Biology subjects

Moratorio, G.

Publications and source records attributed to Moratorio, G..

10 recordsLinked to original sources

bioRxiv2026

End-to-end plaque counting and virus titration from laboratory plate images with deep learning

Plaque assays are the gold standard for quantifying infectious virus, yet plaque enumeration is still routinely performed manually, making virus titration labor-intensive, subjective, and difficult to standardize across analysts and laboratories. Existing automated methods primarily address individual tasks, such as plaque segmentation or counting, but do not provide an integrated workflow from plate images to biological quantification. In this paper we present Titra, an end-to-end workflow for automated cytopathic effect (CPE)-based virus titration from standard photographs of plaque assay plates. The workflow combines automatic well detection, plaque segmentation, post-processing for instance separation, plaque counting, and plaque-forming units per millilitre (PFU/mL) estimation within a single web-based platform that also enables experiment management and expert review. The approach was evaluated using images from three viral species (Mayaro virus, Coxsackievirus B3, and vaccinia virus), two plate formats (6- and 12-well), and heterogeneous image acquisition conditions, including both a newly curated dataset and the public VACVPlaque dataset. Automated plaque counts showed strong agreement with manual annotations (Pearson correlation coefficients of 0.98 for MAYV/CVB3 and 0.88 for VACV), while PFU/mL estimates closely matched manual calculations for the MAYV/CVB3 dataset (Pearson r=0.975). Comparative experiments against U-Net, StarDist, HSD-WBR, and PyPlaque demonstrated competitive segmentation and counting performance, with the proposed approach achieving the highest Dice and mAP on the public VACVPlaque dataset.

bioinformatics↗

bioRxiv2026

Intra-host IRES heterogeneity shapes hepatitis C virus translation through epistatic and population-level effects

Hepatitis C virus (HCV) circulates within each patient as a diverse population of closely related genomes, yet RNA functional properties are commonly inferred from a single consensus or dominant genome. The contribution of non-coding intra-host variability, particularly within the internal ribosome entry site (IRES), to translational efficiency remains poorly defined. Here we investigated how naturally occurring HCV IRES variation influences viral RNA translation. Complete IRES sequences from chronically infected patients were analyzed using molecular cloning, bicistronic reporters, full-length replication-deficient viral RNAs and reconstructed intra-host populations. We found that natural IRES mutations displayed context-dependent effects, and combinations of mutations produced translational phenotypes that could not be predicted from the corresponding single mutations, consistent with intragenic epistasis. Moreover, several variants behaved differently in bicistronic reporters and full-length viral RNAs, demonstrating that both genomic and cellular context shape IRES function. Reconstructed genotype 1a populations largely reproduced the activity of their dominant haplotypes. In contrast, reconstructed genotype 3a populations translated substantially more efficiently than their corresponding dominant sequences, showing that low-frequency variants can collectively modulate translation at the population level. These findings demonstrate that the translational phenotype of HCV cannot always be inferred from the dominant sequence alone and identify epistasis, genomic context, and intra-host population composition as interacting determinants of viral RNA translation. ImportanceHepatitis C virus (HCV) exists within each infected person as a diverse population of closely related viruses rather than as a single genetic sequence. This study shows that natural variation in a key RNA region controlling viral protein production can alter how efficiently the virus functions, and that these effects depend on combinations of mutations rather than on individual changes alone. By analyzing complete viral RNAs in addition to widely used reporter systems, we demonstrate that the full viral genome can substantially influence the activity of this regulatory region, providing a more realistic view of how translation occurs during natural infection. Our findings also reveal that rare viral variants can collectively shape the behavior of the viral population, challenging the common practice of relying on a single dominant sequence to represent an infection. These results provide new insight into how genetic diversity drives HCV evolution and adaptation.

molecular biology↗

bioRxiv2026

IFNγ at the crossroads of systemic protection and brain vulnerability to coronavirus infection

The COVID-19 pandemic, together with previous outbreaks caused by highly pathogenic coronaviruses, highlighted the importance of understanding the host determinants that influence pathogenesis, both to improve our understanding of disease mechanisms, and to strengthen preparedness for future outbreaks. In our previous work we demonstrated dysregulation of IFN{gamma} response genes in COVID-19 patients, associated with both age and viral burden, positioning IFN{gamma} as a key mediator of host defense against SARS-CoV-2. Here, we used a preclinical murine model of coronavirus infection based on murine hepatitis virus (MHV-A59), widely validated for studying SARS-CoV-2 pathogenesis, together with IFN{gamma} knockout (IFN{gamma}-KO) mice to investigate the role of this cytokine during infection. We performed a comprehensive morphological, biochemical, hematological and proteomic characterization of infected wild-type (WT) and IFN{gamma}-deficient mice. Plasma proteomics revealed impaired inflammatory and coagulation-related responses in IFN{gamma}-KO animals compared to WT controls. We found increased viral load and infectious particles in peripheral organs, including liver, spleen, heart and muscle in IFN{gamma}-KO mice, whereas these were significantly reduced in the brain. Region-specific analysis further demonstrated decreased viral load in the prefrontal cortex and hippocampus of IFN{gamma}-KO animals. Consistently, bioinformatics analysis of transcriptomics data of IFN{gamma}-treated primary neuron cultures, together with frontal cortex from human COVID-19 patients, revealed activation of neuroinflammatory and neurological disease-associated pathways, supporting a role for IFN{gamma} in driving brain inflammatory responses during coronavirus infection. Together, these findings reveal a dual role for IFN{gamma} during coronavirus infection: it is important for controlling systemic viral dissemination and limiting peripheral tissue damage, yet it may also promote viral susceptibility and exacerbated inflammatory responses in the brain. These results support IFN{gamma} modulation as a potential therapeutic strategy to prevent or mitigate neurological complications associated with COVID-19. Author SummaryViral infections are controlled by the immune system, but the same responses that protect the body can sometimes contribute to tissue damage and disease. We investigated how a key immune molecule, called Interferon Gamma, influences the outcome of Coronavirus infection in different parts of the body. Using a mouse model of Coronavirus infection with Interferon Gamma deficiency, we found that this molecule plays two opposing roles: it helps control the spread of the virus throughout the body, but it also increases vulnerability of the brain to infection and inflammation. When this immune signal was absent, animals showed higher amounts of virus in several peripheral organs but surprisingly had lower levels of virus in specific brain regions. We further found that this immune response was associated with changes in inflammation and neurological processes in human data. Our findings reveal that antiviral immunity is not always uniformly protective and that its effects depend on the tissue involved. Understanding this balance may help guide future approaches to reduce virus-associated complications while preserving the protective functions of the immune response.

microbiology↗

bioRxiv2026

Potent broad-spectrum antiviral activity of the marine natural product Plitidepsin

Viruses pose a critical global health threat, yet therapeutic options remain limited. Finding drugs with broad-spectrum antiviral activity is essential to confront this threat. Here, we investigated whether plitidepsin, a marine-derived anticancer drug targeting the host eukaryotic elongation factor 1A (eEF1A), has such broad-spectrum activity. Using in vitro infection models and complementary assays (MTT, plaque-forming assays, RT-qPCR, Western blot, flow cytometry), we demonstrated that plitidepsin exhibits potent dose-dependent antiviral activity against Mayaro virus (MAYV) and Chikungunya virus (CHIKV). The compound achieved 4-6 log10 reduction in viral titers at nanomolar concentrations across multiple cell lines and viral strains. Plitidepsin protected human dermal fibroblasts from viral cytopathic effects and disrupted both entry and post-entry replication stages by suppressing viral protein expression (E1, nsP1) and RNA synthesis. The compound also demonstrated antiviral activity against other medically important arboviruses, including Una, Punta Toro, Zika, and Oropouche viruses, as well as RNA and DNA viruses such as influenza A virus, vesicular stomatitis virus, and human cytomegalovirus. These findings establish plitidepsin as a potent host-directed antiviral agent with reduced likelihood of resistance development and therapeutic potential against multiple viral families.

microbiology↗

bioRxiv2026

Extended tissue tropism and vertical transmission in a murine model of Mayaro virus infection

Mayaro virus (MAYV) is an emerging mosquito-borne alphavirus associated with acute febrile illness and persistent arthralgia, with increasing reports in Latin America and potential for geographic expansion. However, its dissemination dynamics, tissue tropism and transmission routes remain incompletely defined. Here, we developed a murine model to characterize systemic viral dissemination, tissue tropism and the impact of gestational stage on maternal-fetal outcomes. A nanoluciferase-expressing MAYV reporter enabled non-invasive in vivo imaging, revealing rapid and widespread systemic dissemination under controlled conditions. Complementary infection with wild-type (WT) virus confirmed broad tropism across lymphoid, musculoskeletal and reproductive tissues. At 24 hours post-infection (hpi), viral distribution was relatively homogeneous across tissues, whereas at 48 hpi, tissue-specific differences emerged, with increased viral loads in selected organs, including the spleen and male reproductive tissues. Transient sex-dependent differences were observed at 24 hpi but were not sustained at later time points. Hematological and biochemical analyses revealed early systemic alterations consistent with changes in leukocyte distribution during acute infection. Infectious viral particles were detected in reproductive tissues of both sexes, including in sperm, supporting the biological plausibility of non-vector transmission, although detection in exposed animals was limited. Gestational stage influenced infection outcomes: early gestation (infected at 7.5-8.5 dpc; analyzed at 9.5-10.5 dpc) was associated with detection of infectious virus in fetal tissues, whereas mid-gestation (infected at 13.5-14.5 dpc; analyzed at 15.5-16.5 dpc) showed no detectable infectious virus in fetuses despite evidence of viral antigen persistence and sustained infectious viral presence in maternal and placental compartments. Together, these findings provide a preclinical framework for investigating MAYV pathogenesis and underscore the role of tissue tropism and gestational context in shaping infection dynamics and maternal-fetal involvement. Author summaryMayaro virus (MAYV) is an emerging mosquito-borne virus that causes fever and long-lasting joint pain, with increasing reports in Latin America and potential for wider geographic spread. Despite its growing relevance, key aspects of MAYV pathogenesis remain to be fully defined, including the mechanisms underlying systemic dissemination and tissue tropism, as well as its potential to affect pregnancy or be transmitted through non-vector routes. Here, we used a mouse model with a luminescent reporter virus to track MAYV dissemination in vivo, revealing rapid and widespread distribution across multiple tissues. In studies using wild-type virus, infectious virus was detected not only in muscle and immune-related organs but also in reproductive tissues of both females and males. Importantly, gestational stage influenced viral distribution. When infection occurred early in gestation, infectious viral particles were detected in fetal tissues. In contrast, at later stages of pregnancy, infectious virus was no longer detectable in fetuses, despite evidence of viral antigen persistence and sustained infectious viral presence in maternal and placental tissues. Together, these findings advance our understanding of MAYV infection, highlight gestational timing as a factor associated with fetal exposure and suggest the possibility of transmission through routes other than mosquitoes, providing a framework for future studies on this emerging virus.

microbiology↗

bioRxiv2023

No evidence for highly pathogenic avian influenza virus H5N1 (clade 2.3.4.4b) in the Antarctic region during the austral summer 2022/23

The current highly pathogenic avian influenza H5N1 panzootic has substantial impacts on wild birds and marine mammals. Although major outbreaks occurred in South America, incursion to Antarctica emerged late in the breeding season of 2023/2024 and was confined the wider region of the Antarctic Peninsula. To infer potential underlying processes, we compiled H5N1 surveillance from Antarctica and Sub-Antarctic Islands prior to the first confirmed cases.

microbiology↗

bioRxiv2020

Evaluation Of SYBR Green Real Time PCR For Detecting SARS-CoV-2 From Clinical Samples

The pandemic caused by SARS-CoV-2 has triggered an extraordinary collapse of healthcare systems and hundred thousand of deaths worldwide. Following the declaration of the outbreak as a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30th, 2020, it has become imperative to develop diagnostic tools to reliably detect the virus in infected patients. Several methods based on real time reverse transcription polymerase chain reaction (RT-qPCR) for the detection of SARS-CoV-2 genomic RNA have been developed. In addition, these methods have been recommended by the WHO for laboratory diagnosis. Since all these protocols are based on the use of fluorogenic probes and one-step reagents (cDNA synthesis followed by PCR amplification in the same tube), these techniques can be difficult to perform given the limited supply of reagents in low and middle income countries. In the interest of economy, time and availability of chemicals and consumables, the SYBR Green-based detection was implemented to establish a convenient assay. Therefore, we adapted one of WHO recommended Taqman-based one-step real time PCR protocols (from the University of Hong Kong) to SYBR Green. Our results suggest that SYBR-Green detection represents a reliable cost-effective alternative to increase the testing capacity.

molecular biology↗

bioRxiv2020

Multiple introductions, regional spread and local differentiation during the first week of COVID-19 epidemic in Montevideo, Uruguay

BackgroundAfter its emergence in China in December 2019, the new coronavirus disease (COVID-19) caused by SARS-CoV-2, has rapidly spread infecting more than 3 million people worldwide. South America is among the last regions hit by COVID-19 pandemic. In Uruguay, first cases were detected on March 13 th 2020 presumably imported by travelers returning from Europe. MethodsWe performed whole-genome sequencing of 10 SARS-CoV-2 from patients diagnosed during the first week (March 16th to 19th) of COVID-19 outbreak in Uruguay. Then, we applied genomic epidemiology using a global dataset to reconstruct the local spatio-temporal dynamics of SARS-CoV-2. ResultsOur phylogeographic analysis showed three independent introductions of SARS-CoV-2 from different continents. Also, we evidenced regional circulation of viral strains originally detected in Spain. Introduction of SARS-CoV-2 in Uruguay could date back as early as Feb 20th. Identification of specific mutations showed rapid local genetic differentiation. ConclusionsWe evidenced early independent introductions of SARS-CoV-2 that likely occurred before first cases were detected. Our analysis set the bases for future genomic epidemiology studies to understand the dynamics of SARS-CoV-2 in Uruguay and the Latin America and the Caribbean region.

microbiology↗

bioRxiv2019

Midgut barriers prevent the replication and dissemination of the yellow fever vaccine in Aedes aegypti

BackgroundTo be transmitted to vertebrate hosts via the saliva of their vectors, arthropod-borne viruses have to cross several barriers in the mosquito body, including the midgut infection and escape barriers. Yellow fever virus (YFV) belongs to the genus Flavivirus, which includes human viruses transmitted by Aedes mosquitoes, such as Dengue and Zika viruses. The live-attenuated YFV-17D vaccine has been used safely and efficiently on a large scale since the end of World War II. Early studies have shown, using viral titration from salivary glands of infected mosquitoes, that YFV-17D can infect Aedes aegypti midgut, but does not disseminate to other tissues.\n\nMethodology/Principal FindingsHere, we re-visited this issue using a panel of techniques, such as RT-qPCR, Western blot, immunofluorescence and titration assays. We showed that YFV-17D replication was not efficient in Aedes aegypti midgut, as compared to the clinical isolate YFV-Dakar. Viruses that replicated in the midgut failed to disseminate to secondary organs. When injected into the thorax of mosquitoes, viruses succeeded in replicating into midgut-associated tissues, suggesting that, during natural infection, the block for YFV-17D replication occurs at the basal membrane of the midgut. Our NGS analysis revealed that YFV-Dakar genome exhibited a greater diversity than the vaccine strain; a trait that may contribute to its ability to infect and disseminate efficiently in Ae. aegypti.\n\nConclusions/SignificanceThe two barriers associated with Ae. aegypti midgut prevent YFV-17D replication. Our study contributes to our basic understanding of vector-pathogen interactions and may also aid in the development of non-transmissible live virus vaccines.\n\nAuthor summaryMost flaviviruses, including yellow fever virus (YFV), are transmitted between hosts by mosquito bites. The yellow fever vaccine (YFV-17D) is one of the safest and most effective live virus vaccine ever developed. It is also used as a platform for engineering vaccines against other health-threatening flaviviruses, such as Japanese encephalitis, West Nile, Dengue and Zika viruses. We studied here the replication and dissemination of YFV-17D in mosquitoes. Our data showed that YFV-17D replicates poorly in mosquito midgut and is unable to disseminate to secondary organs, as compare to a YFV clinical isolate. Our study contributes to our basic understanding of the interactions between viruses and their vectors, which is key for conceiving new approaches in inhibiting virus transmission and designing non-transmissible live virus vaccines.

microbiology↗

bioRxiv2018

DISSEQT - DIStribution based modeling of SEQuence space Time dynamics

Rapidly evolving microbes are a challenge to model because of the volatile, complex and dynamic nature of their populations. We developed the DISSEQT pipeline (DIStribution-based SEQuence space Time dynamics) for analyzing, visualizing and predicting the evolution of heterogeneous biological populations in multidimensional genetic space, suited for population-based modeling of deep sequencing and high-throughput data. DISSEQT is openly available on GitHub (https://github.com/rasmushenningsson/DISSEQT.jl) and Synapse (https://www.synapse.org/#!Synapse:syn11425758), covering the entire workflow from read alignment to visualization of results. DISSEQT is centered around robust dimension and model reduction algorithms for analysis of genotypic data with additional capabilities for including phenotypic features to explore dynamic genotype-phenotype maps. We illustrate its utility and capacity with examples from evolving RNA virus populations, which present on of the highest degrees of population heterogeneity found in nature. Using DISSEQT, we empirically reconstruct the evolutionary trajectories of evolving populations in sequence space and genotype-phenotype fitness landscapes. We show that while sequence space is vastly multidimensional, the relevant genetic space of evolving microbial populations is of intrinsically low dimension. In addition, evolutionary trajectories of these populations can be faithfully monitored to identify the key minority genotypes contributing most to evolution. Finally, we show that empirical fitness landscapes, when reconstructed to include minority variants, can predict phenotype from genotype with high accuracy.

evolutionary biology↗