Search Results (52)

Retroviruses are single-stranded RNA viruses that package two copies of their positively stranded RNA genomes as a non-covalent dimer into newly formed virions. This process is evolutionarily conserved, and disruption of genome dimerization results in production of non-infectious virus particles. Genome dimers can be packaged as homodimers, containing two identical RNAs, or heterodimers, consisting of two genetically distinct copies. Genome dimerization generates genetic diversity, and different retroviruses have preferences for the type of genome dimers packaged into virions. We developed a novel imaging approach to specifically label and detect retroviral genome heterodimers in cells using a modified bimolecular fluorescence complementation (BiFC) technique. This method utilizes viral genomes encoding two different RNA stem-loop cassettes that each specifically binds to an RNA-binding protein conjugated to a split fluorophore. When two genetically different genomes are within close proximity, the fluorophore halves come together to reconstitute fluorescence. These BiFC-labeled RNA dimers can be visualized and tracked in living cells and interact with retroviral Gag proteins. This method has the advantage of low background fluorescence and can be applied to the study of dimeric or double-stranded RNAs of viruses and other organisms. Full article

The objective of this study was to evaluate the time-course of incubation for the potential preventative mitigation of megaviruses using Termin-8 (a formaldehyde-based product) and Finio (non-formaldehyde solution) from Anitox. Emiliania huxleyi virus (EhV), an algal surrogate for African swine fever virus (ASFV), was treated with the recommended concentrations of Termin-8 (0.1% to 0.3%) and Finio (0.05% to 0.2%), and both viability qPCR (V-qPCR) and standard PCR (S-qPCR) were used to quantify EhV concentrations at 1 h, 5 h, 24 h and day 7 post-inoculation. Overall, Finio, and to a lesser extent Termin-8, at their highest treatment concentrations, showed the greatest log reduction of 4.5 and 2 log₁₀ units, respectively, at 1 h post-inoculation. Although Termin-8 efficacy did not improve with time, due to its fixing of viral particles and rendering them non-infectious, treatment with Finio showed 100% viable viral inactivation (>5 log₁₀ reduction units) at the lowest concentration after 7 days of exposure. Our results demonstrate that both Termin-8 and Finio can be used as effective chemical mitigants against megaviruses such as EhV and ASFV and can be used as effective preventive or mitigation strategies to prevent the transmission of ASFV by reducing particle viability in contaminated feed, although additional research is warranted. Full article

Background/Objectives: The use of virus-like particles (VLPs) in vaccinology has expanded significantly in recent years. VLPs have the advantage of being non-infectious while effectively stimulating B cell responses through the repetitive presentation of epitope motifs on their surface. Since VLPs are often derived from human-infecting viruses, preexisting immunity may influence the immune response they elicit, warranting further investigation. Methods: We have developed a 60-mer VLP derived from human adenovirus type 3, a common pathogen. We investigated the impact of pre-existing adenovirus immunity on the immunization outcome against the linear S14P5 epitope of SARS-CoV-2, which was engineered into the particle (Ad-VLP-S14P5). To this end, antibody responses to S14P5 were evaluated following immunization with Ad-VLP-S14P5 in either naive or vector-primed mice. Results: Mice with pre-existing anti-vector immunity exhibited significantly greater anti-S14P5 antibody responses compared to vector-naive animals, demonstrating a beneficial impact of prior anti-adenovirus responses. However, the addition of an oil-in-water adjuvant for the immunizations abolished this positive impact, even leading to a deleterious effect of the pre-existing anti-vector immunity. Conclusions: The data suggest that the immune status against immunizing VLPs must be taken into consideration when designing immunization protocols. Importantly, the effects of prior immunity may vary depending on the nature of the protocol, including factors such as adjuvant use. Full article

Background/Objectives: Virus-like particles (VLPs) represent an attractive platform for delivering vaccine formulations, combining a high biosafety profile with a potent immune-stimulatory ability. VLPs are non-infectious, non-replicating, self-assembling nanostructures that can be exploited to efficiently expose membrane-tethered glycoproteins such as the SARS-CoV-2 Spike (S) protein, the main target of approved preventive vaccines. Here, we describe the development and preclinical validation of Simian Immunodeficiency Virus (SIV)-based GFP-labeled VLPs displaying S from the B.1.617.2 (Delta) variant (VLP/S-Delta) for inducing persistent anti-SARS-CoV-2 neutralizing antibodies (nAbs) and S-specific T cell responses in mice. Methods: SIV-derived VLP/S-Delta were produced by co-transfecting a plasmid expressing SIVGag-GFP, required for VLP assembly and quantification by flow virometry, a plasmid encoding the Delta S protein deleted in the cytoplasmic tail (CT), to improve membrane binding, and a VSV.G-expressing plasmid, to enhance VLP uptake. Recovered VLPs were titrated by flow virometry and characterized in vitro by transmission electron microscopy (TEM) and confocal microscopy (CLSM). BALB/c mice were immunized intramuscularly with VLP/S-Delta following a prime–boost regimen, and humoral and cellular immune responses were assessed. Results: VLP/S-Delta were efficiently pseudotyped with CT-truncated S-Delta. After BALB/c priming, VLP/S-Delta elicited both specific anti-RBD IgGs and anti-Delta nAbs that significantly increased after the boost and were maintained over time. The prime–boost vaccination induced similar levels of cross-nAbs against the ancestral Wuhan-Hu-1 strain as well as cross-nAbs against Omicron BA.1, BA.2 and BA.4/5 VoCs, albeit at lower levels. Moreover, immunization with VLP/S-Delta induced S-specific IFNγ-producing T cells. Conclusions: These data suggest that SIV-based VLPs are an appropriate delivery system for the elicitation of efficient and sustained humoral and cellular immunity in mice, paving the way for further improvements in the immunogen design to enhance the quality and breadth of immune responses against different viral glycoproteins. Full article

Since the discovery of the Australia antigen, now known as the hepatitis B surface antigen (HBsAg), significant research has been conducted to elucidate its physical, chemical, structural, and functional properties. Subviral particles (SVPs) containing HBsAg are highly immunogenic, non-infectious entities that have not only revolutionized vaccine development but also provided critical insights into HBV immune evasion and viral assembly. Recent advances in cryo-electron microscopy (cryo-EM) have uncovered the heterogeneity and dynamic nature of spherical HBV SVPs, emphasizing the essential role of lipid–protein interactions in maintaining particle stability. In this review, recent progress in understanding the molecular architecture of HBV SVPs is consolidated, focusing on their symmetry, lipid organization, and disassembly–reassembly dynamics. High-resolution structural models reveal unique lipid arrangements that stabilize hydrophobic residues, preserve antigenicity, and contribute to SVP functionality. These findings highlight the significance of hydrophobic interactions and lipid–protein dynamics in HBV SVP assembly and stability, offering valuable perspectives for optimizing SVP-based vaccine platforms and therapeutic strategies. Full article

Epidemiological, histological, and immunogenetic studies suggest that podoconiosis (a non-infectious tropical lymphoedema affecting approximately 4 million people globally) is an HLA class II-associated inflammatory condition that develops in response to an unknown trigger found in volcanic red clay soils. Silicate particles of the kaolinite and aluminum types have been identified in femoral lymph node biopsy samples from endemic area residents, suggesting a possible role in the pathogenesis of podoconiosis. We measured in vitro peripheral blood mononuclear cell cytokine responses (TNF-α, IL-1β, and IFN-γ) to stimulation with the minerals kaolinite, chlorite, and beryllium sulfate (all at 100 µM) using ELISA. Real time PCR was used to measure gene expression of signature cytokines in fresh whole blood, comparing podoconiosis patients and endemic healthy controls. Our results showed that the levels of TNF-α and IL-1β from in vitro cell cultures were significantly higher in unstimulated samples from patients compared to controls (p = 0.04 and p = 0.005, respectively). The minerals kaolinite and chlorite induced two and three-fold higher levels of IL-1β following 24 h of stimulation in healthy controls compared to patients, respectively. We did not find significant differences in mRNA expression of the cytokine genes assayed, though a slight fold increment in IL-1β and TGF-β was observed. In conclusion, our data suggest that the immune system is in a state of persistent activation in vivo in podoconiosis patients, and additional studies of immune regulation and exhaustion are needed to further characterize immune dysfunction in the pathogenesis of the disease. A better understanding of the underlying processes could lead to the development of a ‘biosignature’ detectable in the early reversible stages that could ultimately contribute to the elimination of this preventable, disabling, neglected tropical disease. Full article

HIV-1 virion maturation is an essential step in the viral replication cycle to produce infectious virus particles. Gag and Gag-Pol polyproteins are assembled at the plasma membrane of the virus-producer cells and bud from it to the extracellular compartment. The newly released progeny virions are initially immature and noninfectious. However, once the Gag polyprotein is cleaved by the viral protease in progeny virions, the mature capsid proteins assemble to form the fullerene core. This core, harboring two copies of viral genomic RNA, transforms the virion morphology into infectious virus particles. This morphological transformation is referred to as maturation. Virion maturation influences the distribution of the Env glycoprotein on the virion surface and induces conformational changes necessary for the subsequent interaction with the CD4 receptor. Several host factors, including proteins like cyclophilin A, metabolites such as IP6, and lipid rafts containing sphingomyelins, have been demonstrated to have an influence on virion maturation. This review article delves into the processes of virus maturation and Env glycoprotein recruitment, with an emphasis on the role of host cell factors and environmental conditions. Additionally, we discuss microscopic technologies for assessing virion maturation and the development of current antivirals specifically targeting this critical step in viral replication, offering long-acting therapeutic options. Full article

Virus-like particles (VLPs) are non-infectious and serve as promising vaccine platforms because they mimic the membrane-embedded conformations of fusion glycoproteins on native viruses. Here, we employed SARS-CoV-2 VLPs (SMEN) presenting ancestral, Beta, or Omicron spikes to identify the variant spike that elicits potent and cross-protective immune responses in the highly sensitive K18-hACE2 challenge mouse model. A combined intranasal and intramuscular SMEN vaccine regimen generated the most effective immune responses to significantly reduce disease burden. Protection was primarily mediated by antibodies, with minor but distinct contributions from T cells in reducing virus spread and inflammation. Immunization with SMEN carrying ancestral spike resulted in 100, 75, or 0% protection against ancestral, Delta, or Beta variant-induced mortality, respectively. However, SMEN with an Omicron spike provided only limited protection against ancestral (50%), Delta (0%), and Beta (25%) challenges. By contrast, SMEN with Beta spikes offered 100% protection against the variants used in this study. Thus, the Beta variant not only overcame the immunity produced by other variants, but the Beta spike also elicited diverse and effective humoral immune responses. Our findings suggest that leveraging the Beta variant spike protein can enhance SARS-CoV-2 immunity, potentially leading to a more comprehensive vaccine against emerging variants. Full article

Introduction: Vault particles are large cytoplasmic ribonucleoprotein particles that participate in inflammation. The aim of this study was to assess the diagnostic and prognostic value of major vault protein (MVP) in patients with inflammation, in order to determine whether MVP could be used as a biomarker for infection or inflammation. We also aimed to compare the diagnostic impact of MVP compared to other conventional measurements, such as CRP or white blood cell (WBC) counts. Methods: CRP and MVP levels were measured in 111 sera samples from 85 patients with inflammation admitted to a tertiary-care hospital and 26 healthy individuals during an 18-month period (2019–2020), using nephelometry and a custom MVP sandwich ELISA assay, respectively. In addition, WBC counts were measured using a commercial assay. Results: MVP levels were found to be elevated in patients with inflammation compared to healthy individuals (p < 0.0001). Moreover, MVP levels were higher in patients with inflammation due to an infectious etiology compared to those with non-infectious etiology (p = 0.0006). MVP levels significantly decreased during the first four days of infection in response to antibiotic treatment, while CRP levels showed a less-sensitive decline. An ROC curve analysis demonstrated that MVP and CRP have similarly high diagnostic accuracy, with AUCs of 0.955 and 0.995, respectively, followed by WBCs with an AUC of 0.805. Conclusions: The ROC curves demonstrated that MVP has the potential to serve as a diagnostic biomarker for inflammation and infection. Additionally, MVP levels may reflect the efficacy of antibiotic treatment. Full article

As the Global Polio Eradication Initiative (GPEI) strategizes towards the final steps of eradication, routine immunization schedules evolve, and high-quality vaccination campaigns and surveillance systems remain essential. New tools are consistently being developed, such as the novel oral poliovirus vaccine to combat outbreaks more sustainably, as well as non-infectiously manufactured vaccines such as virus-like particle vaccines to eliminate the risk of resurgence of polio on the eve of a polio-free world. As the GPEI inches towards eradication, re-strategizing in the face of evolving challenges and preparing for unknown risks in the post-certification era are critical. Full article

Inactivated poliovirus vaccine (IPV), available since 1955, became the first vaccine to be used to protect against poliomyelitis. While the immunogenicity of IPV to prevent paralytic poliomyelitis continues to be irrefutable, its requirement for strong containment (due to large quantities of live virus used in the manufacturing process), perceived lack of ability to induce intestinal mucosal immunity, high cost and increased complexity to administer compared to oral polio vaccine (OPV), have limited its use in the global efforts to eradicate poliomyelitis. In order to harvest the full potential of IPV, a program of work has been carried out by the Global Polio Eradication Initiative (GPEI) over the past two decades that has focused on: (1) increasing the scientific knowledge base of IPV; (2) translating new insights and evidence into programmatic action; (3) expanding the IPV manufacturing infrastructure for global demand; and (4) continuing to pursue an ambitious research program to develop more immunogenic and safer-to-produce vaccines. While the knowledge base of IPV continues to expand, further research and product development are necessary to ensure that the program priorities are met (e.g., non-infectious production through virus-like particles, non-transmissible vaccine inducing humoral and intestinal mucosal immunity and new methods for house-to-house administration through micro-needle patches and jet injectors), the discussions have largely moved from whether to how to use this vaccine most effectively. In this review, we summarize recent developments on expanding the science base of IPV and provide insight into policy development and the expansion of IPV manufacturing and production, and finally we provide an update on the current priorities. Full article

Chronic wasting disease (CWD) is a highly infectious, fatal prion disease that affects cervid species. One promising method for CWD surveillance is the use of detection dog–handler teams wherein dogs are trained on the volatile organic compound signature of CWD fecal matter. However, using fecal matter from CWD-positive deer poses a biohazard risk; CWD prions can bind to soil particles and remain infectious in contaminated areas for extended periods of time, and it is very difficult to decontaminate the affected areas. One solution is to use noninfectious training aids that can replicate the odor of fecal matter from CWD-positive and CWD-negative deer and are safe to use in the environment. Trained CWD detection dogs’ sensitivity and specificity for different training aid materials (cotton, GetXent tubes, and polydimethylsiloxane, or PDMS) incubated with fecal matter from CWD-positive and CWD-negative deer at two different temperatures (21 °C and 37 °C) for three different lengths of time (6 h, 24 h, and 48 h) were evaluated. Cotton incubated at 21 °C for 24 h was identified as the best aid for CWD based on the dogs’ performance and practical needs for training aid creation. Implications for CWD detection training and for training aid selection in general are discussed. Full article

Hepatitis E virus is a prominent cause of viral hepatitis worldwide. In Western countries, most infections are asymptomatic. However, acute self-limiting hepatitis and chronic cases in immunocompromised individuals can occur. Studying HEV is challenging due to its difficulty to grow in cell culture. Consequently, the detection of the virus mainly relies on RT-qPCR, which cannot differentiate between infectious and non-infectious particles. To overcome this problem, methods assessing viral integrity offer a possible solution to differentiate between intact and damaged viruses. This study aims at optimizing existing HEV cell culture models and RT-qPCR-based assays for selectively detecting intact virions to establish a reliable model for assessing HEV infectivity. In conclusion, these newly developed methods hold promise for enhancing food safety by identifying approaches for inactivating HEV in food processing, thereby increasing food safety measures. Full article

Hepatitis E virus (HEV) is a positive-sense single-stranded RNA virus and a major cause of acute viral hepatitis. HEV is responsible for 20 million infections worldwide in humans every year. HEV-3 and HEV-4 are zoonotic and are responsible for most of the HEV cases in developed countries. Consumption of contaminated pig meat or pig products is considered to be the main transmission route of HEV HEV-3 in Europe. Prevalence studies for HEV generally use PCR methods to detect the presence or absence of genomic RNA. However, these methods do not discriminate infectious virus particles from non-infectious material. Previously developed HEV cell culture systems only worked with high efficiency after cell line adaptation of the subjected virus strains. In this manuscript, the development of a culture system for the detection of infectious HEV strains is described. For this purpose, we optimized the isolation and the growth of primary hepatocytes from young piglets. Subsequently, the isolated hepatocytes were used to culture HEV of different origins, such as liver tissue samples and sausage samples. This method can be applied to better assess the risk of infection through consumption of food products associated with HEV RNA contamination. Full article

The outbreak of COVID-19 started in December 2019 and spread rapidly all over the world. It became clear that the development of an effective vaccine was the only way to stop the pandemic. It was the first time in the history of infectious diseases that the process of the development of a new vaccine was conducted on such a large scale and accelerated so rapidly. At the end of 2020, the first COVID-19 vaccines were approved for marketing. At the end of March 2023, over three years after the outbreak of the COVID-19 pandemic, 199 vaccines were in pre-clinical development and 183 in clinical development. The candidate vaccines in the clinical phase are based on the following platforms: protein subunit, DNA, RNA, non-replication viral vector, replicating viral vector, inactivated virus, virus-like particles, live attenuated virus, replicating viral vector combined with an antigen-presenting cell, non-replication viral vector combined with an antigen-presenting cell, and bacterial antigen-spore expression vector. Some of the new vaccine platforms have been approved for the first time for human application. This review presents COVID-19 vaccines currently available in the world, procedures for assurance of the quality and safety of the vaccines, the vaccinated population, as well as future perspectives for the new vaccine platforms in drug and therapy development for infectious and non-infectious diseases. Full article