The emergence of severe acute respiratory syndrome coronavirus 2, the causative agent of coronavirus disease 2019, has resulted in the largest pandemic in recent history. Current therapeutic strategies to mitigate this disease have focused on the development of vaccines and on drugs that inhibit the viral 3CL protease or RNA-dependent RNA polymerase enzymes. A less-explored and potentially complementary drug target is Nsp15, a uracil-specific RNA endonuclease that shields coronaviruses and other nidoviruses from mammalian innate immune defenses. Here, we perform a high-throughput screen of over 100,000 small molecules to identify Nsp15 inhibitors. We characterize the potency, mechanism, selectivity, and predicted binding mode of five lead compounds. We show that one of these, IPA-3, is an irreversible inhibitor that might act via covalent modification of Cys residues within Nsp15. Moreover, we demonstrate that three of these inhibitors (hexachlorophene, IPA-3, and CID5675221) block severe acute respiratory syndrome coronavirus 2 replication in cells at subtoxic doses. This study provides a pipeline for the identification of Nsp15 inhibitors and pinpoints lead compounds for further development against coronavirus disease 2019 and related coronavirus infections.

Objective Peroxisome injury occurs in the central nervous system (CNS) during multiple virus infections that result in neurological disabilities. We investigated host neuroimmune responses and peroxisome biogenesis factors during severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection using a multiplatform strategy. Methods Brain tissues from coronavirus disease 2019 (COVID‐19) (n = 12) and other disease control (ODC) (n = 12) patients, as well as primary human neural cells and Syrian hamsters, infected with a clinical variant of SARS‐CoV‐2, were investigated by droplet digital polymerase chain reaction (ddPCR), quantitative reverse transcriptase PCR (RT‐qPCR), and immunodetection methods. Results SARS‐CoV‐2 RNA was detected in the CNS of 4 patients with COVID‐19 with viral protein (NSP3 and spike) immunodetection in the brainstem. Olfactory bulb, brainstem, and cerebrum from patients with COVID‐19 showed induction of pro‐inflammatory transcripts ( IL8, IL18, CXCL10, NOD2 ) and cytokines (GM‐CSF and IL‐18) compared to CNS tissues from ODC patients ( p < 0.05). Peroxisome biogenesis factor transcripts ( PEX3, PEX5L, PEX11β, and PEX14 ) and proteins (PEX3, PEX14, PMP70) were suppressed in the CNS of COVID‐19 compared to ODC patients ( p < 0.05). SARS‐CoV‐2 infection of hamsters revealed viral RNA detection in the olfactory bulb at days 4 and 7 post‐infection while inflammatory gene expression was upregulated in the cerebrum of infected animals by day 14 post‐infection ( p < 0.05). Pex3 transcript levels together with catalase and PMP70 immunoreactivity were suppressed in the cerebrum of SARS‐CoV‐2 infected animals ( p < 0.05). Interpretation COVID‐19 induced sustained neuroinflammatory responses with peroxisome biogenesis factor suppression despite limited brainstem SARS‐CoV‐2 neurotropism in humans. These observations offer insights into developing biomarkers and therapies, while also implicating persistent peroxisome dysfunction as a contributor to the neurological post‐acute sequelae of COVID‐19. ANN NEUROL 2023;94:531–546

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) poses a persistent threat to global public health. Although primarily a respiratory illness, extrapulmonary manifestations of COVID-19 include gastrointestinal, cardiovascular, renal and neurological diseases. Recent studies suggest that dysfunction of the endothelium during COVID-19 may exacerbate these deleterious events by inciting inflammatory and microvascular thrombotic processes. Although controversial, there is evidence that SARS-CoV-2 may infect endothelial cells by binding to the angiotensin-converting enzyme 2 (ACE2) cellular receptor using the viral Spike protein. In this review, we explore current insights into the relationship between SARS-CoV-2 infection, endothelial dysfunction due to ACE2 downregulation, and deleterious pulmonary and extra-pulmonary immunothrombotic complications in severe COVID-19. We also discuss preclinical and clinical development of therapeutic agents targeting SARS-CoV-2-mediated endothelial dysfunction. Finally, we present evidence of SARS-CoV-2 replication in primary human lung and cardiac microvascular endothelial cells. Accordingly, in striving to understand the parameters that lead to severe disease in COVID-19 patients, it is important to consider how direct infection of endothelial cells by SARS-CoV-2 may contribute to this process.

In the present report, we describe two small molecules with broad-spectrum antiviral activity. These drugs block the formation of the nodosome. The studies were prompted by the observation that infection of human fetal brain cells with Zika virus (ZIKV) induces the expression of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a host factor that was found to promote ZIKV replication and spread. A drug that targets NOD2 was shown to have potent broad-spectrum antiviral activity against other flaviviruses, alphaviruses, enteroviruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). Another drug that inhibits receptor-interacting serine/threonine protein kinase 2 (RIPK2), which functions downstream of NOD2, also decreased the replication of these pathogenic RNA viruses. The antiviral effect of this drug was particularly potent against enteroviruses. The broad-spectrum action of nodosome-targeting drugs is mediated in part by the enhancement of the interferon response. Together, these results suggest that further preclinical investigation of nodosome inhibitors as potential broad-spectrum antivirals is warranted.

An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the 10-100 ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be pre-designed for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.

Zika virus (ZIKV) infection during pregnancy can result in congenital Zika syndrome which is characterized by microcephaly and other neurodevelopmental disorders. In this chapter, we describe methods to model ex vivo ZIKV infection in astrocytes and tissue explants from human fetal brain. These cell- and tissue-based platforms have been useful to elucidate mechanisms of ZIKV persistence and might lead to important clues about virus-induced neuropathogenesis. In addition, these ex vivo model systems allow researchers to conduct drug discovery and development experiments in more representative settings of the developing human brain.

Before the emergence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causative agent of the current COVID-19 (coronavirus disease 2019) pandemic [...]

Zika virus (ZIKV) has emerged as an important human pathogen that can cause congenital defects in the fetus and neurological conditions in adults. The interferon (IFN) system has proven crucial in restricting ZIKV replication and pathogenesis. The canonical IFN response is triggered by the detection of viral RNA through RIG-I like receptors followed by activation of the adaptor protein MAVS on mitochondrial membranes. Recent studies have shown that a second organelle, peroxisomes, also function as a signaling platforms for the IFN response. Here, we investigated how ZIKV infection affects peroxisome biogenesis and antiviral signaling. We show that ZIKV infection depletes peroxisomes in human fetal astrocytes, a brain cell type that can support persistent infection. The peroxisome biogenesis factor PEX11B was shown to inhibit ZIKV replication, likely by increasing peroxisome numbers and enhancing downstream IFN-dependent antiviral signaling. Given that peroxisomes play critical roles in brain development and nerve function, our studies provide important insights into the roles of peroxisomes in regulating ZIKV infection and potentially neuropathogenesis.

Zika virus (ZIKV) is an emerging pathogen that can cause microcephaly and other neurological defects in developing fetuses. The cellular response to ZIKV in the fetal brain is not well understood. Here, we show that ZIKV infection of human fetal astrocytes (HFAs), the most abundant cell type in the brain, results in elevated expression and secretion of fibroblast growth factor 2 (FGF2). This cytokine was shown to enhance replication and spread of ZIKV in HFAs and human fetal brain explants. The proviral effect of FGF2 is likely mediated in part by suppression of the interferon response, which would represent a novel mechanism by which viruses antagonize host antiviral defenses. We posit that FGF2-enhanced virus replication in the fetal brain contributes to the neurodevelopmental disorders associated with in utero ZIKV infection. As such, targeting FGF2-dependent signaling should be explored further as a strategy to limit replication of ZIKV.

Zika virus (ZIKV) infection and persistence during pregnancy can lead to microcephaly and other fetal neurological disorders collectively known as Congenital Zika Syndrome. The immunological and virological events that contribute to the establishment of persistent ZIKV infection in humans are unclear though. Here we show that human fetal astrocytes (HFAs), the most abundant cell type in the central nervous system, become persistently infected with ZIKV resulting in continuous viral shedding for at least one month; a process that is facilitated by TIM/TAM receptors. HFAs are relatively resistant to ZIKV-induced apoptosis, a factor that may be important for chronic infection of these cells. Once infection was established, interferon treatment did not reduce virus replication. Moreover, the fact that the innate immune system was highly activated in persistently infected HFAs indicates that the virus can thrive in the presence of a sustained antiviral response. RNAseq analyses of persistently infected cells revealed that ZIKV alters host gene expression in a manner that could affect developmental processes. Conversely, data from sequencing of ZIKV genomes in persistently infected HFAs suggest that adaptive mutations were not required for establishing chronic infection. Based on these results, we postulate that HFAs are reservoirs for ZIKV in the fetal brain and that moderate apoptosis combined with inefficient antiviral response from these cells may contribute to the establishment of chronic brain infection associated with the ZIKV neurodevelopmental abnormalities.

Zika virus is a teratogenic mosquito-transmitted flavivirus that is associated with birth defects in newborns and Guillain–Barré syndrome in adults. The virus can also be sexually transmitted, but currently, very little is known about the cell types supporting virus replication and persistence in human testes. Using primary cell cultures, we observed that Sertoli but not Leydig cells are highly susceptible to Zika virus infection, a process that is dependent on the TAM family receptor Axl. In cell culture, Sertoli cells could be productively infected with Zika virus for at least 6-weeks. Infection of Sertoli cells resulted in dramatic changes to the transcriptional profile of these cells. The most upregulated mRNA in infected cells was basic fibroblast growth factor (FGF2), a cytokine that was found to enhance Zika virus replication and support viral persistence. Together these findings provide key insights into understanding how Zika virus persists in the male reproductive tract and in turn may aid in developing antiviral therapies or strategies to minimize sexual transmission of this pathogen.

As you will see, all three referees acknowledge the potential interest of the findings. However, all three referees have raised a number of concerns and suggestions to improve the manuscript or to strengthen the data and the conclusions drawn. In particular point 2 by referee #1 is important and needs to be addressed experimentally. In general, the data on the N25-STAT2 interaction need to be strengthened (see also referee #3), as well as on the proteasomal degradation of STAT2 (referee #2, point 3). Point 3 of referee #1 needs to be addressed, if such animals are available, as well as the last point of referee #2, that is on a similar line (i.e. more evidence needs to be provided that shows why ZIKV cannot antagonize murine STAT2).

Zika virus is an emerging mosquito‐borne pathogen that is associated with Guillain–Barré syndrome in adults and microcephaly and other neurological defects in newborns. Despite being declared an international emergency by the World Health Organization, comparatively little is known about its biology. Here, we investigate the strategies employed by the virus to suppress the host antiviral response. We observe that once established, Zika virus infection is impervious to interferon treatment suggesting that the virus deploys effective countermeasures to host cell defences. This is confirmed by experiments showing that Zika virus infection impairs the induction of type‐I interferon as well as downstream interferon‐stimulated genes. Multiple viral proteins affect these processes. Virus‐mediated degradation of STAT2 acts to reduce type‐I and type‐III interferon‐mediated signaling. Further, the NS5 of Zika virus binds to STAT2, and its expression is correlated with STAT2 degradation by the proteasome. Together, our findings provide key insights into how Zika virus blocks cellular defense systems. This in turn is important for understanding pathogenesis and may aid in designing antiviral therapies. Zika virus (ZIKV) is a mosquito‐borne pathogen that causes Guillain–Barré syndrome in adults and microcephaly in newborns. This report shows that ZIKV infection inhibits the induction of type‐I interferons by downregulating IRF3 and antiviral NF‐κB‐mediated signaling and targets STAT2 for proteasomal degradation. Zika virus (ZIKV) is a mosquito‐borne pathogen that causes Guillain–Barré syndrome in adults and microcephaly in newborns. This report shows that ZIKV infection inhibits the induction of type‐I interferons by downregulating IRF3 and antiviral NF‐κB‐mediated signaling and targets STAT2 for proteasomal degradation.

Introduccion : El dengue es causado por el virus dengue (VDEN) que es considerado el virus trasmitido a humanos por mosquitos mas importante del mundo en terminos de morbilidad y mortalidad. En la compleja fisiopatologia del dengue se ha demostrado la participacion de la apoptosis. Objetivos : Correlacionar los niveles plasmaticos de proteinas apoptoticas y la clasificacion de dengue con el recuento de celulas sanguineas en pacientes con dengue de Brasil. Materiales y Metodos : En 62 pacientes brasilenos con infeccion confirmada por VDEN se determino por ELISA los niveles en plasma de tres proteinas inductoras de apoptosis (TNF-α, FasL, TRAIL) y una proteina anti-apoptotica (Survivin). Esos valores se correlacionaron a la cuantificacion de celulas sanguineas obtenida en los hemogramas y por citometria de flujo. Resultados : El unico caso que falleciera presento el nivel plasmatico mas alto de FasL. Los niveles circulantes de TRAIL se correlacionaron positivamente con el recuento de plaquetas. Nuestros resultados indican que posiblemente TRAIL induce apoptosis en linfocitos T CD4+ y CD8+ y que ademas podria poseer un efecto antiviral. En cambio, el Survivin podria brindar proteccion contra la apoptosis en leucocitos y particularmente en los monocitos CD14+. Conclusiones : Nuestro trabajo es el primero en el mundo que estudia la posible regulacion de apoptosis de leucocitos por el TRAIL y el Survivin en pacientes con dengue. Estos resultados apoyan que la apoptosis esta implicada en la fisiopatologia del dengue.

Introduccion: Se considera una prioridad para el diseno de una vacuna eficaz y segura contra el dengue, distinguir entre una respuesta inmune protectora y una patogenica frente a la infeccion natural. Las citoquinas se han asociado a la patogenesis del cuadro grave, sin embargo, muchos de los resultados de estudios realizados en diferentes partes del mundo son contradictorios o no reproducibles. El objetivo del presente trabajo es seleccionar los modelos mas utiles para el estudio de las citoquinas en la patogenesis del dengue. Material y metodos: Se determinaron los niveles de citoquinas proinflamatorias y antiinflamatorias en muestras de sueros de pacientes de dengue y se midio la expresion genica de esos mediadores en tejidos de fallecidos por dengue. Ademas se cuantifico el ARNm de las citoquinas en celulas sanguineas cultivadas in vitro con virus dengue y se evaluo la asociacion de variantes alelicas de los genes de las citoquinas estudiadas con las formas clinicas de la infeccion por dengue. Resultados: Los estudios in vivo sugirieron una aparente asociacion de las citoquinas antiinflamatorias con la gravedad del cuadro clinico, mientras los experimentos in vitro y el estudio del polimorfismo de genes evidenciaron el papel de los mediadores proinflamatorios en la patogenia del dengue grave y la infeccion secundaria heterotipica. Conclusiones: Son necesarios distintos modelos con enfoques complementarios para discernir el papel de los mediadores de respuesta inmune en la patogenesis de la enfermedad por dengue. Estudios con modelos unicos pueden llevar a conclusiones erradas.

Despite being the most significant arboviral disease worldwide, dengue has no antiviral treatment or reliable severity predictors. It has been shown that apoptotic cells from blood and tissues may be involved in the complex pathogenesis of dengue. However, very little is known about the interplay between proapoptotic and antiapoptotic mediators in this disease. Therefore, plasma levels of the three proapoptotic mediators Fas ligand (FasL), tumor necrosis factor‐α (TNF‐α), and TNF‐related apoptosis‐inducing ligand (TRAIL) were measured in dengue patients. Patients were classified according to the World Health Organization classification of dengue revised in 2009. Additionally, inhibitors of apoptosis protein (IAPs) were determined in plasma (Survivin) and peripheral blood mononuclear cells (PBMCs) lysates (cIAP‐1, cIAP‐2, XIAP). Levels of apoptotic proteins in plasma were correlated with counts of blood cells. FasL and TRAIL levels were elevated in dengue patients without warning signs when compared to patients with severe dengue and controls. Dengue patients with warning signs showed decreased levels of Survivin compared to patients with severe dengue and controls. TRAIL was inversely correlated with counts of lymphocyte subsets. In contrast, Survivin was positively correlated with leukocyte counts. There was a trend of elevated IAPs levels in PBMCs of patients with severe dengue. The results suggest a likely antiviral effect of TRAIL in dengue. It appears that TRAIL might be involved with apoptosis induction of lymphocytes, whereas IAPs might participate in protecting leukocytes from apoptosis. Further research is needed to explore the interactions between pro and antiapoptotic molecules and their implications in dengue pathogenesis. J. Med. Virol. 86:1437–1447, 2014. © 2013 Wiley Periodicals, Inc.

Background: Microsporidiosis is a life threatening opportunistic infection of AIDS patients. The infection is usually restricted. to specific anatomical areas, but could become systemic depending on the involved species. Genital microsporidiosis in female patients is rare.Objective: To report genital microsporidiosis in female AIDS patients.Methods: Tissues samples from the genital tract (ovary, fallopian tubes and uterus) of eight deceased women who died of wasting syndrome associated to AIDS and disseminated microsporidiosis at the Institute of Tropical Medicine Pedro Kouri were collected between 1997 and 2005. Using an indirect immunohistochemistry assay the microsporidia species involved in those cases were identified.Results: We report several cases of microsporidial infection of the female genital tract. Six out of eight women with the disseminated form of the disease showed the presence of microsporidia in the genital tract. Encephalitozoon cuniculi and Encephalitozoon hellem were identified in the internal lining epithelium of the fallopian tubes and endometrium.Conclusions: Microsporidia species could disseminate to other organs and become systemic in severe immunocompromised cases. To our knowledge this is the greatest number of female genital tract microsporidiosis cases so far reported in humans. (C) 2011 Revista Iberoamericana de Micologia. Published by Elsevier Espana, S.L. All rights reserved.

Background: An outbreak of acute hemorrhagic conjunctivitis occurred in Cuba in 2008 and 2009.Objective: To determinate the etiological agent associated with the Cuban outbreaks of acute hemorrhagic conjunctivitis during 2008 and 2009.Study design: Conjunctival swabs and/or faecal samples from 382 patients with clinical diagnosis suggestive of acute hemorrhagic conjunctivitis were subject to viral culture in HEp-2 human laryngeal epidermoid carcinoma cells. Positive samples were identified by a specific Coxsackievirus A24 variant PCR and the 3C protease region of 16 isolates was sequenced for phylogenetic analysis.Results: Enterovirus cytopathic effect was observed in 138 cases (36%). A higher percent of CA24v was recovered from faecal samples, 19 out of 45 cases (42.2%), than from conjunctival swabs, 127 out of 355 samples (35.8%). All isolates were identified as Coxsackievirus A24 variant. Phylogenetic analysis revealed that 2008 and 2009 Cuban outbreaks were caused by the same virus strains and that isolates were closely related to those from Taiwan (2006-2007), China (2007-2008) and Singapore (2005) with a bootstrap value of 71%.Conclusions: Outbreaks of acute hemorrhagic conjunctivitis occurred in Cuba in 2008 and 2009 were caused by Coxsackievirus A24 variant. The faecal-oral route is another mode of transmission of CA24v in the acute hemorrhagic conjunctivitis outbreaks. Phylogenetic analysis of Cuban CA24v strains involved in an acute hemorrhagic conjunctivitis outbreak in 2008 and 2009 confirms a new introduction of the CA24 variant into the Americas from South-east Asia. (C) 2011 Elsevier B. V. All rights reserved.

Dengue virus is the most significant virus transmitted by arthropods worldwide and may cause a potentially fatal systemic disease named dengue hemorrhagic fever. In this work, dengue virus serotype 4 was detected in the tissues of one fatal dengue hemorrhagic fever case using electron immunomicroscopy and molecular methods. This is the first report of dengue virus polypeptides findings by electron immunomicroscopy in human samples. In addition, not-previously-documented virus-like particles visualized in spleen, hepatic, brain, and pulmonary tissues from a dengue case are discussed.

Background: Dengue is the most important arthropod borne viral disease worldwide in terms of morbidity and mortality and is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Brazil is responsible for approximately 80% of dengue cases in the Americas, and since the introduction of dengue in 1986, a total of 5,944,270 cases have been reported including 21,596 dengue hemorrhagic fever and 874 fatal cases. DENV can infect many cell types and cause diverse clinical and pathological effects. The goal of the study was to investigate the usefulness of NS1 capture tests as an alternative tool to detect DENV in tissue specimens from previously confirmed dengue fatal cases (n = 23) that occurred in 2002 in Brazil.Methodology/Principal Findings: A total of 74 tissue specimens were available: liver (n = 23), lung (n = 14), kidney (n = 04), brain (n = 10), heart (n = 02), skin (n = 01), spleen (n = 15), thymus (n = 03) and lymph nodes (n = 02). We evaluated three tests for NS1 antigen capture: first generation Dengue Early ELISA (PanBio Diagnostics), Platelia NS1 (BioRad Laboratories) and the rapid test NS1 Ag Strip (BioRad Laboratories). The overall dengue fatal case diagnosis based on the tissues analyzed by Dengue Early ELISA, Platelia NS1 and the NS1 Ag Strip was 34.7% (08/23), 60.8% (14/23) and 91.3% (21/23), respectively. The Dengue Early ELISA detected NS1 in 22.9% (17/74) of the specimens analyzed and the Platelia NS1 in 45.9% (34/74). The highest sensitivity (78.3%; 58/74) was achieved by the NS1 Ag Strip, and the differences in the sensitivities were statistically significant (p < 0.05). The NS1 Ag Strip was the most sensitive in liver (91.3%; 21/23), lung (71.4%; 10/14), kidney (100%; 4/4), brain (80%; 8/10), spleen (66.6%, 10/15) and thymus (100%, 3/3) when compared to the other two ELISA assays.Conclusions/Significance: This study shows the DENV NS1 capture assay as a rapid and valuable approach to postmortem dengue confirmation. With an increasing number of DHF and fatal cases, the availability of new approaches useful for cases confirmation plays an important tool for the disease surveillance.