@article{dodkins_delaney_overton_scholle_frias-de-diego_crisci_huq_jordan_kimata_findley_et al._2023, title={A rapid, high-throughput, viral infectivity assay using automated brightfield microscopy with machine learning}, volume={28}, ISSN={2472-6303}, url={http://dx.doi.org/10.1016/j.slast.2023.07.003}, DOI={10.1016/j.slast.2023.07.003}, abstractNote={Infectivity assays are essential for the development of viral vaccines, antiviral therapies, and the manufacture of biologicals. Traditionally, these assays take 2-7 days and require several manual processing steps after infection. We describe an automated viral infectivity assay (AVIATM), using convolutional neural networks (CNNs) and high-throughput brightfield microscopy on 96-well plates that can quantify infection phenotypes within hours, before they are manually visible, and without sample preparation. CNN models were trained on HIV, influenza A virus, coronavirus 229E, vaccinia viruses, poliovirus, and adenoviruses, which together span the four major categories of virus (DNA, RNA, enveloped, and non-enveloped). A sigmoidal function, fit between virus dilution curves and CNN predictions, results in sensitivity ranges comparable to or better than conventional plaque or TCID50 assays, and a precision of ∼10%, which is considerably better than conventional infectivity assays. Because this technology is based on sensitizing CNNs to specific phenotypes of infection, it has potential as a rapid, broad-spectrum tool for virus characterization, and potentially identification.}, number={5}, journal={SLAS Technology}, publisher={Elsevier BV}, author={Dodkins, Rupert and Delaney, John R. and Overton, Tess and Scholle, Frank and Frias-De-Diego, Alba and Crisci, Elisa and Huq, Nafisa and Jordan, Ingo and Kimata, Jason T. and Findley, Teresa and et al.}, year={2023}, month={Jul}, pages={324–333} }
 @article{frias-de-diego_gilbertie_scholle_dejarnette_crisci_2022, title={Effect of BIO-PLYTM, a Platelet-Rich Plasma Derived Biologic on PRRSV-2-Infected Macrophages}, volume={14}, ISSN={1999-4915}, url={http://dx.doi.org/10.3390/v14122666}, DOI={10.3390/v14122666}, abstractNote={Porcine Reproductive and Respiratory Syndrome (PRRS) is the one of the most devastating diseases impacting the swine industry worldwide. Control and prevention methods rely on biosafety measures and vaccination. As an RNA virus with a high rate of mutation, vaccines are only partially effective against circulating and newly emerging strains. To reduce the burden of this disease, research on alternative control methods is needed. Here, we assess the in vitro antiviral effect of a novel platelet-rich plasma-derived biologic termed BIO-PLYTM (for the BIOactive fraction of Platelet-rich plasma LYsate) from both swine and equine origin. Our results show that BIO-PLYTM significantly reduces the amount of PRRSV viral load determined by RT-qPCR and the number of infectious viral particles measured by TCID50 in infected porcine alveolar and parenchymal macrophages. This study also showed limited toxicity of BIO-PLYTM in vitro and aspects of its immunomodulatory capacity evaluating the regulation of reactive oxygen species and cytokines production in infected cells. Finally, this study presents promising data on the effect of BIO-PLYTM on other RNA viruses such as human A influenza viruses and coronavirus.}, number={12}, journal={Viruses}, publisher={MDPI AG}, author={Frias-De-Diego, Alba and Gilbertie, Jessica M. and Scholle, Frank and Dejarnette, Sarah and Crisci, Elisa}, year={2022}, month={Nov}, pages={2666} }
 @article{pecoraro_leal_frias-de-diego_browning_odle_crisci_2022, title={The health benefits of selenium in food animals: a review}, volume={13}, ISSN={2049-1891}, url={http://dx.doi.org/10.1186/s40104-022-00706-2}, DOI={10.1186/s40104-022-00706-2}, abstractNote={Selenium is an essential trace mineral important for the maintenance of homeostasis in animals and humans. It evinces a strong antioxidant, anti-inflammatory and potential antimicrobial capacity. Selenium biological function is primarily achieved by its presence in selenoproteins as a form of selenocysteine. Selenium deficiency may result in an array of health disorders, affecting many organs and systems; to prevent this, dietary supplementation, mainly in the forms of organic (i.e., selenomethionine and selenocysteine) inorganic (i.e., selenate and selenite) sources is used. In pigs as well as other food animals, dietary selenium supplementation has been used for improving growth performance, immune function, and meat quality. A substantial body of knowledge demonstrates that dietary selenium supplementation is positively associated with overall animal health especially due to its immunomodulatory activity and protection from oxidative damage. Selenium also possesses potential antiviral activity and this is achieved by protecting immune cells against oxidative damage and decreasing viral replication. In this review we endeavor to combine established and novel knowledge on the beneficial effects of dietary selenium supplementation, its antioxidant and immunomodulatory actions, and the putative antimicrobial effect thereof. Furthermore, our review demonstrates the gaps in knowledge pertaining to the use of selenium as an antiviral, underscoring the need for further in vivo and in vitro studies, particularly in pigs.}, number={1}, journal={Journal of Animal Science and Biotechnology}, publisher={Springer Science and Business Media LLC}, author={Pecoraro, B.M. and Leal, D.F. and Frias-De-Diego, A. and Browning, M. and Odle, J. and Crisci, E.}, year={2022}, pages={58} }
 @article{frias-de-diego_crisci_2022, title={Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays}, volume={2}, ISSN={1940-087X}, url={http://dx.doi.org/10.3791/63063}, DOI={10.3791/63063}, abstractNote={Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are the two main methods to calculate the titer of a virus stock and are often based on microscopy detection or cell staining for visualization. In the case of TCID50 assay, objective visualization is commonly based on immunocytochemical (ICC) staining of intracellular virus to calculate titers combined with visual CPE detection via microscopy. However, ICC staining is costly and time consuming. In this study, we compared visual CPE observation via microscopy, ICC staining and crystal violet staining to determine the titers of two CPE-forming viruses, Influenza A virus (IAV) of swine origin and Porcine Reproductive and Respiratory Syndrome virus (PRRSV). We show that both crystal violet and ICC staining are more accurate than visual CPE detection, presenting nearly identical levels of precision on both IAV and PRRSV. For this reason, here we present crystal violet staining as a faster and more affordable way to determine viral titrations on a TCID50 assay for CPE-forming viruses titrated in cell lines.}, number={180}, journal={Journal of Visualized Experiments}, publisher={MyJove Corporation}, author={Frias-De-Diego, Alba and Crisci, Elisa}, year={2022}, month={Feb} }
 @article{machado_corbellini_frias-de-diego_dieh_santos_jara_costa_2021, title={Impact of changes of horse movement regulations on the risks of equine infectious anemia: A risk assessment approach}, volume={190}, ISSN={["1873-1716"]}, url={https://doi.org/10.1016/j.prevetmed.2021.105319}, DOI={10.1016/j.prevetmed.2021.105319}, abstractNote={Equine infectious anemia virus (EIAV) is a transboundary disease affecting a large number of equines worldwide. In this study, we assessed the transmission risk of EIAV in Rio Grande do Sul, Brazil. Serum samples from 1010 animals from 341 farms were initially analyzed using agar gel immunodiffusion to detect viral antibodies, and no antibody-positive animals were found. A risk assessment stochastic model was applied to generate the expected number of potential infections per month and to estimate the time to new infections. Our results estimated 6.5 months as the interval for new infections in the worst-case scenario. Among the variables evaluated, the number of transported animals and the test sensitivity influenced the model the most. These results were then used to revisit the impact of EIAV control regulations, which triggered a change in the diagnostic testing required for animal movement, in which the validity of a negative test for EIAV was extended from 60 to 180 days. Finally, revisiting the annual average of infected farms before and after the new regulation, the number of infected farms increased pre-implementation, and then, the number of culled animals increased, which should impact future EIAV incidence in this region. Our results demonstrated the importance of constant reviews of disease control programs and provided quantitative-based knowledge for decision-makers in official veterinary services.}, journal={PREVENTIVE VETERINARY MEDICINE}, publisher={Elsevier BV}, author={Machado, Gustavo and Corbellini, Luis Gustavo and Frias-De-Diego, Alba and Dieh, Gustavo Nogueira and Santos, Diego Viali and Jara, Manuel and Costa, Eduardo de Freitas}, year={2021}, month={May} }
 @article{kick_amaral_frias-de-diego_cortes_fogle_crisci_almond_käser_2021, title={The Local and Systemic Humoral Immune Response Against Homologous and Heterologous Strains of the Type 2 Porcine Reproductive and Respiratory Syndrome Virus}, volume={12}, ISSN={1664-3224}, url={http://dx.doi.org/10.3389/fimmu.2021.637613}, DOI={10.3389/fimmu.2021.637613}, abstractNote={The humoral immune response plays a crucial role in the combat and protection against many pathogens including the economically most important, highly prevalent, and diverse pig pathogen PRRSV – the Porcine Reproductive and Respiratory Syndrome Virus. In addition to viremia and viral shedding analyses, this study followed the local and systemic humoral immune response of pigs for 63 days upon inoculation with one of three types of Type-2 PRRSV (PRRSV-2) strains – one modified live virus (MLV) vaccine strain, and two lineage 1 PRRSV-2 strains, NC134 and NC174. The local response was analyzed by quantifying immunoglobulin (Ig)A in nasal swabs. The systemic response was studied by the quantification of IgG with ELISA and homo- and heterologous neutralizing antibodies (NAs) utilizing a novel method of flow cytometry. In all PRRSV-2 inoculated groups, viral nasal shedding started at 3 dpi, peaked between 3 and 7 days post inoculation, and was cleared at 28–35 dpi with sporadic rebounds thereafter. The local IgA response started 4–7 days after viral shedding occurred and showed a bi-phasic course with peaks at 14 dpi and at 28–35 dpi. Of note, the NC134 and NC174 strains induced a much stronger local IgA response. As reported earlier, main viremia lasted from 7 dpi to 28 dpi (NC174), 42 dpi (NC134) or until the end of the study (MLV). Similar to the local IgA response, the systemic IgG response started 4–7 days after viremia; but in contrast to viremia, serum IgG levels stayed high for all PRRSV-2 inoculated groups until the end of the study. A significant finding was that while the serum NA response in the MLV group was delayed by 28 days, serum NAs in pigs infected with our two NC134 and NC174 strains could be detected as early as 7 dpi (NC134) and 14 dpi (NC174). Compared to homologous NA responses, the NA responses against heterologous strains was strong but slightly delayed between our lineage 1 one strains or non-existent between the MLV and lineage 1 strains. This study improves our understanding of the relationship between local and systemic infections and the humoral immune response induced by PRRSV-2 infection or MLV vaccination. Our data also provide novel insights into the timeline of the development of homologous and heterologous NA levels – by both MLV vaccination or infection with two strains from the currently prevalent PRRSV-2 lineage 1.}, journal={Frontiers in Immunology}, publisher={Frontiers Media SA}, author={Kick, Andrew R. and Amaral, Amanda F. and Frias-De-Diego, Alba and Cortes, Lizette M. and Fogle, Jonathan E. and Crisci, Elisa and Almond, Glen W. and Käser, Tobias}, year={2021}, month={Mar} }
 @article{frias-de-diego_jara_pecoraro_crisci_2021, title={Whole Genome or Single Genes? A Phylodynamic and Bibliometric Analysis of PRRSV}, volume={8}, ISSN={2297-1769}, url={http://dx.doi.org/10.3389/fvets.2021.658512}, DOI={10.3389/fvets.2021.658512}, abstractNote={Diversity, ecology, and evolution of viruses are commonly determined through phylogenetics, an accurate tool for the identification and study of lineages with different pathological characteristics within the same species. In the case of PRRSV, evolutionary research has divided into two main branches based on the use of a specific gene (i.e., ORF5) or whole genome sequences as the input used to produce the phylogeny. In this study, we performed a review on PRRSV phylogenetic literature and characterized the spatiotemporal trends in research of single gene vs. whole genome evolutionary approaches. Finally, using publicly available data, we produced a Bayesian phylodynamic analysis following each research branch and compared the results to determine the pros and cons of each particular approach. This study provides an exploration of the two main phylogenetic research lines applied for PRRSV evolution, as well as an example of the differences found when both methods are applied to the same database. We expect that our results will serve as a guidance for future PRRSV phylogenetic research.}, journal={Frontiers in Veterinary Science}, publisher={Frontiers Media SA}, author={Frias-De-Diego, Alba and Jara, Manuel and Pecoraro, Brittany M. and Crisci, Elisa}, year={2021}, month={Jun} }
 @article{krasteva_jara_frias-de-diego_machado_2020, title={Nairobi Sheep Disease Virus: A Historical and Epidemiological Perspective}, volume={7}, ISSN={["2297-1769"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85089210118&partnerID=MN8TOARS}, DOI={10.3389/fvets.2020.00419}, abstractNote={Nairobi Sheep Disease virus (NSDv) is a zoonotic and tick-borne disease that can cause over 90% mortality in small ruminants. NSDv has historically circulated in East Africa and has recently emerged in the Asian continent. Despite efforts to control the disease, some regions, mostly in warmer climates, persistently report disease outbreaks. Consequently, it is necessary to understand how environmental tolerances and factors that influence transmission may shed light on its possible emergence in other regions. In this study, we quantified the available literature of NSDv from which occurrence data was extracted. In total, 308 locations from Uganda, Kenya, Tanzania, Somalia, India, Sri Lanka and China were coupled with landscape conditions to reconstruct the ecological conditions for NSDv circulation and identify areas of potential disease transmission risk. Our results identified areas suitable for NSDv in Ethiopia, Malawi, Zimbabwe, Southeastern China, Taiwan, and Vietnam. Unsuitable areas included Democratic Republic of Congo, Zambia, and Southern Somalia. In summary, soil moisture, livestock density, and precipitation predispose certain areas to NSDv circulation. It is critical to investigate the epidemiology of NSDv in order to promote better allocation of resources to control its spread in regions that are more at risk. This will help reduce disease impact worldwide as climate change will favor emergence of such vector-borne diseases in areas with dense small ruminant populations.}, journal={FRONTIERS IN VETERINARY SCIENCE}, author={Krasteva, Stephanie and Jara, Manuel and Frias-De-Diego, Alba and Machado, Gustavo}, year={2020}, month={Jul} }
 @article{jara_frias-de-diego_machado_2020, title={Phylogeography of Equine Infectious Anemia Virus}, volume={8}, ISSN={["2296-701X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85085374042&partnerID=MN8TOARS}, DOI={10.3389/fevo.2020.00127}, abstractNote={Equine Infectious Anemia virus (EIAV) is the causative agent of one of the most impacting infectious diseases affecting equids. EIAV is genetically diverse with several lineages circulating globally. To elucidate EIAV global spread patterns, we studied its spatiotemporal dynamics applying Bayesian phylodynamic analyses, using a worldwide compiled dataset composed of unique sequences of the gag gene. In addition, we performed a scoping review of 1.470 publications on EIAV to characterize the spatiotemporal trends in EAIV research. Phylogeographic reconstruction suggested Hungary as the most likely country of origin for current EIAV circulation (root state posterior probability = 0.21), and one of the most important centers of diversification for the disease. Historical EIAV spread was predominantly characterized by long-distance spread across continents. As a result, we found that the American and Asian circulating EIAV (i.e., Mongolia) are more related to European lineages than to other Asian countries, being Europe the continent with the highest EIAV phylogenetic diversity. Our bibliometric analysis showed a continuous increment in the number of publications per year, where the United States and China appeared as the countries with highest EIAV-related scientific production. This study provides a historical geographic mapping of the EIAV linages’ spread patterns. In addition, we identified important asymmetry between the current research effort and the availability of genetic data which restricted our abilities to quantify for example the time frame of virus dispersal.}, journal={FRONTIERS IN ECOLOGY AND EVOLUTION}, author={Jara, Manuel and Frias-De-Diego, Alba and Machado, Gustavo}, year={2020}, month={May} }
 @article{frias-de-diego_jara_escobar_2019, title={Papillomavirus in Wildlife}, volume={7}, ISSN={["2296-701X"]}, DOI={10.3389/fevo.2019.00406}, abstractNote={Papillomaviruses (PV) are associated with epithelial malignancies in animals, including cancer in humans. Limited knowledge exists regarding the evolutionary history of non-human PV. We assessed the phylogeography of PV with emphasis in wildlife hosts. We explored the phylogenetic, geographic, and environmental relationships of PV and hosts applying Bayesian inference and spatial analyses of virus and hosts. We found that the available wildlife PV data support previous reports on the higher incidence of fibropapillomatosis over carcinoma in humans and wildlife, being mammals the most common host. We also found geographic bias on the available wildlife papillomavirus (WPV) information towards the Northern Hemisphere, which may have influenced our results to show Europe as the most likely origin of the available WPV lineages. Therefore, we highlight the need for detailed studies on the presence of WPV in regions and species not included in this study (e.g., reptiles from the tropics) to better inform sites of WPV origin, susceptible species, and spillover potential. Future studies of the clinical and subclinical occurrence, distribution, and phylogenetic signatures of WPV may help understand the spread, virulence, and epidemiology of PV in general. From an evolutionary perspective, our overview suggests that WPV are a promising host-pathogen system to untangle questions regarding co-evolution due to its large geographic distribution and occurrence in a wide variety of aquatic and terrestrial wildlife species.}, journal={FRONTIERS IN ECOLOGY AND EVOLUTION}, author={Frias-De-Diego, Alba and Jara, Manuel and Escobar, Luis E.}, year={2019}, month={Oct} }
 @article{silva_souza_baldissera_von laer_lovato_sarturi_herrmann_moura_favaretto_frias-de-diego_et al._2019, title={Relation of reproductive disturbance in sheep and Leptospira interrogans serovar Icterohaemorrhagiae infection: Impacts on cellular oxidation status}, volume={130}, ISSN={["1096-1208"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85062451784&partnerID=MN8TOARS}, DOI={10.1016/j.micpath.2019.02.029}, abstractNote={This study evaluated the seropositivity of Brucella abortus and Leptospira interrogans in ewes with reproductive disturbances in southern Brazil and verified the creatine kinase (CK) activity and oxidation status via assessment of superoxide dismutase, glutathione peroxidase and glutathione transferase in serum of seropositive animals for L. interrogans serovar Icterohaemorrhagiae. For Leptospira infection 381 animals with clinical history of reproductive disturbance from Planalto Serrano de Santa Catarina (Brazil) were analyzed, showing an occurrence for L. interrogans of 20.2% from which 81.8% were seropositive for L. interrogans Icterohaemorrhagiae. Serovars Wolfii, Grippothyphosa, Bratislava, Canicola and Butembo were also identified. In the case of B. abortus, positive cases were identified by buffered acidified antigen, finding 14 positive samples, but none of them were positive after a second test (2-mercaptoethanol), showing the absence of relationship between infection with B. abortus and abortion in the tested individuals. Serum reactive oxygen species (ROS) levels and CK activity were found higher in animals positive for Leptospira infection, presenting higher titrations (1:320) than non-infected individuals. Serum glutathione peroxidase activity was higher in positive animals with titrations 1:160 and 1:320, while serum glutathione S-transferase was higher in positive individuals only for titrations 1:320. Serum superoxide dismutase showed lower activity in infected animals with titrations of 1:320. Our results show the region of Planalto Serrano de Santa Catarina with a high occurrence levels of sheep infected by L. interrogans serovar Icterohaemorrhagiae, from which animals with high titrations (1:320) present oxidative stress elicited by excessive ROS production, triggering the stimulation of antioxidant systems to counter this excess. In summary, ovine with higher titrations (1:320) present oxidative damage that can contribute to disease pathophysiology.}, journal={MICROBIAL PATHOGENESIS}, author={Silva, Aleksandro S. and Souza, Carine F. and Baldissera, Matheus D. and Von Laer, Ana Eucares and Lovato, Luciane T. and Sarturi, Janine A. and Herrmann, Geder P. and Moura, Anderson B. and Favaretto, Jorge A. and Frias-De-Diego, Alba and et al.}, year={2019}, month={May}, pages={65–70} }