soot emissions when comparing blends and neat biodiesel to conventional B7, granting advantages not only with regard to greenhouse gas emissions. Combustion parameters were also assessed, showing that combustion stability and combustion noise were not negatively affected by biodiesel use. Combustion duration was reduced when using WCOME and its blend, even if the center of combustion was slightly shifted along the expansion stroke. The main contribution of this investigation to the scientific and technical knowledge on biodiesel application to internal combustion engines is related to the development of tests on diesel–biodiesel blends with high WCOME content or neat WCOME, identifying their effects on NO_X emissions, the definition of integrated strategies of HP EGR system, fuel rail pressure, and VNT for the simultaneous reduction in NO_X and soot emissions, and the detailed assessment of the influence of biodiesel on a wide range of combustion parameters. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) modified live virus (MLV) vaccination is used to control PRRSV. In China, farms conduct random sampling from sow herds every 4 to 6 months. They use the enzyme-linked immunosorbent assay (ELISA) method to monitor the immune status of the herd by tracking the positive rate or the sample-to-positive ratio. However, in farms that implement mass vaccination and have stable production, the positive rate of ELISA antibodies has decreased, especially in high-parity sows. This poses a considerable challenge to the current monitoring approach of PRRSV immunity. It remains unclear whether this reflects insufficient sensitivity of the kits for these special scenarios or the fact that the sows have truly lost immunity. In this study, 233 samples from four farms (A–D) across different regions of China were acquired. They were tested using four representative ELISA kits, two targeting the nucleocapsid protein (N) and two targeting the glycoprotein (GP) to evaluate PRRS immune status. The respective sample positive rates in A–D were 57.1–100%, 50.9–100%, 50–100%, and 75.7–100% using the kits. The positive rates using the four ELISA kits were 50.0–75.7%, 70.0–75.7%, 82.5–97.1%, and 100%, respectively, with poor agreement among them. The positive rates and humoral antibody levels for parity 1 and 2 sows were significantly lower than those with higher parities (>4). Eighty-eight ELISA-negative samples identified using ELISA kit A were verified using a viral neutralizing test (VNT), with only 15.9% of the samples testing negative. In conclusion, the ELISA antibody negativity issue existed, mostly occurring in specific farms tested using a specific kit. However, the low correlation with the VNT results and the poor agreements among the kits suggest that relying on one ELISA test is insufficient to monitor the immune status of PRRSV MLV-vaccinated herds. Full article

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 19 (COVID-19) pandemic, significantly impacting global health, economies, and social stability. In February 2020, the first cases of SARS-CoV-2 infections in animals were documented, highlighting the potential risks posed by regular human–animal interactions in facilitating viral transmission. In consequence, it is essential to validate surveillance methods for SARS-CoV-2 in animals. In the present study, 101 sera from different animal species (36 cats, 41 dogs, 4 ferrets, 10 wild boar, 6 domestic goats, and 4 lions) were tested using three different ELISA kits to evaluate humoral responses against SARS-CoV-2. ELISA results were compared and correlated with a pseudovirus neutralization test (pVNT), considered as the reference assay. ELISA-1, targeting the receptor binding domain (RBD) neutralizing antibodies (nAbs) of SARS-CoV-2, exhibited the highest diagnostic performance, and proved to be a reliable tool for initial screenings in high-throughput animal studies. In contrast, ELISA-2 (also targeting RBD nAbs) and ELISA-3 (targeting nucleoprotein antibodies) demonstrated lower sensitivity for detecting seropositive animals. Full article

This study aimed to assess West Nile virus (WNV) and Usutu virus seroprevalence among the dog population in the Danube region, Bulgaria, to confirm the results of ELISA by the virus neutralisation test (VNT), as well as to analyse several risk factors of seropositivity in dogs. To implement this, a total of 201 blood samples were collected from dogs in four districts bordering the Danube River. All the samples were tested for anti-WNV protein E antibodies using competitive ELISA. Neutralising antibodies against WNV and Usutu virus were tested in all the ELISA-positive samples. The results show a WNV seroprevalence of 45.3% (n = 91, CI = 36.45–55.59) by ELISA, whereas the virus neutralisation test indicated a seroprevalence of 21.9% (n = 44, CI = 15.91–29.39). Neutralising antibodies against Usutu virus were detected for the first time in Bulgaria, with a prevalence of 6% (n = 12, CI = 3.09–10.43). Compared to VNT, ELISA demonstrated 100.0% sensitivity and 70.1% specificity. The region (p < 0.0187), the district (p = 0.0258) and the ages of the dogs (p = 0.0180) were identified as statistically significant risk factors associated with WNV seropositivity. This study provides indirect evidence of WNV and Usutu virus circulation among dogs in the Danube region of Bulgaria, highlighting a potential risk for susceptible hosts in the area. Full article

Outbreaks of the West Nile Virus (WNV) have increased significantly in recent years in the Mediterranean region, including Tunisia. To understand the risks for animal and human health and to mitigate the impact of future outbreaks, comprehensive viral surveillance in vertebrate hosts and vectors is needed. We conducted the first serosurvey for the WNV in ruminants in southern Tunisia using the ELISA test and confirmed it with the micro-virus neutralization test (VNT). Antibodies were detected by the ELISA test in camels (38/112), sheep (9/155), and goats (7/58), and six samples were doubtful (five camels and one sheep). The ELISA positive and doubtful sera (n = 60) were further analyzed to confirm the presence of specific anti-WNV and anti-Usutu virus (USUV) antibodies using the micro-virus neutralization test (VNT). Out of the 60 sera, 33 were confirmed for specific WNV antibodies, with an overall seroprevalence of 10.15% [95% CI: 7.09–13.96]. The high seroprevalence observed in camels (22.3%) suggests their potential use as sentinel animals for WNV surveillance in southern Tunisia. The viral genome, and consequently active circulation, could not be detected by real-time RT-qPCR in blood samples. Ongoing surveillance of the WNV in animals, including camels, sheep, and goats, may be used for the early detection of viral circulation and for a rapid response to mitigate potential outbreaks in horses and humans. Full article

Background/Objectives: Foot-and-mouth disease (FMD) is a highly contagious class 1 animal disease that affects cloven-hoofed animals, such as cattle, pigs, and goats. Diagnosis and research on live FMD virus (FMDV) typically require biosafety level 3 facilities, which are challenging to maintain due to strict protocols and high costs. The development of NanoBiT-based assays has accelerated in response to the coronavirus disease pandemic, providing safer alternatives for viral research, and is now applicable for general laboratories. This study aimed to develop a NanoBiT-based virus-like particle (VLP) assay for the rapid and safe screening of neutralizing antibodies against FMDV Asia1 Shamir (AS). Methods: We developed an AS VLP with an inserted HiBiT tag that enabled the detection of entry into LgBiT cells through luminescence signals. Results: HiBiT-tagged AS VLPs mixed with anti-serum and introduced into LgBiT-expressing cells led to a reduction in luciferase activity. Therefore, we established a NanoBiT-based viral neutralizing antibody test (VNT) that demonstrated a high correlation (R² = 0.881) with the traditional gold standard VNT. Conclusions: The assay demonstrated high sensitivity and could be performed in BL-2 facilities, offering a safer and more efficient alternative to traditional assays while reducing the need to handle live viruses in high-containment facilities. This method provides a valuable tool for rapid screening of neutralizing antibodies and can be adapted for broader applications in FMDV research. Full article

Background: Rabies is a preventable zoonotic disease caused by the rabies virus (RABV) with a high mortality rate. Most vaccines on the market or under development have issues, such as low single-dose neutralization titer, complex processes, and high costs. During the COVID-19 pandemic, the successful development of mRNA vaccines opened up a new avenue for preventive vaccines. As a new technology, mRNA has higher scalability. Methods: In this study, we designed an mRNA encoding the RV-G protein, encapsulated by our own muscle-targeting lipid nanoparticles (LNPs), and evaluated the expression of the RV-G protein in vitro, its immunogenicity, and its protection against virus infection in vivo. Results: The results show that RV-G mRNA was significantly expressed in vitro. High Virus-IgG binding titers and virus-neutralizing antibody titers (VNT) were induced by immunization with RV-G mRNA-LNP. Additionally, our results showed that the RV-G mRNA vaccine is better than commercially available vaccines in mice. Conclusions: Our research highlights the potential of the mRNA-LNP platform in developing next-generation rabies vaccines. Full article