Biology subjects

Alpizar, Y. A.

Publications and source records attributed to Alpizar, Y. A..

7 recordsLinked to original sources

bioRxiv2026

Cross-protection against Bundibugyo by Ebola and Sudan vaccines

Ebola outbreaks continue to expand across Central Africa, yet available countermeasures remain limited and virus-specific. Whether available vaccines developed against the related Ebola virus (EBOV) and Sudan virus (SUDV) can confer cross-protection against Bundibugyo virus (BDBV) is incompletely understood. Here, we developed a BDBV surrogate challenge model and evaluated cross-protective immunity conferred by a set of monovalent vaccine candidates representing all three Orthoebolavirus species affecting humans. A yellow fever 17D-vectored BDBV vaccine (YF-BDB) expressing the BDBV glycoprotein (GP) as antigen conferred homologous protection in Ifnar-/- mice, preventing systemic viral dissemination and providing survival following surrogate BDBV challenge. Intriguingly, also vaccination with YF-EBO or YF-SUD protected mice from such BDBV surrogate challenge. Conversely, while YF-BDB and YF-EBO fully protected against respective heterologous EBOV or BDBV infection, they conferred only partial protection against lethal challenge in a comparable SUDV model. Serological analyses revealed comparable titers of cross-reactive antibodies across all vaccine groups. However, neutralization, especially cross-neutralization, was limited suggesting a major role for non-neutralizing mechanisms in protection. These findings demonstrate asymmetry in cross-protective immunity among Orthoebolavirus vaccines and indicate that SUDV GP-based antigens may provide broader protection than EBOV- or BDBV-targeted candidates. However, our data add to the preliminary yet limited evidence that EBOV GP-based vaccines might offer at least some degree of protection against BDBV, the virus driving current Public Health Emergency of International Concern (PHIEC) in the Central-East Africa region.

microbiology↗

bioRxiv2025

Conserved phosphorylatable residues in motif G of positive-stranded virus RdRps regulate polymerase activity and suggest targets for drug design.

RNA viruses rely on an RNA-dependent RNA polymerase (RdRp) to replicate their genome. RdRps share a conserved core replicase structure described as a right hand within which RNA replication occurs. RNA polymerases contain a series of conserved motifs (A-G) that are essential for catalysis. Motif G, located at the RNA entry channel, guides the incoming RNA into the catalytic centre and and holds it in place during catalysis. Although RdRp phosphorylation has been reported, it has been scarcely studied. In most studied cases, phosphomimetic mutations reduced viral replication. In this study, we identified Theilers murine encephalomyelitis virus (TMEV) polymerase (3Dpol) residues that undergo some extent of phosphorylation in infected cells. Among these residues, Thr109 and Ser110 located in motif G are highly conserved in the sequences of picornavirus polymerases and in the structure of many positive-stranded virus polymerases, including nsp12 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using mutagenesis and reporter viruses, we show that phosphomimetic mutation of either residue abrogates viral replication, for both TMEV and SARS-CoV-2. Mutations of 3Dpol residues 109 and 110 into all other possible residues shows that, besides negatively charged phosphomimetic residues, bulky residues strongly inhibit replication, suggesting that phosphorylation inhibits polymerase activity by steric hindrance and/or through charge repulsion with RNA entering the catalytic core. Because these phosphorylatable residues are surface-exposed and conserved among viral polymerases, they represent promising targets for the rational design of broad-spectrum antiviral agents. ImportanceRNA viruses require an RNA-dependent RNA polymerase to replicate their genome. We identified in Theilers murine encephalomyelitis virus polymerase (TMEV 3Dpol) residues that undergo some extent of phosphorylation in infected cells. Among these residues, Thr109 and Ser110 are located in the entry channel of the polymerase, a region important for directing the RNA into the polymerase and locking it in place during catalysis. Incidentally, Thr109 and Ser110 are highly conserved in the sequences of picornavirus polymerases and in the structures of many positive-stranded virus polymerases including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp12. Our study revealed that, in both TMEV 3Dpol and SARS-CoV-2 nsp12, mutation of either residue into a negatively charged amino acid that mimics phosphorylation abrogates viral replication, suggesting phosphorylation would block polymerase activity. As these phosphorylated residues are accessible and conserved, they provide important candidate targets for the design of antiviral molecules.

microbiology↗

bioRxiv2025

Sex determination of Syrian hamster pups using placental tissues

Molecular sex determination in Syrian hamsters (Mesocricetus auratus) has been limited by incomplete annotation of Y-linked loci in currently available genome assemblies. Here, we evaluate the Y-linked gene PRSSLY, which encodes a testis-specific serine protease-like protein, as a molecular marker for genetic sexing of Syrian hamster embryonic and placental tissues. Primers flanking a conserved PRSSLY coding region produced a male-specific amplicon showing 100% concordance with results from the established KDM5C/KDM5D PCR assay in E15.5 tail biopsies. SYBR Green-based qPCR enables the accurate detection of PRSSLY, characterized by a unique melt-curve profile, exclusively in male samples, allowing for efficient and sensitive mid-throughput analysis. Among 417 placental samples from 39 dams tested, which naturally contain a mixture of maternal and embryonic genomes, the sex of individual pups was classified with 85% specificity, with the remaining ambiguous samples confirmed as male by PCR and agarose gel electrophoresis. This assay provides a robust and reproducible approach for accurate sex genotyping in developmental and reproductive studies using Syrian hamsters.

genetics↗

bioRxiv2025

A high-throughput cell-based platform for Rhinovirus C research and antiviral drug discovery.

Rhinoviruses (RVs) are the most common cause of upper respiratory infections, yet RV-C is particularly associated with severe respiratory disease, including exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Despite its clinical significance, RV-C research and antiviral development have been hindered by the lack of scalable and biologically relevant models for high-throughput screening (HTS). Here, we present the development, optimization, and validation of a scalable high-content imaging-based platform for RV-C research. Our approach combines engineered HeLa cells expressing CDHR3C529Y, a genetically stable mGreenLantern-reporter RV-C15 virus, and a phenotypic selection strategy to identify monoclonal cell lines with maximal and sustained permissiveness. This approach enabled assay optimization in both 96- and 384-well formats, achieving robust performance (Z' > 0.7) and reproducibility across more than 100 runs with reference antiviral compounds. The platform was readily adapted to RV-A16, RV-B14, and additional RV-C types (C11 and C41), demonstrating broad applicability across rhinovirus species. A pilot screen of 10 240 compounds identified confirmed RV-C inhibitors, highlighting the readiness of the platform for integration into drug discovery pipelines. This platform provides a robust and scalable tool for systematic antiviral discovery and a foundation for mechanistic studies of RV-C replication. Significance statementRhinovirus C (RV-C) is strongly associated with severe respiratory disease such as asthma and COPD exacerbations, yet its study has been limited by the absence of scalable cell-based systems. We developed a high-content imaging-based high-throughput screening platform that supports efficient RV-C replication in engineered CDHR3-expressing cells and enables systematic antiviral discovery. The platform integrates a genetically stable fluorescent reporter virus with phenotypically selected permissive monoclonal cell lines, achieving high reproducibility and scalability in both 96- and 384-well formats. Its adaptability across multiple rhinovirus species establishes a unified framework for antiviral testing and mechanistic investigation. A pilot screen of 10K compounds confirmed the applicability of the platform to drug discovery campaigns.

microbiology↗

bioRxiv2024

Antigenic imprinting dominates humoral responses to new variants of SARS-CoV-2 in a hamster model of COVID-19

The emergence of SARS-CoV-2 variants escaping immunity challenges the efficacy of current vaccines. Here, we investigated humoral recall responses and vaccine-mediated protection in Syrian hamsters immunized with the third-generation Comirnaty(R) Omicron XBB.1.5-adapted COVID-19 mRNA vaccine, followed by infection with either antigenically closely (EG.5.1) or distantly related (JN.1) Omicron subvariants. Vaccination with YF17D vector encoding a modified Gamma spike (YF-S0*) served as a control for pre-Omicron SARS-CoV-2 immunity. Our results show that both Comirnaty(R) XBB.1.5 and YF-S0* induce robust, however, poorly cross-reactive, neutralizing antibody (nAb) responses. In either case, total antibody and nAb levels increased following infection. Intriguingly, the specificity of these boosted nAbs did not match the respective challenge virus but was skewed towards the primary antigen used for immunization, suggesting a marked impact of antigenic imprinting; confirmed by antigenic cartography. Furthermore, limited cross-reactivity and rapid decline of nAbs induced by Comirnaty(R) XBB.1.5 with EG.5.1 and, more concerning, JN.1. raises doubts about sustained vaccine efficacy against recent circulating Omicron subvariants. Future vaccine design may have to address two major issues: (i) to overcome original antigenic sin that limits the breadth of a protective response towards emerging variants, and (ii) to achieve sustained immunity that lasts for at least one season.

immunology↗

bioRxiv2024

Depletion of skin bacteria by topical antibiotic treatment accelerates onset of Zika virus disease in mice.

Mosquito-borne flaviviruses have emerged as global health threats due to their rapid spread and high disease burden. The initial site of virus replication is the mosquito biting site in the host skin, yet the role of host skin bacteria remains unclear. Here, we observed accelerated progression of Zika virus (ZIKV)-induced disease in mice with depleted skin bacteria by topical antibiotic treatment prior to ZIKV infection. Increased viral loads in the blood, draining lymph node, spleen and brain of antibiotic-treated mice suggested faster ZIKV dissemination. Flow cytometry revealed increased frequencies of T cells in the lymph nodes, along with enhanced activation of T cells in the brain upon antibiotic treatment. The antibiotic-induced phenotype was rescued by restoring the skin bacteria prior to infection. Our findings demonstrate an increased susceptibility to ZIKV infection in mice with depleted skin microbiomes, suggesting a potential role of skin bacteria in modulating the infection outcome.

microbiology↗

bioRxiv2023

Stretch-injury promotes activation of microglia with enhanced phagocytic and synaptic stripping activities

Microglial cells must act as the first line of defense of the central nervous system, but they can be exposed to various mechanical signals that may trigger their activation. While the impact of chemical signaling on brain cells has been studied in detail, our current understanding of the mechanical signaling in microglia is still limited. To address this challenge, we exposed microglial cells to a single mechanical stretch and compared their behavior to chemical activation by lipopolysaccharide treatment. Here we show that stretching microglial cells results in their activation, demonstrating a strong mechanosensitivity. Stretched microglial cells exhibited higher Iba1 protein levels, a denser actin cytoskeleton and migrated more persistently. In contrary to LPS-treated cells, stretched microglia maintain a robust secretory profile of chemokines and cytokines, except for TNF-, highlighting the relevance of this model. Interestingly, a single stretch injury results in more compacted chromatin and DNA damage, suggesting possible long-term genomic instabilities in stretched microglia. Using neuronal networks in compartmentalized microfluidic chambers, we found that stretched microglial cells exhibit enhanced phagocytic and synaptic stripping activities. Altogether, our results propose that the immune potential of microglial cells can be unlocked by stretching events to maintain brain tissue homeostasis after mechanical injury.

biophysics↗