Search Results (189)

The ability to study mature neuronal cells ex vivo is complicated by their non-dividing nature and difficulty in obtaining large numbers of primary cells from organisms. Thus, numerous transformed progenitor models have been developed that can be routinely cultured, then scaled, and differentiated to mature neurons. In this paper, we present a new method for differentiating one such model, the Lund human mesencephalic (LUHMES) dopaminergic neurons. This method is two days faster than some established protocols, results in nearly five times greater numbers of mature neurons, and involves fewer handling steps that could introduce technical variability. Moreover, it overcomes the problem of cell aggregate formation that commonly impedes high-resolution imaging, cell dissociation, and downstream analysis. While recently established for herpes simplex virus type 1, we demonstrate that LUHMES neurons can facilitate studies of other herpesviruses, as well as RNA viruses associated with childhood encephalitis and hemorrhagic fever. This protocol provides an improvement in the generation of large-scale neuronal cultures, which may be readily applicable to other neuronal 2D cell culture models and provides a system for studying neurotrophic viruses. We named this method the Streamlined Protocol for Enhanced Expansion and Differentiation Yield, or SPEEDY, method. Full article

Research on highly pathogenic biosafety level 4 (BSL-4) viruses that are classified as Select Agents involves transferring inactivated materials to lower containment levels for further analysis. Compliance with Select Agent and BSL-4 safety regulations necessitates the validation and verification of inactivation procedures. To streamline this process, it would be beneficial to use surrogate BSL-2 viruses for inactivation studies. This not only simplifies BSL-4 work but also enables the testing and validation of inactivation procedures in research facilities that lack access to high-containment laboratories yet may receive samples containing highly pathogenic viruses that require efficient and complete inactivation. In this study, we used Hazara virus (HAZV) as a surrogate virus for Crimean–Congo hemorrhagic fever virus to show the efficacy of various inactivation methods. We demonstrate the successful inactivation of HAZV using TRIzol/TRIzol LS and aldehyde fixation. Importantly, the parameters of the aldehyde inactivation of cell pellets differed from those of the monolayers, highlighting the importance of inactivation validation. As part of this study, we also defined specific criteria that must be met by a BSL-2 virus to be used as a surrogate for a closely related BSL-4 virus. Defining these criteria helps identify suitable nonpathogenic surrogates for developing inactivation procedures for highly pathogenic viruses. Full article

Orthoflaviviruses (formerly flaviviruses) are known for their role in numerous diseases affecting both humans and animals. Despite the worldwide distribution of orthoflaviviruses, individual species are only found in endemic or epidemic regions. However, in recent decades, certain orthoflaviviruses have spread beyond their traditional geographic boundaries, even crossing continents. Given the long-distance movements of birds, the knowledge of zoonotic orthoflaviviruses associated with birds is essential because of their possible introduction into new regions, as was the case with West Nile virus and Usutu virus. A thorough literature review was conducted on zoonotic orthoflaviviruses related to birds, including lesser-known (re-)emerging and neglected orthoflaviviruses that are limited to specific regions and/or avian hosts but have the potential to spread to a wider geographical area and pose a higher risk of transmission to humans. Several of these viruses possess significant zoonotic potential and can cause a wide spectrum of diseases in humans, ranging from mild febrile illnesses (Zika virus) to severe neuroinvasive diseases (tick-borne encephalitis, West Nile, Japanese encephalitis virus) and hemorrhagic fevers (yellow fever, dengue virus). Geographic distribution, hosts, vectors, incidence of human infections, and impact on human and animal health of zoonotic flaviviruses related to birds are critically reviewed. The viruses have been categorized based on the role of birds as an orthoflavivirus host and the clinical presentation in human infections. Full article

Tick-borne viruses (TBVs), notably Orthonairovirus haemorrhagiae (Crimean–Congo hemorrhagic fever virus, CCHFV), are emerging global health threats intensified by climate change. Rising temperatures and altered precipitation patterns are expanding the habitats of key tick vectors, increasing their survival and reproductive success. The African continent is characterized by many different climatic zones, and climatic shifts have increased or changed CCHFV transmission patterns, becoming greater risk to humans and livestock. Beyond Africa, CCHFV spread in Europe, the Middle East, and Asia and has been facilitated by factors such as livestock movement, deforestation, and migratory birds. Climate-driven shifts in tick seasonality, behavior, and vector competence may further enhance viral transmission. Addressing these challenges requires integrated responses, including enhanced surveillance, predictive modeling, and climate-adaptive vector control strategies. A One Health approach—linking environmental, animal, and human health domains—is essential. Innovative strategies such as anti-tick vaccines and sustainable vector control methods offer promise in reducing the burden of these diseases. Proactive, collaborative efforts at regional and international levels are crucial in tackling this growing public health challenge. Full article

Zoonotic viral diseases have continued to threaten global public health in recent decades, with rodent-borne viruses being significant contributors. Infection by rodent-carried hantaviruses (HV) can result in hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) in humans, with varying degrees of morbidity and mortality. However, no Food and Drug Administration (FDA) vaccines or therapeutics have been approved for the treatment of these diseases. In an effort to identify antiviral bioactive molecules, we isolated four oligomeric phloroglucinols from Callistemon rigidus leaves, including two new phloroglucinol trimers, callistemontrimer A and B, along with two previously characterized phloroglucinol dimers, rhodomyrtosone B and rhodomyrtone. We evaluated the anti-Hantaan virus (HTNV) activity of these compounds. Notably, callistemontrimer A demonstrated higher anti-HTNV activity compared to ribavirin. Mechanistic studies revealed that callistemontrimer A exerted its antiviral effects by inhibiting viral replication, likely through interaction with RNA-dependent RNA polymerase (RdRp) of HTNV, as supported by molecular docking analysis. These results highlight oligomeric phloroglucinols as promising lead candidates for the development of anti-HV therapeutics. Full article

Background: African Swine Fever (ASF) is a viral hemorrhagic disease characterized by diverse clinical and pathological manifestations depending on the virulence of isolates/strains and the immunological status of pigs. The use of modified live viruses (MLVs) is currently the most common approach in developing vaccines against ASF. However, despite the availability of dozens of MLV candidates that meet basic safety and efficacy criteria—such as the absence of severe clinical signs and survival after challenge with a virulent strain—no broadly accepted vaccine has yet been developed. Here, we propose viremia testing as an essential criterion for evaluating candidate ASF vaccines, with levels exceeding 10⁴ HAD₅₀/TCID₅₀ and lasting longer than 21–28 days post vaccination considered unfavorable indicators. Methods: We analyzed ASF MLV vaccines obtained through the deletion of one, two, or more genes, focusing on viremia kinetics after vaccination and challenge with virulent ASFV strains. Post mortem data were used to assess viral persistence in organs. Results: Most MLV candidates, especially those with single-gene deletions, demonstrated relatively high viremia levels after vaccination and challenge. Viral persistence was frequently detected in organs upon necropsy. MLVs with an additional EP402R gene deletion showed low viremia after vaccination but high levels after challenge. Nevertheless, several candidates with favorable viremia profiles were identified, including those obtained via targeted deletions or serial passaging in cell cultures. Conclusions: Incorporating viremia assessment as a primary screening criterion can significantly narrow down the selection of promising MLV candidates and help accelerate the development of effective emergency vaccines for use in ASF-affected regions. Full article

Since the emergence of Junín virus in 1953, pathogenic New World arenaviruses have remained a public health concern. These viruses, which also include Machupo virus, Guanarito virus, Sabiá virus, and Chapare virus, cause acute viral hemorrhagic fever and neurological complications, resulting in significant morbidity and mortality. Given the dearth of licensed therapeutics or vaccines against these pathogens, animal models of infection that recapitulate human manifestations of disease remain critically important to the development of efficacious medical countermeasures. Rodents and non-human primates have been successfully used to model human New World arenaviral infections, with guinea pigs, rhesus macaques, and cynomolgus macaques being the most successful models of infection for most major pathogenic New World arenaviruses. Here, we provide a highly comprehensive review of publicly reported animal models of pathogenic New World arenavirus infections, with a discussion of advantages and disadvantages for each model. Full article

Arboviruses, transmitted by blood-sucking arthropods, are responsible for significant human and animal diseases, including fever, hemorrhagic fever, and encephalitis, posing a serious threat to global public health. Nevertheless, research on the mechanisms of arbovirus infection and the development of therapeutic interventions has been impeded. This delay is primarily due to the limitations inherent in current in vitro research models, including cell cultures and animal models. The simplicity of cell types and interspecies differences present significant obstacles to advancing our understanding of arbovirus infection mechanisms and the development of effective drugs. Human brain organoids, derived from human pluripotent stem cells or human embryonic stem cells and cultured in three-dimensional systems, more accurately replicate the extensive neuronal cellular diversity and key characteristics of human neurodevelopment. These organoids serve as an ideal model for investigating the intricate interactions between viruses and human hosts, and providing a novel platform for the development of antiviral drugs. In this review, we summarize how brain organoid models complement classical approaches to accelerate research into the infection mechanisms of arboviruses, with a particular focus on the types of neural cells, key factors, and cellular signaling pathways involved in the arbovirus infection of brain organoids that have been reported. Furthermore, we examine the development of brain organoids, address their current limitations, and propose future directions to enhance the application of brain organoids in the study of arboviral infectious diseases. Full article

We have reviewed the primary literature on the virucidal efficacy of microbicidal active ingredients, formulated microbicides, and physical inactivation approaches (heat, irradiation) for hemorrhagic fever viruses (HFVs) (arenaviruses, filoviruses, flaviviruses, hantaviruses, nairoviruses, and phenuiviruses), and for two non-typical HFV paramyxoviruses. As each of these HFVs are large, lipid-enveloped RNA viruses, their susceptibilities to virucidal agents are informed by the so-called hierarchy of susceptibility of pathogens to microbicides. The unique susceptibility of lipid-enveloped viruses to most classes of microbicides is based on the common mechanisms of action of envelope-disrupting microbicides. Despite this, due to the relatively great lethality of these viruses, it is prudent (where possible) to confirm the expected efficacies of inactivation approaches in testing involving the HFVs themselves (as opposed to less lethal surrogate viruses) using field-relevant methods. Empirical data for virucidal activities of microbicidal active ingredients, formulated microbicides, and physical inactivation approaches, such as heat, ultraviolet light, and gamma irradiation, that were collected specifically for HFVs have been reviewed and summarized in this paper. These empirical data for surface and hand hygiene approaches, liquid inactivation approaches, and approaches for rendering diagnostic samples safe to handle inform non-pharmaceutical interventions intended to mitigate transmission risk associated with these HFVs. Full article

Handling cultured isolates and clinical, environmental, or wildlife surveillance samples containing Risk Group 3 and 4 pathogens presents considerable biosafety challenges in minimizing human exposure during processing and transport. Safe handling typically requires high- or maximum-containment facilities, demanding substantial logistical planning and resources. We evaluated PrimeStore Molecular Transport Medium (PS-MTM), a guanidine-based solution created to kill pathogens and preserve nucleic acids at ambient temperatures, for inactivating Crimean-Congo hemorrhagic fever, eastern equine encephalitis, Ebola, Hendra, Japanese encephalitis, Lassa, Marburg, Nipah, Rift Valley fever, and West Nile viruses. To mimic diagnostic conditions, human whole blood spiked with any of these viruses was incubated with PS-MTM for 20-, 30-, or 60-min. Samples with titers up to 10⁷ PFU/mL exposed to PS-MTM at all time points resulted in complete loss of infectivity judged by plaque assays. A 30-min incubation provided a 50% safety margin over the minimum inactivation time and was used for quantification with the tissue culture infectious dose (TCID₅₀) assay, enabling evaluation of PS-MTM’s activity for viruses that do or do not produce well-defined plaques. Results confirmed that PS-MTM inactivated all tested viruses at titers up to 10⁷ TCID₅₀/mL, underscoring its reliability for enhancing biosafety in diagnostics, outbreak management, and surveillance. Full article

Background/Objectives: African swine fever (ASF), a fatal febrile hemorrhagic disease in domestic pigs and Eurasian wild boars, is caused by ASF virus (ASFV). ASF continues to spread across the globe, causing a significant impact on the world’s pig industry. Recently, highly virulent chimeric ASFV (chASFV) strains with recombined genomes of the p72 genotype I and II viruses have been reported in China, Vietnam and Russia. Methods: In order to understand the propensity of ASFV genome for recombination, we attempted to experimentally generate chASFVs both in vitro and in vivo employing two distinct attenuated ASFV strains: OUR T88/3 (genotype I) and AQSΔB119L (genotype II). Results: When IPKM cells were co-infected with ASFV OUR T88/3 and AQSΔB119L strains, three genetically distinct chASFV emerged. When pigs were inoculated with the individual chASFV isolates, all pigs developed acute ASF. When four pigs were co-infected with ASFV OUR T88/3 and AQSΔB119L, all of them developed acute ASF and died or were euthanized. Three chASFV strains were successfully isolated from splenic homogenates from each pig. Conclusions: Our research indicates that genotype I and II chASFV with diverse genomes can be easily generated experimentally both in vitro and in vivo. Full article

A novel ephemeral fever rhabdovirus and a CHeRI orbivirus of a previously unidentified genetic lineage were isolated in mosquito cell line C6/36 cells as co-infecting agents from the spleen tissue of a dead farmed white-tailed deer (WTD; Odocoileus virginianus) in Florida. We designated the ephemeral fever rhabdovirus as Hardee County ephemerovirus 1, strain CHeRI ephemerovirus 1. The genetic sequences of the CHeRI orbivirus isolated in this work differ significantly from those of three previously described CHeRI orbivirus lineages. We designated this new virus as CHeRI orbivirus 4, strain CHeRI orbivirus 4-1. Whereas it remains unknown whether one, both, or none of the viruses contributed to the pathology, gross observations revealed that the dead WTD had severely congested and hemorrhagic lungs, and that its heart, kidneys, and spleen were also congested. Full article

Crimean–Congo hemorrhagic fever virus (CCHFV) causes severe or fatal infections in humans and is geographically widespread. The virus has coevolved with its tick vectors, establishing persistent infections critical to its transmission. This study explored the mechanisms underpinning these persistent infections, using tick cell lines and the Hazara virus (HAZV) as a biosafety level 2 (BSL-2) model for CCHFV. Initially, an RT-qPCR protocol was developed to detect HAZV in tick cells. The study then focused on the production of virus-derived DNA (vDNAs) by tick cells as a defensive response to infection. These vDNAs regulate viral particle production, enabling tick cells to maintain viability and establish persistent infections. The experiments characterized vDNAs production, viral titers, and subcellular localization, and they examined the effect of the reverse transcriptase inhibitor azidothymidine triphosphate (AZT). The results showed that all tested tick cell lines supported HAZV replication, achieving persistent infections without cytopathic effects. vDNAs was detected in both the cytoplasm and nucleus, and its formation was dependent on HAZV infection. Importantly, vDNAs presence was linked to infection persistence; cells treated with AZT exhibited a marked reduction in vDNAs production and an associated increase in viral particle production, which correlated with higher cell death. These findings underscore the critical role of vDNAs in balancing viral replication and promoting long-term cell survival in tick cells, highlighting their importance in the coevolution of tick-borne viruses and their vectors. Full article

Statins are among the most frequently prescribed medications. In addition to their well-established effectiveness in lowering total cholesterol, LDL, and triglycerides, statins have been described to have immunomodulatory and anti-inflammatory properties and have been associated with improved endothelial functions. Given the common use of statins, we sought to evaluate the effect of statins on some viral infections encountered by residents in tropical areas or by international travelers. A literature search was performed in PubMED/MEDLINE focusing on keywords that included statins and the viruses of interest, including SARS-CoV-2, influenza, yellow fever, dengue, Zika, tick-borne encephalitis, hemorrhagic fever viruses, hepatitis A, norovirus, hepatitis B, hepatitis C, measles, and herpesviruses; findings were synthesized for each virus into a summary. The effects of statins on viral infections vary depending on the specific virus. While some studies indicate potential benefits in chronic HBV and HCV infections, evidence regarding SARS-CoV-2 and influenza remains inconclusive due to mixed findings from observational studies and randomized controlled trials. The role of statins in other viral infections is largely unexplored, with preclinical data available for only a few viruses. Given the conflicting evidence, further prospective studies and randomized controlled trials are warranted to elucidate statins’ role in viral infections, particularly in modulating inflammation, endothelial dysfunction, and immune responses. Future research should aim to define the optimal patient populations, target viruses, statin types, and treatment durations that may confer benefits in specific viral infections. Full article

Omsk hemorrhagic fever virus (OHFV) is the etiological agent of a poorly studied acute viral disease, causing several epidemic waves observed in the western Siberia regions of Omsk, Kurgan, Novosibirsk, and Tyumen. OHFV is a flavivirus and shares structural and morphological features with tick-borne encephalitis (TBE) complex viruses. The disease’s symptoms show high variability, from flu-like symptoms, hyperesthesia, and petechial rush in the upper body to high fever and hemorrhagic manifestations, with a fatality rate of about 1%. The real number of OHFV-infected people is still unknown due to the difficulties in diagnosis and the presence of asymptomatic patients that lead to an underestimation of the total cases. Little is known about the viral infection dynamics at the molecular and cellular levels, the viral involvement in immune escape, cellular pathways alteration, or metabolic influence. It is noteworthy that no clinical trials have currently been performed for effective and specific drug treatments. In this review, we will give an overview of OHFV interactions with humans and animals, diagnostic tools, and drug treatments. We aim to highlight the importance of a frequently undiagnosed or misdiagnosed viral infection that might also even cause severe clinical manifestations such as meningitis and hemorrhage, in order to point out the need to develop new research studies, new diagnostic tools, and new treatments for OHFV. Full article