Search Results (658)

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

The Akabane virus (AKAV) is a significant member of the Orthobunyavirus genus, with its envelope glycoprotein Gc, focusing on its molecular structural features, immunoregulatory mechanisms, and application value in pathogen diagnosis and vaccine design. As a key structural protein of AKAV, Gc mediates virus adsorption and neutralizing antibody recognition through the N-terminal highly variable region (HVR), while the C-terminal conserved region (CR) dominates the membrane fusion process, and its glycosylation modification has a significant regulatory effect on protein function. In clinical diagnostics, serological assays based on Gc proteins (e.g., ELISA, immunochromatographic test strips) have been standardized; in vaccine development, the neutralizing epitope of Gc proteins has become a core target for subunit vaccine design. Follow-up studies were deeply needed to analyze the structure-function interaction mechanism of Gc proteins to provide theoretical support for the construction of a new type of AKAV prevention and control system. 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

Background: Kidney transplant recipients (KTRs) exhibit a significantly diminished immune response to Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) vaccines compared with the general population, primarily due to ongoing immunosuppressive therapy. This study evaluated the immunogenicity of a third SARS-CoV-2 mRNA vaccine dose in KTRs and assessed the association between antibody response and protection against SARS-CoV-2 breakthrough infection. Additionally, the clinical and immunological correlates of post-vaccination SARS-CoV-2 infection were examined. Methods: A prospective cohort of 135 KTRs received a third vaccine dose approximately six months following the second dose. Plasma samples were collected at baseline (pre-vaccination), six months after the second dose, and six weeks following the third dose. Humoral responses were assessed using SARS-CoV-2-specific Immunoglobulin G (IgG) titers and virus neutralization assays against wild-type (WT) and viral strains, including multiple Omicron sub-lineages. Results: After the third vaccine dose, 74% of the KTRs had detectable SARS-CoV-2-specific IgG antibodies, compared with 48% following the second dose. The mean IgG titers increased approximately ten-fold post-booster. Despite this increase, neutralizing activity against the Omicron variants remained significantly lower than that against the WT strain. KTRs who subsequently experienced a SARS-CoV-2 breakthrough infection demonstrated reduced neutralizing antibody activity across all variants tested. Additionally, individuals receiving triple immunosuppressive therapy had a significantly higher risk of SARS-CoV-2 breakthrough infection compared with those on dual or monotherapy. A multivariate machine learning analysis identified age and neutralizing activity against WT, Delta, and Omicron BA.2 as the most robust correlates of SARS-CoV-2 breakthrough infection. Conclusions: A third SARS-CoV-2 mRNA vaccine dose significantly improves SARS-CoV-2-specific IgG levels in KTRs; however, the neutralizing response against Omicron variants remains suboptimal. Diminished neutralizing capacity and intensified immunosuppression are key determinants of SARS-CoV-2 breakthrough infection in this immunocompromised population. Full article

Background: An effective vaccine against Nipah virus (NiV) is crucial due to its high fatality rate and recurrent outbreaks in South and Southeast Asia. Vaccine development is challenged by the lack of validated accessible neutralization assays, as virus culture requires BSL-4 facilities, restricting implementation in resource-limited settings. To address this, we standardized and validated a pseudotyped virus neutralization assay (PNA) for assessing NiV-neutralizing antibodies in BSL-2 laboratories. Methods: The NiV-PNA was validated following international regulatory standards, using a replication-defective recombinant Vesicular stomatitis virus (rVSV) backbone dependent pseudotyped virus. Assessments included sensitivity, specificity, dilutional linearity, relative accuracy, precision, and robustness. The assay was calibrated using the WHO International Standard for anti-NiV antibodies and characterized reference sera to ensure reliable performance. Findings: Preliminary evaluation of the developed NiV-PNA showed 100% sensitivity and specificity across 10 serum samples (5 positive, 5 negative), with a positive correlation to a calibrated reference assay (R² = 0.8461). Dilutional linearity (R² = 0.9940) and accuracy (98.18%) were confirmed across the analytical titer range of 11-1728 IU/mL. The assay also exhibited high precision, with intra-assay and intermediate precision geometric coefficients of variation of 6.66% and 15.63%, respectively. Robustness testing demonstrated minimal variation across different pseudotyped virus lots, incubation times, and cell counts. Conclusions: The validated NiV-PNA is a reproducible and scalable assay platform for quantifying NiV neutralizing antibodies, offering a safer alternative to virus culture. Its validation and integration into the CEPI Centralized Laboratory Network will enhance global capacity for vaccine evaluation and outbreak preparedness. Full article

GW4869, a cell-permeable, selective inhibitor of neutral sphingomyelinase is a pharmacological agent that blocks the production and release of extracellular vesicles (EVs). Our previous studies have shown that GW4869 inhibits flaviviral loads in tick, mosquito and mammalian cells, including murine cortical neurons. Yet the mechanism(s) of GW4869 inhibitor upon viral infections were not addressed. In the current study, we focused on how GW4869 interferes with Langat Virus (LGTV, a tick-borne flavivirus) replication in ISE6 tick cells. First, we found that GW4869 is neither cytotoxic at tested doses of 50, 100, and 150 µM in tick cells, nor does it directly bind to the free LGTV present in cell culture supernatants. When tick cells were treated with GW4869, followed by infection with viral stock at dilutions of 10⁻², 10⁻³, 10⁻⁴ (the infectious dose determination by viral dilution assay), it affected LGTV replication in tick cells. A reduction in viral burden was noted in GW4869-treated tick cells, which constituted more than half the amount of decrease when compared to the mock control. Next, GW4869 treatment not only resulted in decreased LGTV transcript levels in tick cells and EVs derived from these infected cells, but also revealed diminished EVs concentrations. Enhanced IsSMase transcripts in the LGTV-infected group was noted upon GW4869 treatment, thus suggesting a host response to perhaps inhibit virus replication. In addition, GW4869 treatment reduced LGTV loads in density gradient EVs fractions, which correlated with decreased EVs concentration in those fractions. These data not only indicate that GW4869 affects LGTV replication, but that it also interferes with EV secretion and release from tick cells. Lastly, we found that GW4869 inhibits LGTV replication in tick cells but does not directly affect the infectivity of LGTV viral particles. Overall, our study suggests that GW4869 is a potential therapeutic inhibitor in controlling tick-borne diseases. Full article

Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of yet. Here, we variably fused the wildtype SARS-CoV-2 spike, its receptor-binding domain (RBD) and nucleocapsid (NC) to the minimal CD16b-GPI anchor acceptor sequence for expression on the surface of VNP. Moreover, a CD16b-GPI-anchored single-chain version of IL-12 was tested for its adjuvanticity. VNPs expressing RBD::CD16b-GPI alone or in combination with IL-12::CD16b-GPI were used to immunize BALB/c mice intramuscularly and subsequently to investigate virus-specific humoral and cellular immune responses. CD16b-GPI-anchored viral molecules and IL-12-GPI were well-expressed on HEK-293T-producer cells and purified VNPs. After the immunization of mice with VNPs, RBD-specific antibodies were only induced with RBD-expressing VNPs, but not with empty control VNPs or VNPs solely expressing IL-12. Mice immunized with RBD VNPs produced RBD-specific IgM, IgG_2a and IgG₁ after the first immunization, whereas RBD-specific IgA only appeared after a booster immunization. Protein/peptide microarray and ELISA analyses confirmed exclusive IgG reactivity with folded but not unfolded RBD and showed no specific IgG reactivity with linear RBD peptides. Notably, booster injections gradually increased long-term IgG antibody avidity as measured by ELISA. Interestingly, the final immunization with RBD–Omicron VNPs mainly enhanced preexisting RBD Wuhan Hu-1-specific antibodies. Furthermore, the induced antibodies significantly neutralized SARS-CoV-2 and specifically enhanced cellular cytotoxicity (ADCC) against RBD protein-expressing target cells. In summary, VNPs expressing viral proteins, even in the absence of adjuvants, efficiently induce functional SARS-CoV-2-specific antibodies of all three major classes, making this technology very interesting for future vaccine development and boosting strategies with low reactogenicity. Full article

Wildlife serves as a significant reservoir for various pathogens transmissible to domestic animals and humans. Vector-borne diseases represent an increasing concern in Europe, affecting both animal and human health. This pilot study investigated the circulation of endemic and emerging vector-borne viruses in wild ungulates in Hungary, utilizing a One Health approach. Serum samples were obtained from European fallow deer (Dama dama), red deer (Cervus elaphus), and roe deer (Capreolus capreolus) during routine national game management activities between 2020 and 2023. Samples were analyzed for antibodies against the Bluetongue virus (BTV), West Nile virus (WNV), and Epizootic hemorrhagic disease virus (EHDV) using ELISA and neutralization tests. The results revealed a WNV seroprevalence of 22.3% in fallow deer and 31.8% in red deer, while BTV seroprevalence was 2.5% in fallow deer. All samples were negative for EHDV antibodies. These findings confirm the circulation of WNV and BTV in Hungarian wild ungulates. While the study’s design precludes statistical analysis due to non-random sampling, it demonstrates the potential of integrating wild ungulate serosurveillance into disease monitoring programs, leveraging established wildlife management activities for a cost-effective and complementary approach to One Health surveillance, particularly considering the ongoing spread of EHDV in Europe and the importance of BTV serotype monitoring for effective vaccination strategies. 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

Zika virus (ZIKV), once considered a relatively benign pathogen, has emerged as a cause of severe neurological complications, including Guillain-Barrè Syndrome and encephalitis. This report presents the case of a 21-year-old Brazilian woman who initially presented with fever, rash, and arthralgia. Seven days later, she developed confusion, speech impairment, and gait disturbance. Following a tonic-clonic seizure, neurological examination revealed dysphonia, dysarthria and facial palsy, suggestive of brainstem involvement. ZIKV infection was detected by positive IgM serology and a plaque reduction neutralization test. The patient was treated with corticosteroids and antiepileptic drugs, leading to substantial clinical improvement, and discharge after 25 days of hospitalization. This case underscores the neuroinvasive potential of ZIKV and highlights the importance of early recognition and management of atypical neurological manifestations. It also reinforces the need to consider ZIKV in the differential diagnosis of encephalitis, particularly in endemic regions, and contributes to the growing understanding of ZIKV neurotropism and possible therapeutic approaches for severe presentations. Full article

Background/Objectives: Rapid development of vaccines against SARS-CoV-2 was pivotal to controlling the COVID-19 pandemic. The emergency also provided a rare opportunity to test novel vaccine platforms such as mRNA in large clinical trials. Most of the early vaccines used SARS-CoV-2 Spike protein as the target antigen. Nevertheless, subsequent studies have shown that Receptor Binding Domain (RBD) of Spike also can yield efficacious vaccines, and we previously demonstrated that chemical conjugation of RBD to a carrier protein, EcoCRM^®, enhanced antibody responses and induced strong virus neutralization activity in mice. Methods: Here, we compared the immunogenicity of this conjugate to that of an approved mRNA vaccine from Pfizer/BioNTech in rhesus macaques over a period of nine months. Results: AS01-adjuvanted RBD conjugate induced a similar or better antibody response, receptor binding inhibition, and virus neutralization activity against different variants of SARS-CoV-2, compared to mRNA. IgG subclass profiles induced by conjugate and mRNA vaccines were initially dominated by IgG1 and IgG3 then switched to IgG2 and IgG4 dominant profiles during the subsequent six-month period. Polyclonal immune sera from the conjugate and mRNA had similar antibody avidity at multiple time points. Conclusions: In summary, antibody responses in rhesus macaques induced by the RBD-EcoCRM conjugate and the Spike mRNA vaccine are very similar. These results demonstrate the potential for the RBD-EcoCRM conjugate as a vaccine against SARS-CoV-2. Full article

Neuroinvasive arboviruses such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Usutu virus (USUV), and Toscana virus (TOSV) have (re-)emerged with increasing incidence and geographic range. We analyzed the epidemiology of arboviral infections in Croatia during the 2024 transmission season. A total of 154 patients with neuroinvasive diseases (NID), 1596 horses, 69 dead birds, and 7726 mosquitoes were tested. Viral RNA was detected using RT-qPCR. IgM/IgG-specific antibodies were detected using commercial ELISA or IFA, with confirmation of cross-reactive samples by virus neutralization test. RT-qPCR-positive samples were Sanger sequenced. Arboviral etiology was confirmed in 33/21.42% of patients with NID. WNV was most frequently detected (17/11.03%), followed by TBEV (10/6.49%), USUV (5/3.24%), and TOSV (1/0.64%). WNV infections were reported in regions previously known as endemic, while in one continental county, WNV was recorded for the first time. USUV infections re-emerged after a six-year absence. In addition to human cases, acute WNV infections were recorded in 11/395 (2.78%) of horses and two dead crows. WNV IgG seropositivity was detected in 276/1168 (23.63%) and TBEV IgG seropositivity in 68/428 (15.88%) horses. None of the tested mosquito pools were positive for WNV and USUV RNA. Phylogenetic analysis showed the circulation of WNV lineage 2 and Usutu Europe 2 lineage. Climate conditions in 2024 in Croatia were classified as extremely warm, which could, at least in part, impact the quite intense arboviral season. The spreading of flaviviruses in Croatia highlights the need for continuous surveillance in humans, animals, and vectors (“One Health”). 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

Background: canine distemper (CD) is a highly contagious and fatal disease caused by canine distemper virus (CDV), posing a significant threat to carnivores. New CDV strain circulation and multi-species infection may lead to the potential dilemma of safety concern and insufficient efficacy of the commercial modified live vaccines. Safe and effective vaccines for canine and wildlife prevention of CD need to be continuously updated and developed. Methods: we developed two DNA vaccines, p17F-LNP and p17H-LNP, encoding the fusion protein (F) or hemagglutinin protein (H) of a field CDV strain (HLJ17) and encapsulated in lipid nanoparticles (LNPs). Serum neutralizing antibody (NAb) was evaluated via neutralization tests, and mouse serum cytokine detection were evaluated via ELISA. Results: immunization of p17F-LNP and p17H-LNP monovalent or bivalent were safe, and induced robust CDV NAb and cytokine responses in mice. LNP encapsulation improved immune responses compared to naked DNA formulation, and the bivalent formulation of p17F-LNP and p17H-LNP (p17F/H-LNP) exhibited synergistic effects with a high level of immune responses. Moreover, two doses of p17F/H-LNP induced long-lasting CDV NAb for over 300 days in dogs, and prime and boost NAb responses in foxes and raccoon dogs. Conclusions: the preliminary findings provided here warrant further development of the p17F/H-LNP vaccine for animal targets against CDV infection. Full article

Human respiratory syncytial virus (RSV) causes acute respiratory illness, attributing to deaths among young children and older adults worldwide. RSV neutralization assay is an important tool to measure RSV neutralization antibody that can prevent infection and severe complication of RSV. Conventional RSV neutralization assays have some limitations of speed and cost, especially for expensive kits, reagents or instruments required for detection. To solve this problem, this paper describes an improved simple and economical RSV neutralization assay protocol using recombinant RSV (rRSV) expressing reporter fluorescent protein to measure RSV growth as reporter activity with plate reader. The condition of 3 days culture demonstrated sufficient fluorescent activity even when small amounts of rRSV were used to inoculate Hep-2 cells. In addition, white 96-well cell culture plate showed better stable reporter activities than black plate. Furthermore, RSV neutralization assay protocol using rRSV-reporter fluorescent protein demonstrated similar signal detection capacity for RSV antibody titer detection compared to other protocols, such as rRSV-Luciferase and ELISA assay. The new RSV neutralization assay protocol can be applied to RSV antibody titration of numerous samples necessary for RSV surveillance or antiviral testing. Full article