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We previously reported that deletion of a 44-nucleotide element in the 3' untranslated region (UTR) of the Chikungunya virus (CHIKV) genome enhances the virulence of CHIKV infection in mice. Here, we find that while this 44-nucleotide deletion enhances CHIKV fitness in murine embryonic fibroblasts in a manner independent of the type I interferon response, the same mutation decreases viral fitness in C6/36 mosquito cells. Further, the fitness advantage conferred by the UTR deletion in mammalian cells is maintained in vivo in a mouse model of CHIKV dissemination. Finally, SHAPE-MaP analysis of the CHIKV 3' UTR revealed this 44-nucleotide element forms a distinctive two-stem-loop structure that is ablated in the mutant 3' UTR without altering additional 3' UTR RNA secondary structures.
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Regiones no Traducidas 3' , Fiebre Chikungunya , Virus Chikungunya , Replicación Viral , Virus Chikungunya/genética , Virus Chikungunya/fisiología , Animales , Ratones , Fiebre Chikungunya/virología , ARN Viral/genética , Virulencia , Línea Celular , Fibroblastos/virología , Aptitud Genética , Humanos , Eliminación de Secuencia , Conformación de Ácido Nucleico , Modelos Animales de EnfermedadRESUMEN
Chikungunya virus (CHIKV) is an enveloped, positive-sense RNA virus that has re-emerged to cause millions of human infections worldwide. In humans, acute CHIKV infection causes fever and severe muscle and joint pain. Chronic and debilitating arthritis and joint pain can persist for months to years. To date, there are no approved antivirals against CHIKV. Recently, the ribonucleoside analog 4'-fluorouridine (4'-FlU) was reported as a highly potent orally available inhibitor of SARS-CoV-2, respiratory syncytial virus, and influenza virus replication. In this study, we assessed 4'-FlU's potency and breadth of inhibition against a panel of alphaviruses including CHIKV, and found that it broadly suppressed alphavirus production in cell culture. 4'-FlU acted on the viral RNA replication step, and the first 4 hours post-infection were the critical time for its antiviral effect. In vitro replication assays identified nsP4 as the target of inhibition. In vivo, treatment with 4'-FlU reduced disease signs, inflammatory responses, and viral tissue burden in mouse models of CHIKV and Mayaro virus infection. Treatment initiated at 2 hours post-infection was most effective; however, treatment initiated as late as 24-48 hours post-infection produced measurable antiviral effects in the CHIKV mouse model. 4'-FlU showed effective oral delivery in our mouse model and resulted in the accumulation of both 4'-FlU and its bioactive triphosphate form in tissues relevant to arthritogenic alphavirus pathogenesis. Together, our data indicate that 4'-FlU inhibits CHIKV infection in vitro and in vivo and is a promising oral therapeutic candidate against CHIKV infection.IMPORTANCEAlphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro, there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4'-fluorouridine (4'-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4'-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4'-FlU and accumulation of both 4'-FlU and its bioactive form in relevant tissues. In summary, 4'-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA.
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Alphavirus , Antivirales , Virus Chikungunya , Replicación Viral , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Ratones , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/fisiología , Alphavirus/efectos de los fármacos , Alphavirus/fisiología , Uridina/análogos & derivados , Uridina/farmacología , Humanos , Fiebre Chikungunya/tratamiento farmacológico , Fiebre Chikungunya/virología , Modelos Animales de Enfermedad , Línea Celular , Chlorocebus aethiops , Femenino , Células VeroRESUMEN
Antigens from protein subunit vaccination traffic from the tissue to the draining lymph node, either passively via the lymph or carried by dendritic cells at the local injection site. Lymph node (LN) lymphatic endothelial cells (LEC) actively acquire and archive foreign antigens, and archived antigen can be released during subsequent inflammatory stimulus to improve immune responses. Here, we answer questions about how LECs achieve durable antigen archiving and whether there are transcriptional signatures associated with LECs containing high levels of antigen. We used single cell sequencing in dissociated LN tissue to quantify antigen levels in LEC and dendritic cell populations at multiple timepoints after immunization, and used machine learning to define a unique transcriptional program within archiving LECs that can predict LEC archiving capacity in independent data sets. Finally, we validated this modeling, showing we could predict antigen archiving from a transcriptional dataset of CHIKV infected mice and demonstrated in vivo the accuracy of our prediction. Collectively, our findings establish a unique transcriptional program in LECs that promotes antigen archiving that can be translated to other systems.
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The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya, eastern- (EEEV), and Venezuelan- (VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology.
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Breast cancer is the second most common cancer globally. Most deaths from breast cancer are due to metastatic disease which often follows long periods of clinical dormancy1. Understanding the mechanisms that disrupt the quiescence of dormant disseminated cancer cells (DCC) is crucial for addressing metastatic progression. Infection with respiratory viruses (e.g. influenza or SARS-CoV-2) is common and triggers an inflammatory response locally and systemically2,3. Here we show that influenza virus infection leads to loss of the pro-dormancy mesenchymal phenotype in breast DCC in the lung, causing DCC proliferation within days of infection, and a greater than 100-fold expansion of carcinoma cells into metastatic lesions within two weeks. Such DCC phenotypic change and expansion is interleukin-6 (IL-6)-dependent. We further show that CD4 T cells are required for the maintenance of pulmonary metastatic burden post-influenza virus infection, in part through attenuation of CD8 cell responses in the lungs. Single-cell RNA-seq analyses reveal DCC-dependent impairment of T-cell activation in the lungs of infected mice. SARS-CoV-2 infected mice also showed increased breast DCC expansion in lungs post-infection. Expanding our findings to human observational data, we observed that cancer survivors contracting a SARS-CoV-2 infection have substantially increased risks of lung metastatic progression and cancer-related death compared to cancer survivors who did not. These discoveries underscore the significant impact of respiratory viral infections on the resurgence of metastatic cancer, offering novel insights into the interconnection between infectious diseases and cancer metastasis.
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Infection by chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes severe polyarthralgia and polymyalgia, which can last in some people for months to years. Chronic CHIKV disease signs and symptoms are associated with the persistence of viral nucleic acid and antigen in tissues. Like humans and nonhuman primates, CHIKV infection in mice results in the development of robust adaptive antiviral immune responses. Despite this, joint tissue fibroblasts survive CHIKV infection and can support persistent viral replication, suggesting that they escape immune surveillance. Here, using a recombinant CHIKV strain encoding the fluorescent protein VENUS with an embedded CD8+ T cell epitope, SIINFEKL, we observed a marked loss of both MHC class I (MHC-I) surface expression and antigen presentation by CHIKV-infected joint tissue fibroblasts. Both in vivo and ex vivo infected joint tissue fibroblasts displayed reduced cell surface levels of H2-Kb and H2-Db MHC-I proteins while maintaining similar levels of other cell surface proteins. Mutations within the methyl transferase-like domain of the CHIKV nonstructural protein 2 (nsP2) increased MHC-I cell surface expression and antigen presentation efficiency by CHIKV-infected cells. Moreover, expression of WT nsP2 alone, but not nsP2 with mutations in the methyltransferase-like domain, resulted in decreased MHC-I antigen presentation efficiency. MHC-I surface expression and antigen presentation was rescued by replacing VENUS-SIINFEKL with SIINFEKL tethered to ß2-microglobulin in the CHIKV genome, which bypasses the requirement for peptide processing and TAP-mediated peptide transport into the endoplasmic reticulum. Collectively, this work suggests that CHIKV escapes the surveillance of antiviral CD8+ T cells, in part, by nsP2-mediated disruption of MHC-I antigen presentation.
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Fiebre Chikungunya , Virus Chikungunya , Humanos , Animales , Ratones , Presentación de Antígeno , Replicación Viral , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Epítopos de Linfocito T , Péptidos/metabolismoRESUMEN
Antigens from viruses or immunizations can persist or are archived in lymph node stromal cells such as lymphatic endothelial cells (LEC) and fibroblastic reticular cells (FRC). Here, we find that, during the time frame of antigen archiving, LEC apoptosis caused by a second, but unrelated, innate immune stimulus such as vaccina viral infection or CpG DNA administration resulted in cross-presentation of archived antigens and boosted memory CD8 + T cells specific to the archived antigen. In contrast to "bystander" activation associated with unrelated infections, the memory CD8 + T cells specific to the archived antigen from the immunization were significantly higher than memory CD8 + T cells of a different antigen specificity. Finally, the boosted memory CD8 + T cells resulted in increased protection against Listeria monocytogenes expressing the antigen from the immunization, but only for the duration that the antigen was archived. These findings outline an important mechanism by which lymph node stromal cell archived antigens, in addition to bystander activation, can augment memory CD8 + T cell responses during repeated inflammatory insults.
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Alphaviruses are mosquito-transmitted pathogens that induce high levels of viremia, which facilitates dissemination and vector transmission. One prevailing paradigm is that, after skin inoculation, alphavirus-infected resident dendritic cells migrate to the draining lymph node (DLN), facilitating further rounds of infection and dissemination. Here, we assess the contribution of infiltrating myeloid cells to alphavirus spread. We observe two phases of virus transport to the DLN, one that occurs starting at 1 h post infection and precedes viral replication, and a second that requires replication in the skin, enabling transit to the bloodstream. Depletion of Ly6C+ monocytes reduces local chikungunya (CHIKV) or Ross River virus (RRV) infection in the skin, diminishes the second phase of virus transport to the DLN, and delays spread to distal sites. Our data suggest that infiltrating monocytes facilitate alphavirus infection at the initial infection site, which promotes more rapid spread into circulation.
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Fiebre Chikungunya , Virus Chikungunya , Animales , Monocitos/patología , Mosquitos Vectores , Fiebre Chikungunya/patología , Células Mieloides , Replicación ViralRESUMEN
Intracellular Salmonella experiencing oxidative stress downregulates aerobic respiration. To maintain cellular energetics during periods of oxidative stress, intracellular Salmonella must utilize terminal electron acceptors of lower energetic value than molecular oxygen. We show here that intracellular Salmonella undergoes anaerobic respiration during adaptation to the respiratory burst of the phagocyte NADPH oxidase in macrophages and in mice. Reactive oxygen species generated by phagocytes oxidize methionine, generating methionine sulfoxide. Anaerobic Salmonella uses the molybdenum cofactor-containing DmsABC enzymatic complex to reduce methionine sulfoxide. The enzymatic activity of the methionine sulfoxide reductase DmsABC helps Salmonella maintain an alkaline cytoplasm that supports the synthesis of the antioxidant hydrogen sulfide via cysteine desulfuration while providing a source of methionine and fostering redox balancing by associated dehydrogenases. Our investigations demonstrate that nontyphoidal Salmonella responding to oxidative stress exploits the anaerobic metabolism associated with dmsABC gene products, a pathway that has accrued inactivating mutations in human-adapted typhoidal serovars.
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Metionina/análogos & derivados , NADPH Oxidasas , Fagocitos , Animales , Ratones , Humanos , Anaerobiosis , Fagocitos/metabolismo , Metionina/metabolismo , Salmonella typhimurium/metabolismo , RespiraciónRESUMEN
Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we found that, during the first 24 hours of infection, CHIKV RNA accumulated in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response - including recruitment of myeloid cells to the LN - was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.
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Fiebre Chikungunya , Virus Chikungunya , Células Endoteliales/metabolismo , Inmunización , Ganglios Linfáticos , AnimalesRESUMEN
Infection by chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes severe polyarthralgia and polymyalgia, which can last in some people for months to years. Chronic CHIKV disease signs and symptoms are associated with the persistence of viral nucleic acid and antigen in tissues. Like humans and nonhuman primates, CHIKV infection in mice results in the development of robust adaptive antiviral immune responses. Despite this, joint tissue fibroblasts survive CHIKV infection and can support persistent viral replication, suggesting that they escape immune surveillance. Here, using a recombinant CHIKV strain encoding a chimeric protein of VENUS fused to a CD8+ T cell epitope, SIINFEKL, we observed a marked loss of both MHC class I (MHC-I) surface expression and antigen presentation by CHIKV-infected joint tissue fibroblasts. Both in vivo and ex vivo infected joint tissue fibroblasts displayed reduced cell surface levels of H2-Kb and H2-Db MHC proteins while maintaining similar levels of other cell surface proteins. Mutations within the methyl transferase-like domain of the CHIKV nonstructural protein 2 (nsP2) increased MHC-I cell surface expression and antigen presentation efficiency by CHIKV-infected cells. Moreover, expression of WT nsP2 alone, but not nsP2 with mutations in the methyltransferase-like domain, resulted in decreased MHC-I antigen presentation efficiency. MHC-I surface expression and antigen presentation could be rescued by replacing VENUS-SIINFEKL with SIINFEKL tethered to ß2-microglobulin in the CHIKV genome, which bypasses the need for peptide processing and TAP-mediated peptide transport into the endoplasmic reticulum. Collectively, this work suggests that CHIKV escapes the surveillance of antiviral CD8+ T cells, in part, by nsP2-mediated disruption of MHC-I antigen presentation.
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Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we find that during the first 24 h of infection, CHIKV RNA accumulates in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response, including recruitment of myeloid cells to the LN, was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, we find that antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.
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The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya (CHIKV), eastern-(EEEV), and Venezuelan-(VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate (HS) impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology. Significance Statement: Previously, evidence of arbovirus-GAG interactions in vivo has been limited to associations between viral residues shown to promote enhanced GAG-binding phenotypes in vitro and in vivo phenotypes of viral dissemination and pathogenesis. By directly manipulating host GAG expression, we identified virion-GAG interactions in vivo and discovered a role for phagocyte-expressed GAGs in viral vascular clearance. Moreover, we observe species-specific differences in viral vascular clearance of enhanced GAG-binding virions between murine and avian hosts. These data suggest species-specific variation in GAG structure is a mechanism to distinguish amplifying from dead-end hosts for arbovirus transmission.
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Chikungunya virus (CHIKV) has recently emerged to cause millions of human infections worldwide. Infection can induce the formation of long intercellular extensions that project from infected cells and form stable non-continuous membrane bridges with neighbouring cells. The mechanistic role of these intercellular extensions in CHIKV infection was unclear. Here we developed a co-culture system and flow cytometry methods to quantitatively evaluate transmission of CHIKV from infected to uninfected cells in the presence of neutralizing antibody. Endocytosis and endosomal acidification were critical for virus cell-to-cell transmission, while the CHIKV receptor MXRA8 was not. By using distinct antibodies to block formation of extensions and by evaluation of transmission in HeLa cells that did not form extensions, we showed that intercellular extensions mediate CHIKV cell-to-cell transmission. In vivo, pre-treatment of mice with a neutralizing antibody blocked infection by direct virus inoculation, while adoptive transfer of infected cells produced antibody-resistant host infection. Together our data suggest a model in which the contact sites of intercellular extensions on target cells shield CHIKV from neutralizing antibodies and promote efficient intercellular virus transmission both in vitro and in vivo.
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Fiebre Chikungunya , Virus Chikungunya , Humanos , Animales , Ratones , Células HeLa , Anticuerpos Neutralizantes , Técnicas de CocultivoRESUMEN
Infectious particles can be shared through aerosols and droplets formed as the result of normal respiration. Whether Abs within the nasal/oral fluids can similarly be shared between hosts has not been investigated. The circumstances of the SARS-CoV-2 pandemic facilitated a unique opportunity to fully examine this provocative idea. The data we show from human nasal swabs provides evidence for the aerosol transfer of Abs between immune and nonimmune hosts.
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COVID-19 , Humanos , Inmunidad Humoral , SARS-CoV-2 , Aerosoles y Gotitas Respiratorias , PandemiasRESUMEN
Arboviruses are public health threats that cause explosive outbreaks. Major determinants of arbovirus transmission, geographic spread, and pathogenesis are the magnitude and duration of viremia in vertebrate hosts. Previously, we determined that multiple alphaviruses are cleared efficiently from murine circulation by the scavenger receptor MARCO (Macrophage receptor with collagenous structure). Here, we define biochemical features on chikungunya (CHIKV), o'nyong 'nyong (ONNV), and Ross River (RRV) viruses required for MARCO-dependent clearance in vivo. In vitro, MARCO expression promotes binding and internalization of CHIKV, ONNV, and RRV via the scavenger receptor cysteine-rich (SRCR) domain. Furthermore, we observe species-specific effects of the MARCO SRCR domain on CHIKV internalization, where those from known amplification hosts fail to promote CHIKV internalization. Consistent with this observation, CHIKV is inefficiently cleared from the circulation of rhesus macaques in contrast with mice. These findings suggest a role for MARCO in determining whether a vertebrate serves as an amplification or dead-end host following CHIKV infection.
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Fiebre Chikungunya , Virus Chikungunya , Animales , Ratones , Viremia , Macaca mulatta , Brotes de Enfermedades , Receptores InmunológicosRESUMEN
Endothelial dysfunction and inflammation contribute to the vascular pathology of coronavirus disease (COVID-19). However, emerging evidence does not support direct infection of endothelial or other vascular wall cells, and thus inflammation may be better explained as a secondary response to epithelial cell infection. In this study, we sought to determine whether lung endothelial or other resident vascular cells are susceptible to productive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and how local complement activation contributes to endothelial dysfunction and inflammation in response to hypoxia and SARS-CoV-2-infected lung alveolar epithelial cells. We found that ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) mRNA expression in lung vascular cells, including primary human lung microvascular endothelial cells (HLMVECs), pericytes, smooth muscle cells, and fibroblasts, was 20- to 90-fold lower compared with primary human alveolar epithelial type II cells. Consistently, we found that HLMVECs and other resident vascular cells were not susceptible to productive SARS-CoV-2 infection under either normoxic or hypoxic conditions. However, viral uptake without replication (abortive infection) was observed in HLMVECs when exposed to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells. Furthermore, we demonstrated that exposure of HLMVECs to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells and hypoxia resulted in upregulation of inflammatory factors such as ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), and IL-6 (interleukin 6) as well as complement components such as C3 (complement C3), C3AR1 (complement C3a receptor 1), C1QA (complement C1q A chain), and CFB (complement factor B). Taken together, our data support a model in which lung endothelial and vascular dysfunction during COVID-19 involves the activation of complement and inflammatory signaling and does not involve productive viral infection of endothelial cells.
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COVID-19 , Humanos , COVID-19/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , SARS-CoV-2/metabolismo , Células Endoteliales/metabolismo , Medios de Cultivo Condicionados , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Pulmón/patología , Inflamación/metabolismo , Proteínas del Sistema Complemento/metabolismoRESUMEN
Alphaviruses are a large group (>30 species) of enveloped, positive-strand RNA viruses. The re-emergence of mosquito-transmitted alphaviruses associated with human diseases ranging from severe and potentially fatal neurological disease to chronic arthritic disease highlights the need to understand the biology and pathogenesis of alphaviruses. Here, we review the development and use of animal models of alphavirus transmission and human disease, and discuss areas for continued refinement of these models including possible avenues for future investigation.
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Infecciones por Alphavirus , Alphavirus , Animales , Humanos , Infecciones por Alphavirus/patología , Alphavirus/genética , Modelos Animales de EnfermedadRESUMEN
Background: There is an urgent need for harmonization between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology platforms and assays prior to defining appropriate correlates of protection and as well inform the development of new rapid diagnostic tests that can be used for serosurveillance as new variants of concern (VOC) emerge. We compared multiple SARS-CoV-2 serology reference materials to the WHO International Standard (WHO IS) to determine their utility as secondary standards, using an international network of laboratories with high-throughput quantitative serology assays. This enabled the comparison of quantitative results between multiple serology platforms. Methods: Between April and December 2020, 13 well-characterized and validated SARS-CoV-2 serology reference materials were recruited from six different providers to qualify as secondary standards to the WHO IS. All the samples were tested in parallel with the National Institute for Biological Standards and Control (NIBSC) 20/136 and parallel-line assays were used to calculate the relevant potency and binding antibody units. Results: All the samples saw varying levels of concordance between diagnostic methods at specific antigen-antibody combinations. Seven of the 12 candidate materials had high concordance for the spike-immunoglobulin G (IgG) analyte [percent coefficient of variation (%CV) between 5 and 44%]. Conclusion: Despite some concordance between laboratories, qualification of secondary materials to the WHO IS using arbitrary international units or binding antibody units per milliliter (BAU/ml) does not provide any benefit to the reference materials overall, due to the lack of consistent agreeable international unit (IU) or BAU/ml conversions between laboratories. Secondary standards should be qualified to well-characterized reference materials, such as the WHO IS, using serology assays that are similar to the ones used for the original characterization of the WHO IS.
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The type III secretion system encoded in the Salmonella pathogenicity island-2 (SPI-2) gene cluster facilitates intracellular growth of nontyphoidal Salmonella by interfering with the maturation of Salmonella-containing vacuoles along the degradative pathway. SPI-2 gene products also protect Salmonella against the antimicrobial activity of reactive oxygen species (ROS) synthesized by the phagocyte NADPH oxidase 2 (NOX2). However, a potential relationship between inflammatory ROS and the activation of transcription of SPI-2 genes by intracellular Salmonella is unclear. Here, we show that ROS engendered in the innate host response stimulate SPI-2 gene transcription. We found that the expression of SPI-2 genes in Salmonella-sustaining oxidative stress conditions involves DksA, a protein otherwise known to regulate the stringent response of bacteria to nutritional stress. We also demonstrate that the J and zinc-2-oxidoreductase domains of DnaJ as well as the ATPase activity of the DnaK chaperone facilitate loading of DksA onto RNA polymerase complexed with SPI-2 promoters. Furthermore, the DksA-driven transcription of SPI-2 genes in Salmonella experiencing oxidative stress is contingent on upstream OmpR, PhoP, and SsrB signaling events that participate in the removal of nucleoid proteins while simultaneously recruiting RNA polymerase to SPI-2 promoter regions. Taken together, our results suggest the activation of SPI-2 gene transcription in Salmonella subjected to ROS produced by the respiratory burst of macrophages protects this intracellular pathogen against NOX2-mediated killing. We propose that Salmonella have co-opted inflammatory ROS to induce SPI-2-mediated protective responses against NOX2 host defenses.