Search Results (98)

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

Background: Zoonotic risks in exposed students are poorly documented in Belgium. According to the literature, even though human tick-borne encephalitis (TBE) infection risk has increased significantly in southern Belgium, no previous human serological survey has demonstrated specific antibodies directed at TBE virus. Methods and principal findings: The aim of this paper was to perform a representative serological survey on sera involving students at the University of Liege, in the southern part of Belgium, to discover possible exposure to TBEV. A total of 207 sera samples were randomly chosen and analyzed using ELISA IgM (with 1 positive student out of 207) and ELISA IgG (with 10 positive students out of 207), subsequent serial immunofluorescence antibody testing (IFAT) IgG (with 8 positive students out of 10 positive in ELISA IgG) and serial IFAT IgM (with 1 negative student out of 1 positive in ELISA IgM), and confirmatory tests, i.e., 50% and 90% plaque reduction neutralization tests (PRNTs) (1 strongly positive student out of 8 positive in IFAT). Conclusions and significance: The exposure of students from the southern part of Belgium (area with increasing risk) to TBEV was assessed for the first time. Antibodies against TBEV could only be demonstrated in 1 out of 207 students. This finding contributes to better decision-making in public health and prevention and management of tick-borne diseases in the context of climate change. Awareness among all students should be prioritized, with prevention measures against tick bites, particularly during forest and recreational activities contributing to risk, to maintain the current low seroprevalence levels. Full article

In December 2023, infections of western equine encephalitis virus (WEEV) within Argentina were reported to the World Health Organization (WHO). By April 2024, more than 250 human infections, 12 of which were fatal, and 2500 equine infections were identified in South America. Laboratory diagnosis and surveillance in affected countries were hindered by a lack of facilities equipped with BSL-3 laboratories, as confirmatory serodiagnosis for WEEV requires live virus in the plaque reduction neutralization test (PRNT). To expand serodiagnosis for WEEV in the Americas, we developed a virus chimera composed of vesicular stomatitis virus (VSV) engineered to display the E2-E1 glycoproteins of WEEV (VSV/WEEV) in place of the VSV glycoprotein (G). PRNT₉₀ and IC₉₀ values of parental WEEV and VSV/WEEV were analogous using sera collected from mice, horses, and chickens. VSV/WEEV rapidly formed plaques with clear borders and reduced the assay readout time by approximately 8 h compared to the parental virus. Overall, we demonstrate that chimeric VSV/WEEV is a suitable surrogate for WEEV in a diagnostic PRNT. Use of chimeric VSV/WEEV in place of authentic WEEV will dramatically expand testing capacity by enabling PRNTs to be performed at BSL-2 containment, while simultaneously decreasing the health risk to testing personnel. Full article

Co-infections of Orthopoxviruses (OPVs), such as vaccinia virus (VACV) and monkeypox virus (MPXV), and the human immunodeficiency virus (HIV) can be associated with severe outcomes. Serro’s dairy region, located in Minas Gerais, southeastern Brazil, is an endemic area for VACV, where zoonotic outbreaks affect rural communities. This epidemiological context is especially relevant for at-risk populations, such as people living with HIV (PLHIV). This study aimed to assess the presence of neutralizing antibodies (NAbs) against OPV in PLHIV in this endemic setting. Serum samples were collected from 177 PLHIV in treatment at the specialized service between December 2021 and August 2022. VACV and MPXV NAbs were measured using the plaque reduction neutralization test (PRNT) and VACV-infected cells. The overall occurrence of OPV NAbs was 27.7%. NAbs were higher in individuals born before 1980 (53.3%) than those born after 1980 (1.1%). Among anti-VACV-seropositive individuals, 40.8% also had MPXV NAbs, suggesting cross-immunity. These findings indicate the circulation of VACV in PLHIV and highlight the increased susceptibility to OPV infections among individuals born after the cessation of smallpox vaccination. The results reinforce the importance of continued surveillance of OPV, especially in endemic regions and vulnerable populations. Full article

Yellow fever (YF) is an acute hemorrhagic zoonotic disease that causes severe liver damage, renal failure, and hemorrhagic shock. No antiviral treatment is available; thus, vaccination is a critical preventive measure. Although the World Health Organization (WHO) revised the guidelines regarding the need for booster vaccination for YF with the rationale that a single vaccination provides sufficient long-term immunogenicity, no studies have evaluated long-term immunity in Japanese adults who received a single dose of YF vaccine. This study evaluated the long-term persistence of immunogenicity in Japanese adults vaccinated with the YF vaccine. This observational study enrolled Japanese adults who received a single YF vaccination >5 years previously. Blood samples were collected after confirming eligibility for the study. The serum levels of anti-yellow fever virus (YFV)-neutralizing antibodies were measured using the 50% plaque reduction neutralization test (PRNT₅₀). The 65 participants comprised 35 males and 30 females, with a median age at vaccination of 34 years. The time between YF vaccination and registration was between 5 and 26 years. All participants remained seropositive even after a long time. Statistical analysis showed no correlation between the time elapsed since YF vaccination and PRNT₅₀. Our results indicate that a single dose of YF vaccine provides adequate long-term immunity in Japanese adults and that booster vaccinations are not routinely required. These findings strongly aid in the development of travel medicine guidelines and the optimization of vaccination strategies by reducing the usage of medical resources and simplifying the health requirements for travelers. Full article

We performed serologic surveillance for selected arthropod-borne viruses (arboviruses) in bats and opossums in the Lacandona Rainforest, Chiapas, Mexico, in 2023–2024. Sera were collected from 94 bats of at least 15 species and 43 opossums of three species. The sera were assayed by the plaque reduction neutralization test (PRNT) for antibodies to eight orthoflaviviruses (dengue viruses 1–4, St. Louis encephalitis virus, T’Ho virus, West Nile virus, and Zika virus) and one alphavirus (chikungunya virus; CHIKV). Twelve (12.8%) bats and 15 (34.9%) opossums contained orthoflavivirus-specific antibodies. One bat (a Jamaican fruit bat) was seropositive for Zika virus, and 11 bats contained antibodies to an undetermined orthoflavivirus, as did the 15 opossums. All bats and most opossums seropositive for an undetermined orthoflavivirus had low PRNT titers, possibly because they had been infected with another (perhaps unrecognized) orthoflavivirus not included in the PRNTs. Antibodies that neutralized CHIKV were detected in three (7.0%) opossums and none of the bats. The three opossums had low CHIKV PRNT titers, and therefore, another alphavirus may have been responsible for the infections. In summary, we report serologic evidence of arbovirus infections in bats and opossums in Chiapas, Mexico. Full article

This paper presents the results of experimental and numerical analysis of fracture mechanics testing of ductile metallic materials using a non-standard procedure with PRNT (pipe ring notched tensile) ring-shaped specimens, introduced in previous publications through analysis of 3D-printed polymer rings. The main focus of this research is the determination of the values of the plastic geometry factor η_pl since the specimen is not a standard one. Toward this aim, the finite element software package Simulia Abaqus was applied to evaluate the J-integral (by using the domain integral method) and the F-CMOD curve so that the plastic geometry factor η_pl can be evaluated for different values of the ratio of crack length to specimen width (a₀/W = 0.45 ÷ 0.55). In this way, a procedure and the possibility of practical implementation on the thin-walled pipelines are established. Full article

The emergence of COVID-19 necessitated the rapid development of vaccines. While highly effective at reducing severe disease and death, breakthrough infections remain a problem as the virus continues to mutate. To help address this issue, we show the utility of a multiplex immunoassay in measuring multiple aspects of the antibody response generated by SARS-CoV-2 vaccines. We use a multiplex immunoassay platform to measure spike-specific IgG concentration, avidity, and receptor-binding inhibition. In addition, we correlate results from an ACE-2 receptor-binding inhibition assay with corresponding data from a SARS-CoV-2 microneutralization assay to establish this inhibitory assay as a potential predictor of virus neutralization. We studied these antibody responses in SARS-CoV-2-naïve and -convalescent vaccinees. Our results showed increased IgG concentrations, avidity, and inhibition following vaccination in both groups. We were also able to differentiate the immune response between the two groups using the multiplex immunoassay platform to look at antibody diversity. The receptor-binding inhibition assay has strong correlations with a cell-based pseudovirus neutralization assay as well as with WT SARS-CoV-2 Washington and Delta variant PRNT₅₀ assays. This suggests that the inhibition assay may be able to simultaneously predict virus neutralization of different SARS-CoV-2 variants. Overall, we show that the developed custom multiplex immunoassay with several experimental variations is a powerful tool in assessing multiple aspects of the SARS-CoV-2 antibody response in vaccinated individuals. Full article

Neutralization assays have become an important tool since the beginning of the COVID-19 pandemic for testing vaccine responses and therapeutic antibodies as well as for monitoring humoral immunity to SARS-CoV-2 in epidemiological studies. The spike glycoprotein (S) present on the viral surface contains a receptor binding domain (RBD) that recognizes the angiotensin-converting enzyme 2 receptor (ACE2) in host cells, allowing virus entry. The gold standard for determining SARS-CoV-2 neutralizing antibodies is the plaque reduction neutralization test (PRNT), which relies on live-virus replication performed exclusively in biosafety level 3 (BSL-3) laboratories. Here, we report the development of a surrogate live-cell imaging-based fluorescent SARS-CoV-2 neutralization assay, applicable to BSL-1 or BSL-2 laboratories, by antibody-mediated blockage of the interaction between recombinant RBD with overexpressed ACE2 receptor in a genetically modified HEK 293T stable cell line. Our approach was able to detect neutralizing antibodies both in COVID-19-positive human serum samples and polyclonal equine formulations against SARS-CoV-2. This new cell-based surrogate neutralization assay represents a virus-free fluorescence imaging alternative to the reported approaches, which can be used to detect antibody-neutralizing capabilities toward SARS-CoV-2. This assay could also be extrapolated in the future to other established and emergent viral agents. Full article

Japanese encephalitis (JE) is a zoonotic disease caused by the Japanese encephalitis virus (JEV), belonging to the Flaviviridae family. Diagnosis of Japanese encephalitis (JE) based on clinical signs alone is challenging due to the high proportion of subclinical cases. The Plaque Reduction Neutralization Test (PRNT) is considered the gold standard for detecting JE-specific antibodies because of its high specificity. However, PRNT is complex, time-consuming, and requires live viruses, limiting its applicability in routine diagnostics. In this study, we compared the sensitivity and correlation of the Hemagglutination Inhibition (HI) assay and PRNT for detecting JE antibodies in avian serum samples. We conducted a comparative analysis of the outcomes obtained from the PRNT and HI using 240 serum samples collected from 30 JEV-immunized avian subjects at various time points. Comparative analysis revealed a significant correlation between the HI and PRNT (R² = 0.9321, p ≤ 0.0001). The Bland–Altman analysis also exhibited favorable concordance between the two assays. Consequently, HI may function as a viable substitute for PRNT in the screening of a substantial number of serum samples. Full article

Background: Available assays to measure pox virus neutralizing antibody titers are laborious and take up to 5 days. In addition, assays to measure T cell responses require the use of specific antigens, which may not be the same for all pox viruses. This study reports the development of robust assays for the measurement of mpox-specific neutralizing antibodies and IFN-γ-producing T-cell responses. Methods: Fourteen samples from 7 volunteers who received Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN) were used. The focused reduction neutralization test (FRNT) was performed using the mpox-specific A29 monoclonal antibody. Optimization and further development of FRNT were conducted using the plaque reduction neutralization test (PRNT) as the gold standard. The mpox-specific IFN-γ ELISPOT assay was optimized using different mpox antigen preparations. Results with pre-vaccination samples were compared with post-vaccination samples using the Wilcoxon matched-pairs test. Results: Pre-vaccination and post-vaccination sera (n = 7) had FRNT50 (i.e., titers that inhibited at least 50% of the virus) of 109.1 ± 161.8 and 303.7 ± 402.8 (mean ± SD), respectively. Regression analysis of fold changes in FRNT50 and PRNT50 showed that the two assays closely agree (n = 25 tests on paired samples, R² of 0.787). Using UV-inactivated mpox as an antigen, the number of IFN-γ spot-forming T cells (SFC) in pre-vaccination samples (16.13 ± 15.86, mean ± SD) was significantly lower than SFC in post-vaccination samples (172.9 ± 313.3, mean ± SD) with p = 0.0078. Conclusions: Our newly developed microneutralization test has a good correlation with PRNT. UV-inactivated mpox is an appropriate antigen for the ELISPOT assay that measures mpox cross-reactive T cells. These assays will be useful in future mpox vaccine studies. Full article

Background/Objectives: Yellow fever virus (YFV) (Flaviviridae, Orthoflavivirus) is the etiologic agent of yellow fever (YF), a vector-borne disease with significant morbidity and mortality across the tropics and neotropics, despite having a highly efficacious and safe vaccine (17D). Vaccination provides lifelong protection from YF disease mediated by humoral immunity. There are several versions of the original 17D vaccine: 17D-204 (marketed in the USA as YF-VAX, in France as Stamaril, and in China as Tiantan-V), 17D-213 (Russian Federation), and 17DD (by FIOCRUZ in Brazil). Vaccines produced in the US, France, Senegal, China, and Russia represent 17D-204-derived strains, whereas the Brazilian 17DD has a unique passage/attenuation history from 17D-204-derived strains. Their functional differences in the neutralization profiles are not known. Methods: The Plaque Reduction Neutralization Test (PRNT) was used to determine the neutralization profiles of sera from 209 patients that were previously vaccinated with the 17DD strain against both 17D-204 and 17DD. Results: Sera exhibited significantly more efficient neutralization of 17DD (mean reciprocal PRNT₅₀ 183, PRNT₈₀ 86, median reciprocal PRNT₅₀ 80, and PRNT₈₀ 40) compared to 17D-204 (mean reciprocal PRNT₅₀ 91, PRNT₈₀ 33, median reciprocal PRNT₅₀ 40, and PRNT₈₀ 10). Conclusions: Our data indicate antigenic differences between 17D and 17DD vaccines. Full article

A 9-year-old male with autism and a history of well-controlled epilepsy presented with acute headache, fever, and generalized tonic-clonic seizures. Initial diagnostics, including imaging and cerebrospinal fluid analysis, were inconclusive. However, further serological testing suggested the presence of the chikungunya virus, establishing a diagnosis of chikungunya-associated neurological manifestation. The patient was treated with anticonvulsants, antibiotics for secondary bacterial pneumonia, and supportive care, leading to a gradual recovery. This case highlights the importance of considering systemic viral infections in pediatric patients with neurological symptoms and underscores the potential for arboviruses like chikungunya to cause neurological manifestation. Full article

Background: Despite its global eradication in 1977, smallpox remains a concern owing to its potential as a biological agent, thereby prompting the ongoing development and utilization of its vaccine. Vaccination with the Vaccinia virus induces immunity against variola virus, the causative agent of smallpox; however, this immunity does not extend to viruses of different genera within the Poxviridae family. In this study, we aimed to assess the efficacy of an enzyme-linked immunosorbent assay (ELISA) method utilizing Vaccinia virus and recombinant A27L antigen for detecting antibodies against smallpox. Methods. An analysis of the serum from 20 individuals pre- and post-vaccination with the CJ strain (CJ50300) revealed neutralizing antibodies, which were confirmed using the plaque reduction neutralization test (PRNT). The ELISA method, validated with a PRNT₅₀ cut-off value of >4, exhibited a sensitivity and specificity of >95% and was particularly reactive with the inactivated virus. Furthermore, adherence to the smallpox vaccination policy revealed significant differences in Orthopoxvirus antibody levels among 300 individuals of different age groups. These findings highlight the reliability and efficacy of the ELISA method in detecting post-vaccination antibodies and contribute significantly to diagnostic methods to prepare for potential smallpox resurgence and bioterrorism threats. Full article

Passive immunisation with normal human immunoglobulin (NHIG) is recommended as post-exposure prophylaxis (PEP) for higher-risk measles contacts where vaccination is contraindicated. However, the concentration of measles-specific antibodies in NHIG depends on antibody levels within pooled donor plasma. There are concerns that measles immunity in the Australian population may be declining over time and that blood donors’ levels will progressively decrease, impacting levels required to produce effective NHIG for measles PEP. A cross-sectional study of Australian plasmapheresis donors was performed using an age-stratified, random sample of recovered serum specimens, collected between October and November 2019 (n = 1199). Measles-specific IgG antibodies were quantified by ELISA (Enzygnost anti-measles virus IgG, Siemens), and negative and equivocal specimens (n = 149) also underwent plaque reduction neutralisation testing (PRNT). Mean antibody levels (optical density values) progressively decreased from older to younger birth cohorts, from 2.09 [±0.09, 95% CI] to 0.58 [±0.04, 95% CI] in donors born in 1940–1959 and 1990–2001, respectively (p < 0.0001). This study shows that mean measles-specific IgG levels are significantly lower in younger Australian donors. While current NHIG selection policies target older donors, as younger birth cohorts become an increasingly larger proportion of contributing donors, measles-specific antibody concentrations of NHIG will progressively reduce. We therefore recommend monitoring measles-specific antibody levels in future donors and NHIG products in Australia and other countries that eliminated measles before the birth of their youngest blood donors. Full article