BACKGROUND: We have previously described a linear increase in thrombotic events since 2020 in an integrated Health consortium in the metropolitan area of Barcelona.

AIM: To describe the characteristics of thrombotic events in relation to COVID-19 vaccination status, SARS-CoV-2 infection, and age.

METHODS: We analyzed anonymized data on infections, hospital admissions, and thrombotic events after March 2020 in the population assigned to the Consorci Sanitari de Terrassa (n=192,651 in 2025). The number of cases of ischemic stroke, ischemic heart disease, retinal vessel thrombosis, and pulmonary thromboembolism were quantified. These were analyzed across different age groups (<60 vs. ≥60 years), COVID-19 vaccination status (vaccinated [V] vs. unvaccinated [UV]), SARS-CoV-2 infection and hospital admission. The dates of infection and thrombotic events were recorded. An odds ratio (OR) was calculated to compare the risk of thrombosis between 2021 and 2024.

RESULTS:

The overall increase in thrombotic events from 2021 to 2024 was 1.20 in vaccinated individuals (V) and 1.81 in non-vaccinated individuals (NoV). The odds ratio (OR) for ischemic stroke was 1.35–3.8 (V vs. NoV) in those aged >60 years, and 1.35–2.0 (V vs. NoV) in those aged <60 years. The OR for ischemic cardiopathy was 1.17–1.75 (V vs. NoV) in individuals aged >60, and 1.6–1.8 (V vs. NoV) in those aged <60. The OR for retinal vessel thrombosis decreased in most groups (OR 0.33–0.75), but vaccinated individuals under 60 years of age showed an OR of 2.5. Pulmonary thromboembolism showed an OR of 0.47–0.87 (V vs. NoV) in individuals aged <60, and 0.90 (V) vs. 3.5 (NoV) in those aged >60.

CONCLUSION: Ischemic stroke and ischemic heart disease exhibited an increased incidence of thrombosis. Potential explanations for this rise, including post-pandemic lifestyle changes, undetected SARS-CoV-2 infections, a growing population without vaccination records, and a more impaired socioeconomic environment, should be explored in future studies.

The seven APOBEC3 proteins are cytidine deaminases, key antiviral innate immune effectors of the human proteome. They bind viral nucleic acid and catalyze C-to-U mutations. From the outbreak of SARS-CoV-2, and still to this day, the SARS-CoV-2 transcriptome is described as being shaped by host factors. Among them, APOBEC3-attributed mutagenesis is the most represented mutagenic bias. Despite some studies investigating the role of APOBEC3 proteins in SARS-CoV-2 replication, no consensus is available. We developed an infection model of immortalized normal human bronchial epithelial cells transduced with both ACE2 and TMPRSS2 lentiviral expressing constructs (HBEC3-KT-AT). HBEC3-KT-AT cells were infected with four different variants of concern (VOCs), namely, Wuhan, Alpha, Delta, and Omicron. Cellular RNA quantification shows that, with all VOCs, APOBEC3G mRNA expression is upregulated in a dose-dependent manner. Cellular protein quantification indicates that, with Wuhan, Alpha, and Omicron VOCs, APOBEC3G protein content is increased in a dose-dependent manner. Strikingly, infection with the Delta VOC does not trigger APOBEC3G protein increase. Experiments are needed to investigate the mechanism underlying the absence of correlation between APOBEC3G mRNA and protein content upon Delta VOC infection. Preliminary data suggest that, upon SARS-CoV-2 infection, APOBEC3G protein is associated with the nucleoprotein. These findings shed light on the interplay between APOBEC3 proteins and SARS-CoV-2 replication.

Abstract

Influenza A represents a global public health threat, with an estimated 290,000 to 650,000 cases annually. Its high genetic variability allows the virus to develop resistance to currently available antivirals, highlighting the urgent need for new therapeutic strategies. This study identified bioactive compounds present in Opuntia sp. with the ability to inhibit the influenza A virus neuraminidase and act as potential antiviral agents. A literature review was conducted to compile the molecules reported on Opuntia sp. extracts. These compounds were subjected to molecular docking analysis against two viral neuraminidases, 3B7E (N1) and 4GZW (N2), using zanamivir and oseltamivir as interaction controls. Compounds C1, C3, and C4 demonstrated more stable binding energies than the controls; therefore, they were selected and purchased for subsequent experimental evaluation. Their cytotoxicity was then assessed in MDCK cells, and the median inhibitory concentration (IC₅₀) was determined through neuraminidase inhibition assays using two influenza A virus strains, A/PR/8/34 H1N1 (human) and A/Chicken/Mexico/31381-7/1994 H5N2, with zanamivir as a control. The results showed that the selected molecules inhibited neuraminidase activity in both viral strains with IC₅₀ values below 100 µM and exhibited low cytotoxicity. These findings support their potential as antiviral candidates against influenza A virus and justify further evaluation in cell-based models for viral replication inhibition assays.

The conservation of marine megafauna including sea turtles is crucial for the maintenance of healthy marine ecosystems. The Eastern Mediterranean coast is a nesting area for two marine turtle species, the green turtle (Chelonia mydas) and the loggerhead turtle (Caretta caretta). In the last decade, a slow shift to Western Mediterranean nesting areas is being observed for the loggerhead turtle. Globally, conservation of sea turtle species is striked by different pressures. Marine turtles are exposed to threats like coastal development, bycatch, maritime traffic or infectious diseases. One of these diseases is fibropapillomatosis (FP), presumably caused by Chelonid herpesvirus 5 (ChHV5). The disease is suspected to be triggered by the interplay of viral, host and environmental factors. FP disease mainly affects green turtles of the Atlantic Ocean, although recent studies suggest the virus is present in Mediterranean loggerhead turtles. The risk of FP disease spread to Mediterranean has been assessed through genetic characterization of host and viral factors in sea turtles rescued in the Strait of Gibraltar.

Acknowledgements: This study has been funded by the subproject ALMA in the framework of the complementary plan on biodiversity, financed by the NextGenerationEU Recovery and Resilience Mechanism, and coordinated under the agreement between Junta de Andalucía and Universidad Pablo de Olavide.

Cetaceans are marine mammals that provide important ecosystem services. Conservation of these iconic species is threatened by factors such as climate change and accidental bycatch. Infectious diseases are another threat, as mass stranding events in cetaceans can be caused by viral infections, mostly by Morbillivirus. Influenza A virus (IAV) have a wide range of avian and mammalian host species, including humans. The virus circulates across different species, leading to disease outbreaks and increasing the risk of panzootics and pandemics. Since the 1970s, IAV has been diagnosed in wild animals belonging to the two living cetacean families, Delphinidae and Phocoenidae. A comprehensive review of IAV infection of cetacean species is necessary to understand the risk of HPAI outbreaks in cetaceans. Documented cases relate to IAV strains H1N3, H13N2, H13N9, and H5N1 and have been reported in cetaceans sampled in the Pacific, Atlantic, and Arcticoceans. The analysis of molecular markers of viral adaptation to mammals in H5N1 clade 2.3.4.4b cetacean isolates reveals mutations are present in three viral proteins: hemagglutinin (HA), polymerase basic protein 2 (PB2), and nucleoprotein (NP). No sustained transmission of the virus between cetaceans to date is suspected from the documented cases or from amino acid sequence phylogenetic analysis. IAV H5N1 appears to be reaching cetaceans after spillover from seabirds and other marine mammals. Increasing worldwide surveillance of IAV infection of cetaceans is crucial, as sentinel species for human pandemic preparedness and key species for marine biodiversity conservation and ecosystem health.

This study has been funded by the subproject ALMA in the framework of the complementary plan on biodiversity, financed by the NextGenerationEU Recovery and Resilience Mechanism, and coordinated under the agreement between Junta de Andalucía and Universidad Pablo de Olavide.

The clinical manifestation of COVID-19 evolved significantly throughout the pandemic, shifting from severe, often fatal pneumonia to a more manageable, multi-organ infection. This change reflects advancements in medical knowledge, improved treatments, and widespread vaccination efforts. However, the contribution of viral evolution, including reduced virulence to evade immunity and enhanced transmissibility, remains under investigation. This study utilizes the K18-hACE2 mouse model that replicates key features of human COVID-19, demonstrating a dose-dependent progression from asymptomatic or mild disease at lower viral doses to severe, lethal outcomes at higher doses. The study compares four representative SARS-CoV-2 variants to identify and characterize biomarkers of pathogenicity and host responses. Mice were infected intranasally with increasing doses of SARS-CoV-2, while sham-infected animals received PBS. Daily monitoring of weight loss and symptoms, along with Kaplan–Meier survival analysis, revealed significant weight loss correlating with poor survival. Following determination of the optimal viral dose, mice were sacrificed at various time points to evaluate disease progression. Viral load was highest in the lungs and brain. Lung pathology, assessed via immunohistochemistry, revealed intra-alveolar hemorrhages, edema, pneumocyte hyperplasia, congestion, inflammation, and multinucleated giant cells. Lipid inflammatory markers were analyzed on plasma samples collected at different time points. Of 58 markers analyzed, 50 were detectable in ≥50% of samples and subjected to statistical analysis, revealing significant shifts. Brain tissues from infected and control mice were analyzed for neurodegenerative markers. IFN-I/III gene expression in lung and spleen cells was assessed at four post-infection time points to investigate innate immune responses. This study provides a comprehensive analysis of SARS-CoV-2 variant pathogenicity and host responses using the K18-hACE2 mouse model. By elucidating the mechanisms of disease progression and immune response, these findings deepen our understanding of COVID-19 and its evolving clinical manifestations. Ongoing investigations aim to identify differences in innate immune responses among SARS-CoV-2 variants.

Zika virus (ZIKV) is a medically important mosquito-borne orthoflavivirus, but no vaccines are currently available to prevent ZIKV-associated disease. In this study, we compared three recombinant chimeric viruses developed as candidate vaccine prototypes (rJEV/ZIKV^MR-766, rJEV/ZIKV^P6-740, and rJEV/ZIKV^PRVABC-59), in which the two neutralizing antibody-inducing prM and E genes from each of three genetically distinct ZIKV strains were used to replace the corresponding genes of the clinically proven live-attenuated Japanese encephalitis virus vaccine SA₁₄-14-2 (rJEV). In WHO-certified Vero cells, rJEV/ZIKV^P6-740 exhibited the slowest viral growth, formed the smallest plaques, and displayed a unique protein expression profile with the highest ratio of prM to cleaved M, when compared to the other two chimeric viruses, rJEV/ZIKV^MR-766 and rJEV/ZIKV^PRVABC-59, as well as their vector, rJEV. In IFNAR^–/– mice, subcutaneous inoculation of rJEV/ZIKV^P6-740 caused a low-level localized infection limited to the spleen, with no clinical signs of infection, weight loss, or mortality; in contrast, the other two chimeric viruses and their vector caused high-level systemic infections involving multiple organs, consistently leading to clear clinical signs of infection, rapid weight loss, and 100% mortality. Subsequently, subcutaneous immunization with rJEV/ZIKV^P6-740 proved highly effective, offering complete protection against a lethal intramuscular ZIKV challenge 28 days after a single-dose immunization. This protection was specific to ZIKV prM/E and likely mediated by neutralizing antibodies targeting ZIKV prM/E. Therefore, our data indicate that the chimeric virus rJEV/ZIKV^P6-740 is a highly promising vaccine prototype for developing a safe and effective vaccine to induce neutralizing antibody-mediated protective immunity against ZIKV.

Retrotransposon reactivation may drive neuro-inflammation and synaptic loss via type I interferon signaling. Lamivudine (3TC), a reverse transcriptase (RT) inhibitor that blocks retrotransposon RT activity, improved cognition in mouse models of accelerated ageing. Here, we evaluated whether 3TC reduces the inflammation from reactivated retrotransposons in individuals with mild cognitive impairment (MCI) due to Alzheimer’s disease (AD)—defined by CSF biomarkers or amyloid PET.

An ongoing single-center, open-label, self-controlled Phase 2a pilot trial enrolled 20 participants aged 55–90 to receive oral 3TC (300 mg/day) for 24 weeks (NCT06519357). Participants met NIA-AA criteria for MCI and were positive for CSF or PET biomarkers for AD. Exclusion criteria included other dementias or unstable medical or psychiatric conditions. Blood was collected during screening, at baseline, and at weeks 1, 4, 12, 24, and 48. Primary outcomes included measuring 10 type I interferon signaling genes (ISGs) via RT-PCR in PBMCs. Safety and tolerability were also evaluated.

Three participants did not complete treatment, but 3TC was safe and well tolerated in all cases. Changes up to week 12 in the 10 ISGs were measured by calculating significant negative slopes using a linear regression model. Of note, 47% of the participants had a statistically significant decrease in IFN-a and IFN-b, consistent with a reduced sensing of retrotransposon RT activity blocked by 3TC. Moreover, 20-15% had a significant reduction in MX1, IFHI1, OAS1, TLR7, and TLR3. About 12% showed reduced IP10, IRF7, and TREX1. In three patients, at least six genes were downregulated. RT-PCR at weeks 24 to 48, as well as quantification of neurocognitive plasma biomarkers, is underway.

3TC lowers IFN-a and IFN-b release in PBMCs from AD patients, potentially reducing immune inflammation driven by retrotransposon RT. Further work should address if a strategy based on 3TC, with higher brain-barrier penetrance than other antiretrovirals, could reduce brain inflammation.

The COVID-19 pandemic underscored the critical role of the SARS-CoV-2 spike (S) protein in facilitating viral entry and modulating immune responses. As mRNA-based vaccines utilize the S protein to elicit immunity, understanding its direct effects on immune cells is essential. In this study, we investigated the impact of an S protein-expressing mRNA vaccine on macrophage differentiation, cytokine production, and phagocytic activity. THP-1 cells were treated with mRNA encoding the S protein, or a scrambled mRNA control, and changes in the cellular transcriptome, cytokine profiles, and phagocytosis were assessed. Proteome changes were also analyzed using Olink proximity extension assay technology. Parallel experiments in primary macrophages from healthy donors were also conducted. Our results demonstrate that S protein expression significantly alters macrophage differentiation, including cytokine secretion and phagocytic capacity, suggesting potential implications for immune modulation by mRNA vaccines. These findings highlight the need to further elucidate the effects of mRNA vaccines on immune cells, particularly macrophages, to optimize vaccine design and mitigate the risks of immune dysregulation.

We recently discovered that hepatitis C virus (HCV) is capped with flavin adenine dinucleotide (FAD) on the 5’ end of its RNA and showed that the RNA-dependent RNA polymerase NS5B is responsible for its incorporation as a non-canonical initiating nucleotide (NCIN). Thus, to elucidate the structural basis of FAD-initiated HCV replication and to shed light on the poorly understood de novo initiation step, we used X-ray crystallography to determine structures of NS5B in complex with RNA and FAD.

Obtaining structures of NS5B with RNA bound is challenging and was previously only possible using a mutated genotype 2a NS5B (strain JFH1) containing five substitutions of which S15G/C223H/V321I are near the active site and therefore could influence substrate recognition. Here, we successfully obtained well-diffracting crystals capable of binding RNA and FAD by using only two protein surface substitutions, thus enabling structural studies in a near-wild-type context. To further enhance physiological relevance, we used the authentic NTP substrate in the active site rather than the commonly used NDP model substrate.

We successfully obtained de novo initiation structures of NS5B-RNA-FAD at high resolutions ranging from 1.7 to 2.2 Å. We found that FAD binding is stabilized by key interactions with Y448 and G449 in NS5B’s beta-loop, as well as stacking and base pairing with the RNA. The structures reveal that the authentic NTP substrate promotes catalytically competent conformations of FAD and the incoming nucleotide. Additionally, we observed structural consequences of S15G/C223H/V321I, including altered active site electrostatics and changes in NS5B–RNA interactions.

Overall, our structures provide new insights into HCV replication, elucidate the role of non-canonical initiating nucleotides in viral RNA synthesis and establish a platform for studying de novo initiation, including nucleotide analog recognition in a physiologically relevant NS5B context.