RESUMEN
Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.
Asunto(s)
Citomegalovirus , Transducción de Señal , Animales , Ratones , Humanos , Citomegalovirus/fisiología , Latencia del Virus , Diferenciación Celular , Células Madre HematopoyéticasRESUMEN
Chikungunya virus (CHIKV) is an emerging/re-emerging mosquito-borne pathogen responsible for explosive epidemics of febrile illness characterized by debilitating polyarthralgia and the risk of lethal infection among the most severe cases. Despite the public health risk posed by CHIKV, no vaccine is currently available. Using a site-directed hydrogen peroxide-based inactivation approach, we developed a new CHIKV vaccine, HydroVax-CHIKV. This vaccine technology was compared to other common virus inactivation approaches including ß-propiolactone (BPL), formaldehyde, heat, and ultraviolet (UV) irradiation. Heat, UV, and BPL were efficient at inactivating CHIKV-181/25 but caused substantial damage to neutralizing epitopes and failed to induce high-titer neutralizing antibodies in vaccinated mice. HydroVax-CHIKV and formaldehyde-inactivated CHIKV retained intact neutralizing epitopes similar to live virus controls but the HydroVax-CHIKV approach demonstrated a more rapid rate of virus inactivation. HydroVax-CHIKV vaccination induced high neutralizing responses to homologous and heterologous CHIKV clades as well as to other alphaviruses including Mayaro virus, O'nyong'nyong virus, and Una virus. Following heterologous infection with CHIKV-SL15649, HydroVax-CHIKV-immunized mice were protected against viremia, CHIKV-associated arthritic disease, and lethal CHIKV infection by an antibody-dependent mechanism. In contrast, animals vaccinated with Heat- or UV-inactivated virus showed no protection against viremia in addition to demonstrating significantly exacerbated CD4+ T cell-mediated footpad swelling after CHIKV infection. Together, these results demonstrate the risks associated with using suboptimal inactivation methods that fail to elicit protective neutralizing antibody responses and show that HydroVax-CHIKV represents a promising new vaccine candidate for prevention of CHIKV-associated disease.
Asunto(s)
Fiebre Chikungunya , Virus Chikungunya , Vacunas Virales , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Fiebre Chikungunya/prevención & control , Epítopos , Formaldehído , Ratones , ViremiaRESUMEN
Distinct cytomegaloviruses (CMVs) are widely distributed across their mammalian hosts in a highly host species-restricted pattern. To date, evidence demonstrating this has been limited largely to PCR-based approaches targeting small, conserved genomic regions, and only a few complete genomes of isolated viruses representing distinct CMV species have been sequenced. We have now combined direct isolation of infectious viruses from tissues with complete genome sequencing to provide a view of CMV diversity in a wild animal population. We targeted Natal multimammate mice (Mastomys natalensis), which are common in sub-Saharan Africa, are known to carry a variety of zoonotic pathogens, and are regarded as the primary source of Lassa virus (LASV) spillover into humans. Using transformed epithelial cells prepared from M. natalensis kidneys, we isolated CMVs from the salivary gland tissue of 14 of 37 (36â%) animals from a field study site in Mali. Genome sequencing showed that these primary isolates represent three different M. natalensis CMVs (MnatCMVs: MnatCMV1, MnatCMV2 and MnatCMV3), with some animals carrying multiple MnatCMVs or multiple strains of a single MnatCMV presumably as a result of coinfection or superinfection. Including primary isolates and plaque-purified isolates, we sequenced and annotated the genomes of two MnatCMV1 strains (derived from sequencing 14 viruses), six MnatCMV2 strains (25 viruses) and ten MnatCMV3 strains (21 viruses), totalling 18 MnatCMV strains isolated as 60 infectious viruses. Phylogenetic analysis showed that these MnatCMVs group with other murid viruses in the genus Muromegalovirus (subfamily Betaherpesvirinae, family Orthoherpesviridae), and that MnatCMV1 and MnatCMV2 are more closely related to each other than to MnatCMV3. The availability of MnatCMV isolates and the characterization of their genomes will serve as the prelude to the generation of a MnatCMV-based vaccine to target LASV in the M. natalensis reservoir.
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Infecciones por Citomegalovirus , Citomegalovirus , Animales , Humanos , Ratones , Filogenia , Secuencia de Bases , MurinaeRESUMEN
Chikungunya virus (CHIKV) infection in humans is rarely fatal but is often associated with chronic joint and muscle pain. Chronic CHIKV disease is highly debilitating and is associated with viral persistence. To date, there are no approved vaccines or therapeutics to prevent or treat CHIKV infections once they are established. Current palliative treatments aim to reduce joint inflammation and pain associated with acute and chronic CHIKV disease. Development of novel therapeutics that reduces viral loads should positively impact virus inflammatory disease and improve patient outcomes following CHIKV infection. Therapies that target multiple aspects of CHIKV replication cycle should be developed since the virus is capable of rapidly mutating around any single therapeutic. This review summarizes the current status of small molecule inhibitor development against CHIKV.
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Fiebre Chikungunya , Virus Chikungunya , Virus , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/genética , Humanos , Replicación ViralRESUMEN
In human cytomegalovirus (HCMV)-seropositive patients, CD34+ hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34+ HPCs to investigate mechanisms of viral latency. However, analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34+ HPCs from different donors, availability of cells, and low frequency of reactivation. In addition, multiple progenitor cell types express surface CD34, and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro In this study, we generated CD34+ progenitor cells from two different embryonic stem cell (ESC) lines, WA01 and WA09, to circumvent limitations associated with primary CD34+ HPCs. HCMV infection of CD34+ HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34+ HPCs. Analysis of HCMV-infected primary or ESC-derived CD34+ HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38+ CD90+ and CD38+/low CD90- HPCs but persistently infected CD38- CD90+ cells to produce infectious virus. These results indicate that ESC-derived CD34+ HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34+ cells.IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34+ hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however, these cells are a heterogeneous pool with donor-to-donor variation in functional, genetic, and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states, which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation.
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Antígenos CD34/metabolismo , Infecciones por Citomegalovirus/virología , Citomegalovirus/aislamiento & purificación , Células Madre Hematopoyéticas/virología , Interacciones Huésped-Patógeno , Células Madre Embrionarias Humanas/virología , Activación Viral , Latencia del Virus , Células Cultivadas , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/patología , Células Madre Hematopoyéticas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Transducción de SeñalRESUMEN
Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.
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Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Genoma Viral/genética , Viremia , Animales , Línea Celular , Cromosomas Artificiales Bacterianos , Citomegalovirus/patogenicidad , ADN Recombinante , Modelos Animales de Enfermedad , Femenino , Fibroblastos/virología , Humanos , Macaca mulatta , Masculino , Mutación , Sistemas de Lectura Abierta/genética , Filogenia , Especificidad de la EspecieRESUMEN
Human cytomegalovirus (HCMV) causes substantial disease in transplant patients and harms the development of the nervous system in babies infected in utero. Thus, there is a major focus on developing safe and effective HCMV vaccines. Evidence has been presented that a major target of neutralizing antibodies (NAbs) is the HCMV pentamer glycoprotein gH/gL/UL128-131. In some studies, most of the NAbs in animal or human sera were found to recognize the pentamer, which mediates HCMV entry into endothelial and epithelial cells. It was also reported that pentamer-specific antibodies correlate with protection against transmission from mothers to babies. One problem with the studies on pentamer-specific NAbs to date has been that the studies did not compare the pentamer to the other major form of gH/gL, the gH/gL/gO trimer, which is essential for entry into all cell types. Here, we demonstrate that both trimer and pentamer NAbs are frequently found in human transplant patients' and pregnant mothers' sera. Depletion of human sera with trimer caused reductions in NAbs similar to that observed following depletion with the pentamer. The trimer- and pentamer-specific antibodies acted in a synergistic fashion to neutralize HCMV and also to prevent virus cell-to-cell spread. Importantly, there was no correlation between the titers of trimer- and pentamer-specific NAbs and transmission of HCMV from mothers to babies. Therefore, both the trimer and pentamer are important targets of NAbs. Nevertheless, these antibodies do not protect against transmission of HCMV from mothers to babies.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Infecciones por Citomegalovirus/transmisión , Citomegalovirus/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Citomegalovirus/química , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/prevención & control , Vacunas contra Citomegalovirus/química , Vacunas contra Citomegalovirus/inmunología , Células Epiteliales/inmunología , Femenino , Humanos , Embarazo , Internalización del VirusRESUMEN
Ischemia-reperfusion injury (IRI) is an important risk factor for accelerated cardiac allograft rejection and graft dysfunction . Utilizing a rat heart isogeneic transplant model, we identified inflammatory pathways involved in IRI in order to identify therapeutic targets involved in disease. Pathway analyses identified several relevant targets, including cytokine signaling by the IL-1 receptor (IL-1R) pathway and inflammasome activation. To investigate the role of IL-1R signaling pathways during IRI, we treated syngeneic cardiac transplant recipients at 1-hour posttransplant with Anakinra, a US Food and Drug Administration (FDA)-approved IL-1R antagonist; or parthenolide, a caspase-1 and nuclear factor kappa-light-chain-enhancer of activated B cells inhibitor that blocks IL-1ß maturation. Both Anakinra and parthenolide significantly reduced graft inflammation and cellular recruitment in the treated recipients relative to nontreated controls. Anakinra treatment administered at 1-hour posttransplant to recipients of cardiac allografts from CMV-infected donors significantly increased the time to rejection and reduced viral loads at rejection. Our results indicate that reducing IRI by blocking IL-1Rsignaling pathways with Anakinra or inflammasome activity with parthenolide provides a promising approach for extending survival of cardiac allografts from CMV-infected donors.
Asunto(s)
Infecciones por Citomegalovirus , Trasplante de Corazón , Daño por Reperfusión , Animales , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/etiología , Rechazo de Injerto/prevención & control , Trasplante de Corazón/efectos adversos , Isquemia , Ratas , Receptores de Interleucina-1 , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/etiología , Daño por Reperfusión/prevención & controlRESUMEN
Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.
Asunto(s)
Virus Chikungunya , Virus de la Encefalitis Equina Venezolana , Quinolonas , Animales , Antivirales/farmacología , Virus de la Encefalitis Equina Venezolana/genética , Caballos , Humanos , Quinolonas/farmacología , Replicación ViralRESUMEN
Cytomegalovirus (CMV) infection is linked to acceleration of solid organ transplant vascular sclerosis (TVS) and chronic rejection (CR). Donor latent CMV infection increases cardiac-resident macrophages and T cells leading to increased inflammation, promoting allograft rejection. To investigate the role of cardiac-resident passenger macrophages in CMV-mediated TVS/CR, macrophages were depleted from latently ratCMV (RCMV)-infected donor allografts prior to transplantation. Latently RCMV-infected donor F344 rats were treated with clodronate, PBS, or control liposomes 3 days prior to cardiac transplant into RCMV-naïve Lewis recipients. Clodronate treatment significantly increased graft survival from post-operative day (POD)61 to POD84 and decreased TVS at rejection. To determine the kinetics of the effect of clodronate treatment's effect, a time study revealed that clodronate treatment significantly decreased macrophage infiltration into allograft tissues as early as POD14; altered allograft cytokine/chemokine protein levels, fibrosis development, and inflammatory gene expression profiles. These findings support our hypothesis that increased graft survival as a result of allograft passenger macrophage depletion was in part a result of altered immune response kinetics. Depletion of donor macrophages prior to transplant is a strategy to modulate allograft rejection and reduce TVS in the setting of CMV + donors transplanted into CMV naïve recipients.
Asunto(s)
Infecciones por Citomegalovirus , Trasplante de Corazón , Animales , Citomegalovirus , Rechazo de Injerto , Humanos , Macrófagos , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Donantes de TejidosRESUMEN
BACKGROUND: Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract has raised concerned for potential damaging effects on function. Animal studies have demonstrated that ZIKV virus can infect and damage the testis and epididymis, and these results has been correlated to lower sperm counts in ZIKV-infected humans. The prostate plays a vital role in the male reproductive tract, with acute and chronic prostatitis linked to male infertility. METHODS: In this study, we evaluated the effects of ZIKV virus on the prostate in mice and nonhuman primates. RESULTS: In mice, ZIKV infected the prostate and triggered inflammation that persisted even after virus clearance. Evidence of chronic prostatitis associated with ZIKV infection remained for several months. Similar histological findings were observed in the prostate of ZIKV-infected rhesus macaques. CONCLUSIONS: These studies establish that ZIKV replicates in the prostate and can cause acute and chronic inflammatory and proliferative changes in mouse and nonhuman primate models.
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Prostatitis/virología , Testículo/virología , Infección por el Virus Zika/complicaciones , Enfermedad Aguda , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Epidídimo , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Prostatitis/patología , Semen/virología , Testículo/patología , Virus Zika , Infección por el Virus Zika/transmisiónRESUMEN
Alphaviruses are arthropod-transmitted RNA viruses that can cause arthralgia, myalgia, and encephalitis in humans. Since the role of cellular kinases in alphavirus replication is unknown, we profiled kinetic changes in host kinase abundance and phosphorylation following chikungunya virus (CHIKV) infection of fibroblasts. Based upon the results of this study, we treated CHIKV-infected cells with kinase inhibitors targeting the Src family kinase (SFK)-phosphatidylinositol 3-kinase (PI3K)-AKT-mTORC signaling pathways. Treatment of cells with SFK inhibitors blocked the replication of CHIKV as well as multiple other alphaviruses, including Mayaro virus, O'nyong-nyong virus, Ross River virus, and Venezuelan equine encephalitis virus. Dissecting the effect of SFK inhibition on alphavirus replication, we found that viral structural protein levels were significantly reduced, but synthesis of viral genomic and subgenomic RNAs was unaffected. By measuring the association of viral RNA with polyribosomes, we found that the SFK inhibitor dasatinib blocks alphavirus subgenomic RNA translation. Our results demonstrate a role for SFK signaling in alphavirus subgenomic RNA translation and replication. Targeting host factors involved in alphavirus replication represents an innovative, perhaps paradigm-shifting, strategy for exploring the replication of CHIKV and other alphaviruses while promoting antiviral therapeutic development.
Asunto(s)
Infecciones por Alphavirus/tratamiento farmacológico , Alphavirus/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , Familia-src Quinasas/genética , Alphavirus/genética , Infecciones por Alphavirus/virología , Animales , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Genoma Viral/efectos de los fármacos , Genoma Viral/genética , Humanos , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , ARN Viral/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Vero , Proteínas Virales/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of N-(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection.IMPORTANCE Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.
Asunto(s)
Alphavirus/metabolismo , Antivirales/farmacología , Fibroblastos/metabolismo , Proteínas de la Membrana/agonistas , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Fibroblastos/patología , Fibroblastos/virología , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/genética , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal/genéticaRESUMEN
Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.
Asunto(s)
Infección por el Virus Zika/patología , Infección por el Virus Zika/virología , Animales , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Hibridación in Situ , Macaca mulatta , Masculino , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Viremia/virología , Virus ZikaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006219.].
RESUMEN
Chikungunya virus (CHIKV) is a re-emerging global pathogen with pandemic potential, which causes fever, rash and debilitating arthralgia. Older adults over 65 years are particularly susceptible to severe and chronic CHIKV disease (CHIKVD), accounting for >90% of all CHIKV-related deaths. There are currently no approved vaccines or antiviral treatments available to limit chronic CHIKVD. Here we show that in old mice excessive, dysregulated TGFß production during acute infection leads to a reduced immune response and subsequent chronic disease. Humans suffering from CHIKV infection also exhibited high TGFß levels and a pronounced age-related defect in neutralizing anti-CHIKV antibody production. In vivo reduction of TGFß levels minimized acute joint swelling, restored neutralizing antibody production and diminished chronic joint pathology in old mice. This study identifies increased and dysregulated TGFß secretion as one key mechanism contributing to the age-related loss of protective anti-CHIKV-immunity leading to chronic CHIKVD.
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Envejecimiento/inmunología , Fiebre Chikungunya/inmunología , Factor de Crecimiento Transformador beta/inmunología , Adulto , Anciano , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Virus Chikungunya , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68-1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells in vitro, RhCMV 68-1 failed to productively infect MCM in vivo, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68-1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68-1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68-1 and 68-1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics.
Asunto(s)
Infecciones por Citomegalovirus/transmisión , Citomegalovirus/genética , Modelos Animales de Enfermedad , Macaca fascicularis/virología , Macaca mulatta/virología , Animales , Infecciones por Citomegalovirus/genética , ADN Viral/análisis , ADN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Especificidad de la EspecieRESUMEN
Human Cytomegalovirus (HCMV) encodes multiple microRNAs (miRNAs) whose functions are just beginning to be uncovered. Using in silico approaches, we identified the Toll-Like Receptor (TLR) innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2). We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1ß, IL-6 and IL-8) upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These results provide a novel mechanism through which a HCMV miRNA regulates the innate immune response by down-regulating TLR-2 expression.
Asunto(s)
Citomegalovirus/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , MicroARNs/metabolismo , Interferencia de ARN , ARN Viral/metabolismo , Transducción de Señal , Receptor Toll-Like 2/antagonistas & inhibidores , Regiones no Traducidas 3' , Células Cultivadas , Fibroblastos/citología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Genes Reporteros , Células HEK293 , Humanos , Inmunidad Innata , Quinasas Asociadas a Receptores de Interleucina-1/genética , Ligandos , MicroARNs/genética , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Mutación , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Interferente Pequeño , ARN Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.
Asunto(s)
Antivirales/farmacología , Fiebre Chikungunya/inmunología , Proteínas de la Membrana/agonistas , Transducción de Señal/inmunología , Tiazinas/farmacología , Alphavirus/inmunología , Infecciones por Alphavirus/inmunología , Animales , Células Cultivadas , Virus Chikungunya/inmunología , Ensayos Analíticos de Alto Rendimiento , Humanos , Immunoblotting , Factor 3 Regulador del Interferón/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Chikungunya virus (CHIKV) is a reemerging alphavirus that causes acute febrile illness and severe joint pain in humans. Although acute symptoms often resolve within a few days, chronic joint and muscle pain can be long lasting. In the last decade, CHIKV has caused widespread outbreaks of unprecedented scale in the Americas, Asia, and the Indian Ocean island regions. Despite these outbreaks and the continued expansion of CHIKV into new areas, mechanisms of chikungunya pathogenesis and disease are not well understood. Experimental animal models are indispensable to the field of CHIKV research. The most commonly used experimental animal models of CHIKV infection are mice and nonhuman primates; each model has its advantages for studying different aspects of CHIKV disease. This review will provide an overview of animal models used to study CHIKV infection and disease and major advances in our understanding of chikungunya obtained from studies performed in these models.