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1.
Immunology ; 159(2): 183-192, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31630392

RESUMO

The plant virus, cowpea mosaic virus (CPMV), has demonstrated a remarkable capacity to induce anti-tumour immune responses following direct administration into solid tumours. The molecular pathways that account for these effects and the capacity of CPMV to activate human cells are not well defined. Here, we examine the ability of CPMV particles to activate human monocytes, dendritic cells (DCs) and macrophages. Monocytes in peripheral blood mononuclear cell cultures and purified CD14+ monocytes were readily activated by CPMV in vitro, leading to induction of HLA-DR, CD86, PD-L1, IL-15R and CXCL10 expression. Monocytes released chemokines, CXCL10, MIP-1α and MIP-1ß into cell culture supernatants after incubation with CPMV. DC subsets (pDC and mDC) and monocyte-derived macrophages also demonstrated evidence of activation after incubation with CPMV. Inhibitors of spleen tyrosine kinase (SYK), endocytosis or endocytic acidification impaired the capacity of CPMV to activate monocytes. Furthermore, CPMV activation of monocytes was partially blocked by a TLR7/8 antagonist. These data demonstrate that CPMV activates human monocytes in a manner dependent on SYK signalling, endosomal acidification and with an important contribution from TLR7/8 recognition.


Assuntos
Comovirus/patogenicidade , Endossomos/virologia , Monócitos/virologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Endossomos/imunologia , Endossomos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia
2.
BMC Infect Dis ; 19(1): 986, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752731

RESUMO

BACKGROUND: Zika virus (ZIKV) infection gained public health concern after the 2015 outbreak in Brazil, when microcephaly rates increased in babies born from infected mothers. It was demonstrated that ZIKV causes a congenital Zika virus syndrome, including various alterations in the development of the central nervous system. Although the infection of cells from the nervous system has been well documented, less is known in respect of ZIKV ability to infect immune cells. Herein, we investigated if peripheral blood mononuclear cells (PBMCs), freshly-isolated from healthy donors, could be infected by ZIKV. METHODS: PBMCs from healthy donors were isolated and cultured in medium with ZIKV strain Rio-U1 (MOI = 0.1). Infection was analyzed by RT-qPCR and flow cytometry. RESULTS: We detected the ZIKV RNA in PBMCs from all donors by RT-qPCR analysis. The detection of viral antigens by flow cytometry revealed that PBMC from more than 50% the donors were infected by ZIKV, with CD3+CD4+ T cells, CD3-CD19+ B cells and CD3+CD8+ T cells being, respectively, the most frequently infected subpopulations, followed by CD14+ monocytes. Additionally, we observed high variability in PBMC infection rates among different donors, either by numbers or type infected cells. CONCLUSIONS: These findings raise the hypothesis that PBMCs can act as a reservoir of the virus, which may facilitate viral dissemination to different organs, including immune-privileged sites.


Assuntos
Leucócitos Mononucleares/virologia , Infecção por Zika virus/virologia , Zika virus/isolamento & purificação , Antígenos CD19/genética , Antígenos CD19/imunologia , Linfócitos B/imunologia , Brasil , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Humanos , Leucócitos Mononucleares/imunologia , Monócitos/imunologia , Monócitos/virologia , Reação em Cadeia da Polimerase em Tempo Real , Zika virus/genética , Zika virus/fisiologia , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/genética , Infecção por Zika virus/imunologia
3.
J Immunol Res ; 2019: 8028725, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31612153

RESUMO

Influenza virus infection is a serious threat to humans and animals, with the potential to cause severe pneumonia and death. Annual vaccination strategies are a mainstay to prevent complications related to influenza. However, protection from the emerging subtypes of influenza A viruses (IAV) even in vaccinated individuals is challenging. Innate immune cells are the first cells to respond to IAV infection in the respiratory tract. Virus replication-induced production of cytokines from airway epithelium recruits innate immune cells to the site of infection. These leukocytes, namely, neutrophils, monocytes, macrophages, dendritic cells, eosinophils, natural killer cells, innate lymphoid cells, and γδ T cells, become activated in response to IAV, to contain the virus and protect the airway epithelium while triggering the adaptive arm of the immune system. This review addresses different anti-influenza virus schemes of innate immune cells and how these cells fine-tune the balance between immunoprotection and immunopathology during IAV infection. Detailed understanding on how these innate responders execute anti-influenza activity will help to identify novel therapeutic targets to halt IAV replication and associated immunopathology.


Assuntos
Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Leucócitos/virologia , Citocinas , Células Dendríticas/imunologia , Células Dendríticas/virologia , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Leucócitos/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Monócitos/imunologia , Monócitos/virologia , Replicação Viral/imunologia
4.
PLoS Pathog ; 15(10): e1007778, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31603920

RESUMO

Type I interferons (T1-IFN) are critical in the innate immune response, acting upon infected and uninfected cells to initiate an antiviral state by expressing genes that inhibit multiple stages of the lifecycle of many viruses. T1-IFN triggers the production of Interferon-Stimulated Genes (ISGs), activating an antiviral program that reduces virus replication. The importance of the T1-IFN response is highlighted by the evolution of viral evasion strategies to inhibit the production or action of T1-IFN in virus-infected cells. T1-IFN is produced via activation of pathogen sensors within infected cells, a process that is targeted by virus-encoded immunomodulatory molecules. This is probably best exemplified by the prototypic poxvirus, Vaccinia virus (VACV), which uses at least 6 different mechanisms to completely block the production of T1-IFN within infected cells in vitro. Yet, mice lacking aspects of T1-IFN signaling are often more susceptible to infection with many viruses, including VACV, than wild-type mice. How can these opposing findings be rationalized? The cytosolic DNA sensor cGAS has been implicated in immunity to VACV, but has yet to be linked to the production of T1-IFN in response to VACV infection. Indeed, there are two VACV-encoded proteins that effectively prevent cGAS-mediated activation of T1-IFN. We find that the majority of VACV-infected cells in vivo do not produce T1-IFN, but that a small subset of VACV-infected cells in vivo utilize cGAS to sense VACV and produce T1-IFN to protect infected mice. The protective effect of T1-IFN is not mediated via ISG-mediated control of virus replication. Rather, T1-IFN drives increased expression of CCL4, which recruits inflammatory monocytes that constrain the VACV lesion in a virus replication-independent manner by limiting spread within the tissue. Our findings have broad implications in our understanding of pathogen detection and viral evasion in vivo, and highlight a novel immune strategy to protect infected tissue.


Assuntos
Quimiocina CCL4/metabolismo , Interferon Tipo I/farmacologia , Proteínas de Membrana/fisiologia , Nucleotidiltransferases/fisiologia , Vírus Vaccinia/efeitos dos fármacos , Vaccinia/prevenção & controle , Carga Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Quimiocina CCL4/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/virologia , Vaccinia/imunologia , Vaccinia/metabolismo , Vaccinia/virologia , Vírus Vaccinia/imunologia , Replicação Viral
5.
Nat Commun ; 10(1): 4430, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562326

RESUMO

Zika virus (ZIKV) invades and persists in the central nervous system (CNS), causing severe neurological diseases. However the virus journey, from the bloodstream to tissues through a mature endothelium, remains unclear. Here, we show that ZIKV-infected monocytes represent suitable carriers for viral dissemination to the CNS using human primary monocytes, cerebral organoids derived from embryonic stem cells, organotypic mouse cerebellar slices, a xenotypic human-zebrafish model, and human fetus brain samples. We find that ZIKV-exposed monocytes exhibit higher expression of adhesion molecules, and higher abilities to attach onto the vessel wall and transmigrate across endothelia. This phenotype is associated to enhanced monocyte-mediated ZIKV dissemination to neural cells. Together, our data show that ZIKV manipulates the monocyte adhesive properties and enhances monocyte transmigration and viral dissemination to neural cells. Monocyte transmigration may represent an important mechanism required for viral tissue invasion and persistence that could be specifically targeted for therapeutic intervention.


Assuntos
Moléculas de Adesão Celular/metabolismo , Monócitos/metabolismo , Monócitos/virologia , Neurônios/metabolismo , Migração Transendotelial e Transepitelial/fisiologia , Infecção por Zika virus/metabolismo , Zika virus/fisiologia , Zika virus/patogenicidade , Animais , Adesão Celular/fisiologia , Sobrevivência Celular , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Cerebelo/patologia , Cerebelo/virologia , Modelos Animais de Doenças , Células-Tronco Embrionárias , Endotélio/virologia , Feminino , Humanos , Monócitos/patologia , Neurônios/patologia , Neurônios/virologia , Organoides/metabolismo , Organoides/patologia , Peixe-Zebra , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia
6.
Acta Virol ; 63(3): 253-260, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507190

RESUMO

The human immunodeficiency virus (HIV) envelope, via a key extracellular amino acid sequence, may simulate the functionality of native undecapeptide substance P (SP) acting through the host's neurokinin 1 (SP preferring) receptor (NK-1R). Human monocytes and macrophages express both NK-1Rs and SP. In HIV/AIDS the NK-1R may function as a chemokine-like G-protein coupled co-receptor that: 1) fuses to the outer envelope of HIV; 2) enables intracellular entry of the envelope-capsid-NK-1R complex; 3) co-opts immune defence via its physiological interaction with the SP-like envelope; 4) may contribute to resistance of CD4/chemokine entry inhibitor type drugs; 5) relaxes the blood-brain barrier to support entry of the HIV into the central nervous system, and 6) mediates most of the common clinical sequelae of HIV/AIDS (encephalopathy and AIDS dementia complex). The data support the idea that NK-1R antagonists could be useful to treat HIV/AIDS. Keywords: human immunodeficiency virus; NK-1 receptor; NK-1 receptor antagonist; aprepitant; fusion protein; virus.


Assuntos
Infecções por HIV , Receptores da Neurocinina-1 , Substância P , Proteínas Virais de Fusão , Dipeptídeos/genética , Dipeptídeos/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/fisiopatologia , Humanos , Macrófagos/virologia , Monócitos/metabolismo , Monócitos/virologia , Antagonistas do Receptor de Neuroquinina-1/uso terapêutico , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Proteínas Virais de Fusão/metabolismo
7.
mSphere ; 4(5)2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533998

RESUMO

Dengue is caused by infection with any one of four dengue viruses (DENV); the risk of severe disease appears to be enhanced by the cross-reactive or subneutralizing levels of antibody from a prior DENV infection. These antibodies opsonize DENV entry through the activating Fc gamma receptors (FcγR), instead of infection through canonical receptor-mediated endocytosis, to result in higher levels of DENV replication. However, whether the enhanced replication is solely due to more efficient FcγR-mediated DENV entry or is also through FcγR-mediated alteration of the host transcriptome response to favor DENV infection remains unclear. Indeed, more efficient viral entry through activation of the FcγR can result in an increased viral antigenic load within target cells and confound direct comparisons of the host transcriptome response under antibody-dependent and antibody-independent conditions. Herein, we show that, despite controlling for the viral antigenic load in primary monocytes, the antibody-dependent and non-antibody-dependent routes of DENV entry induce transcriptome responses that are remarkably different. Notably, antibody-dependent DENV entry upregulated DENV host dependency factors associated with RNA splicing, mitochondrial respiratory chain complexes, and vesicle trafficking. Additionally, supporting findings from other studies, antibody-dependent DENV entry impeded the downregulation of ribosomal genes caused by canonical receptor-mediated endocytosis to increase viral translation. Collectively, our findings support the notion that antibody-dependent DENV entry alters host responses that support the viral life cycle and that host responses to DENV need to be defined in the context of its entry pathway.IMPORTANCE Dengue virus is the most prevalent mosquito-borne viral infection globally, resulting in variable manifestations ranging from asymptomatic viremia to life-threatening shock and multiorgan failure. Previous studies have indicated that the risk of severe dengue in humans can be increased by a specific range of preexisting anti-dengue virus antibody titers, a phenomenon termed antibody-dependent enhancement. There is hence a need to understand how antibodies augment dengue virus infection compared to the alternative canonical receptor-mediated viral entry route. Herein, we show that, besides facilitating viral uptake, antibody-mediated entry increases the expression of early host dependency factors to promote viral infection; these factors include RNA splicing, mitochondrial respiratory chain complexes, vesicle trafficking, and ribosomal genes. These findings will enhance our understanding of how differences in entry pathways can affect host responses and offer opportunities to design therapeutics that can specifically inhibit antibody-dependent enhancement of dengue virus infection.


Assuntos
Anticorpos Antivirais/imunologia , Vírus da Dengue/fisiologia , Interações entre Hospedeiro e Microrganismos , Receptores de IgG/imunologia , Internalização do Vírus , Anticorpos Facilitadores , Antígenos Virais/imunologia , Linhagem Celular , Células Cultivadas , Dengue/virologia , Humanos , Monócitos/imunologia , Monócitos/virologia , Análise de Sequência de RNA , Transcriptoma , Replicação Viral
8.
Emerg Microbes Infect ; 8(1): 920-933, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31237479

RESUMO

In early 2017, an outbreak caused by an unknown and supposedly viral agent in the Marilena region of southern Brazil was investigated. Since the etiological agent causing the outbreak was not identified from human samples, mosquitoes from this region were collected. Three out of 121 mosquito pools collected from the region tested positive for alphavirus in molecular tests. Next generation sequencing results revealed the presence of a novel alphavirus, tentatively named here as Caainguá virus (CAAV). DNA barcoding analyses indicated that different species of Culex are hosts for CAAV. This new virus was basal to the New World encephalitic alphaviruses in a comprehensive and robust phylogenetic approach using complete genomes. Viral particles were observed in the cytosol and inside of intracellular compartments of cells in mosquito-derived cell cultures. Despite being noninfectious in vertebrate derived cell cultures, primary culturing of CAAV in human mononuclear cells suggests monocytes and lymphocytes as CAAV targets. However, the epidemiological link of CAAV on the human outbreak should be further explored.


Assuntos
Alphavirus/isolamento & purificação , Encefalite/virologia , Adulto , Alphavirus/classificação , Alphavirus/genética , Alphavirus/fisiologia , Animais , Brasil/epidemiologia , Culicidae/fisiologia , Culicidae/virologia , Encefalite/epidemiologia , Feminino , Humanos , Linfócitos/virologia , Masculino , Monócitos/virologia , Mosquitos Vetores/fisiologia , Mosquitos Vetores/virologia , Filogenia , Adulto Jovem
9.
Viruses ; 11(2)2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30781810

RESUMO

Antigen-specific T lymphocytes play a critical role in controlling viral infections. However, we report here that preexisting virus-specific T cell responses also contribute to promoting adenovirus (Ad) infection. Previously, we found that CD14+ monocytes from Ad-seropositive individuals exhibited an increased susceptibility to Ad infection, when compared with that of Ad-seronegative individuals. But the underlying mechanisms for this enhancement of viral infection are not completely clarified. In this study, we found that the efficacy of Ad infection into CD14+ monocytes was significantly decreased after CD3+ T lymphocytes depletion from PBMC samples of Ad-seropositive individuals. In contrast, adding virus-specific CD3+ T lymphocytes into PBMC samples of Ad-seronegative individuals resulted in a significant increase of infection efficacy. CD3+ T lymphocytes in PBMC samples from Ad-seropositive individuals were more sensitive to be activated by adenovirus stimulus, characterized by upregulation of multiple cytokines and activation markers and also enhancement of cell proliferation. Further studies demonstrated that GM-CSF and IL-4 can promote Ad infection by up-regulating the expression of scavenger receptor 1 (SR-A) and integrins αVß5 receptor of CD14+ cells. And taken together, these results suggest a novel role of virus-specific T cells in mediating enhancement of viral infection, and provide insights to understand the pathogenesis and complicated interactions between viruses and host immune cells.


Assuntos
Infecções por Adenoviridae/imunologia , Monócitos/imunologia , Monócitos/virologia , Linfócitos T/imunologia , Infecções por Adenoviridae/patologia , Adenovírus Humanos , Células Cultivadas , Citocinas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/farmacologia , Receptores de Lipopolissacarídeos/metabolismo
10.
Viruses ; 11(2)2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30781856

RESUMO

One of several mechanisms that leads to the development of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) is called antibody-dependent enhancement (ADE). Monocytes can be infected by the ADE phenomenon, which occurs in dengue secondary infection. This study aimed to investigate the proteins involved in ADE of DENV infection in the human monocytic cell line U937. The phosphoproteins were used to perform and analyze for protein expression using mass spectrometry (GeLC-MS/MS). The differential phosphoproteins revealed 1131 altered proteins compared between isotype- and DENV-specific antibody-treated monocytes. The altered proteins revealed 558 upregulated proteins and 573 downregulated proteins. Protein disulfide isomerase (PDI), which is an enzyme that had a high-ranking fold change and that catalyzes the formation, breakage, and rearrangement of disulfide bonds within a protein molecule, was selected for further study. PDI was found to be important for dengue virus infectivity during the ADE model. The effect of PDI inhibition was also shown to be involved in the early stage of life cycle by time-of-drug-addition assay. These results suggest that PDI is important for protein translation and virion assembly of dengue virus during infection in human monocytes, and it may play a significant role as a chaperone to stabilize dengue protein synthesis.


Assuntos
Anticorpos Facilitadores , Vírus da Dengue/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Monócitos/imunologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Anticorpos Antivirais , Linhagem Celular , Vírus da Dengue/fisiologia , Regulação para Baixo , Humanos , Monócitos/virologia , Fosfoproteínas/imunologia , Transdução de Sinais , Espectrometria de Massas em Tandem , Células U937 , Regulação para Cima
11.
J Infect Dis ; 220(1): 32-40, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-30785182

RESUMO

Zika virus (ZIKV) is a clinically important flavivirus that can cause neurological disturbances in newborns. Here, we investigated comparatively the outcome of in vitro infection of newborn monocytes by ZIKV. We observed that neonatal cells show defective production of interleukin 1ß, interleukin 10, and monocyte chemoattractant protein 1 in response to ZIKV, although they were as efficient as adult cells in supporting viral infection. Although CLEC5A is a classical flavivirus immune receptor, it is not essential to the cytokine response, but it regulates the viral load only in adult cells. Greater expression of viral entry receptors may create a favorable environment for viral invasion in neonatal monocytes. We are the first to suggest a role for CLEC5A in human monocyte infectivity and to show that newborn monocytes are interesting targets in ZIKV pathogenesis, owing to their ability to carry the virus with only a partial triggering of the immune response, creating a potentially favorable environment for virus-related pathologies in young individuals.


Assuntos
Citocinas/imunologia , Monócitos/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Células Cultivadas , Humanos , Recém-Nascido , Lectinas Tipo C/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Monócitos/virologia , Receptores de Superfície Celular/imunologia , Carga Viral/imunologia , Infecção por Zika virus/virologia
12.
J Gen Virol ; 100(4): 656-661, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30767852

RESUMO

The release of porcine endogenous retrovirus (PERV) particles from pig cells is a potential risk factor during xenotransplantation by way of productively infecting the human transplant recipient. Potential countermeasures against PERV replication are restriction factors that block retroviral replication. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of dNTPs in non-cycling cells starving retroviral reverse transcription. We investigated the antiviral activity of human SAMHD1 against PERV and found that SAMHD1 potently restricts its reverse transcription in human monocytes, monocyte-derived dendritic cells (MDDC), or macrophages (MDM) and in monocytic THP-1 cells. Degradation of SAMHD1 by SIVmac Vpx or CRISPR/Cas9 knock-out of SAMHD1 allowed for PERV reverse transcription. Addition of deoxynucleosides alleviated the SAMHD1-mediated restriction suggesting that SAMHD1-mediated degradation of dNTPs restricts PERV replication in these human immune cells. In conclusion, our findings highlight SAMHD1 as a potential barrier to PERV transmission from pig transplants to human recipients during xenotransplantation.


Assuntos
Retrovirus Endógenos/fisiologia , Xenoenxertos/metabolismo , Xenoenxertos/virologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Animais , Sistemas CRISPR-Cas/fisiologia , Linhagem Celular , Células HEK293 , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Monócitos/metabolismo , Monócitos/virologia , Transcrição Reversa/fisiologia , Suínos , Células THP-1 , Transplante Heterólogo/métodos , Replicação Viral/fisiologia
13.
Arch Virol ; 164(3): 739-745, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30631959

RESUMO

African swine fever (ASF) is a contagious viral disease of wild and domestic pigs that is present in many parts of Africa, Asia and Europe, including Sardinia (Italy). Deletions in the EP402R and B602L genes have been found in almost all ASF virus (ASFV) strains circulating in Sardinia from 1990 onwards, and modern Sardinian strains (isolated after 1990) might have acquired some selective advantage compared to historical ones (isolated before 1990). Here, we analysed the host cell responses of wild boars and domestic pigs upon infection with virus variants. Higher intracellular levels of the late protein p72 were detected after infection with the modern strain 22653/14 compared to the historical strain Nu81.2, although both isolates grew at the same rate in both monocytes and monocyte-derived macrophages. Higher cytokine levels in the supernatants of ASFV-infected pig monocytes compared to pig macrophages and wild-boar cells were detected, with no differences between isolates.


Assuntos
Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/virologia , Macrófagos/virologia , Monócitos/virologia , Febre Suína Africana/metabolismo , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/crescimento & desenvolvimento , Animais , Células Cultivadas , Citocinas/metabolismo , Itália , Macrófagos/metabolismo , Monócitos/metabolismo , Sus scrofa , Suínos , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
J Virol ; 93(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651370

RESUMO

Equine herpesvirus 1 (EHV1) replicates in the respiratory epithelium and disseminates through the body via a cell-associated viremia in leukocytes, despite the presence of neutralizing antibodies. "Hijacked" leukocytes, previously identified as monocytic cells and T lymphocytes, transmit EHV1 to endothelial cells of the endometrium or central nervous system, causing reproductive (abortigenic variants) or neurological (neurological variants) disorders. In the present study, we questioned the potential route of EHV1 infection of T lymphocytes and how EHV1 misuses T lymphocytes as a vehicle to reach the endothelium of the target organs in the absence or presence of immune surveillance. Viral replication was evaluated in activated and quiescent primary T lymphocytes, and the results demonstrated increased infection of activated versus quiescent, CD4+ versus CD8+, and blood- versus lymph node-derived T cells. Moreover, primarily infected respiratory epithelial cells and circulating monocytic cells efficiently transferred virions to T lymphocytes in the presence of neutralizing antibodies. Albeit T-lymphocytes express all classes of viral proteins early in infection, the expression of viral glycoproteins on their cell surface was restricted. In addition, the release of viral progeny was hampered, resulting in the accumulation of viral nucleocapsids in the T cell nucleus. During contact of infected T lymphocytes with endothelial cells, a late viral protein(s) orchestrates T cell polarization and synapse formation, followed by anterograde dynein-mediated transport and transfer of viral progeny to the engaged cell. This represents a sophisticated but efficient immune evasion strategy to allow transfer of progeny virus from T lymphocytes to adjacent target cells. These results demonstrate that T lymphocytes are susceptible to EHV1 infection and that cell-cell contact transmits infectious virus to and from T lymphocytes.IMPORTANCE Equine herpesvirus 1 (EHV1) is an ancestral alphaherpesvirus that is related to herpes simplex virus 1 and causes respiratory, reproductive, and neurological disorders in Equidae. EHV1 is indisputably a master at exploiting leukocytes to reach its target organs, accordingly evading the host immunity. However, the role of T lymphocytes in cell-associated viremia remains poorly understood. Here we show that activated T lymphocytes efficiently become infected and support viral replication despite the presence of protective immunity. We demonstrate a restricted expression of viral proteins on the surfaces of infected T cells, which prevents immune recognition. In addition, we indicate a hampered release of progeny, which results in the accumulation of nucleocapsids in the T cell nucleus. Upon engagement with the target endothelium, late viral proteins orchestrate viral synapse formation and viral transfer to the contact cell. Our findings have significant implications for the understanding of EHV1 pathogenesis, which is essential for developing innovative therapies to prevent the devastating clinical symptoms of infection.


Assuntos
Infecções por Herpesviridae/imunologia , Herpesvirus Equídeo 1/imunologia , Doenças dos Cavalos/imunologia , Cavalos/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Células Endoteliais/imunologia , Células Endoteliais/virologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Infecções por Herpesviridae/virologia , Doenças dos Cavalos/virologia , Cavalos/virologia , Evasão da Resposta Imune/imunologia , Monócitos/imunologia , Monócitos/virologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Linfócitos T/virologia , Proteínas Virais/imunologia , Viremia/imunologia , Viremia/virologia , Replicação Viral/imunologia
15.
Genes Immun ; 20(3): 214-223, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29728610

RESUMO

Recently, deficiency in the cytosolic DNA sensor RNA Polymerase III was described in children with severe primary varicella-zoster virus (VZV) infection in the CNS and lungs. In the present study we examined adult patients with VZV CNS infection caused by viral reactivation. By whole exome sequencing we identified mutations in POL III genes in two of eight patients. These mutations were located in the coding regions of the subunits POLR3A and POLR3E. In functional assays, we found impaired expression of antiviral and inflammatory cytokines in response to the POL III agonist Poly(dA:dT) as well as increased viral replication in patient cells compared to controls. Altogether, this study provides significant extension on the current knowledge on susceptibility to VZV infection by demonstrating mutations in POL III genes associated with impaired immunological sensing of AT-rich DNA in adult patients with VZV CNS infection.


Assuntos
RNA Polimerase III/genética , Infecção pelo Vírus da Varicela-Zoster/genética , Adulto , Idoso , Células Cultivadas , Citocinas/metabolismo , Suscetibilidade a Doenças , Feminino , Humanos , Masculino , Monócitos/imunologia , Monócitos/virologia , Mutação , RNA Polimerase III/metabolismo , Infecção pelo Vírus da Varicela-Zoster/imunologia , Infecção pelo Vírus da Varicela-Zoster/virologia , Replicação Viral
16.
Arch Immunol Ther Exp (Warsz) ; 67(1): 27-40, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30238127

RESUMO

Diseases caused by dengue virus (DENV) are a major public health problem worldwide, considered one of the infections with more prevalence in tropical and subtropical zones of the world. Despite the intense research in the pathogenesis of DENV, this feature is not well understood. One of the main target cells for DENV infection is monocytes; these phagocytes can play a dual role, since they are essential to control viremia, but they also participate in the induction of tissue damage during DENV infection. Monocytes produce different pro-inflammatory cytokines and chemokines in response to infection, and also mediate endothelial damage. In peripheral blood, monocytes can be divided into three different subpopulations, namely classical, intermediate and non-classical, which differ in frequency, cytokine production, among others. Studies in the last years suggest that non-classical monocytes have higher affinity for microvasculature endothelium compared to other type of monocytes, which implies that they could be more involved in the increase of endothelial permeability observed during DENV infection. This review provides a general view of the role of monocytes and their subpopulations in DENV pathogenesis and its effect in viral replication. Finally, the potential contribution of these phagocytes in the alterations of endothelial permeability is discussed.


Assuntos
Vírus da Dengue/patogenicidade , Dengue/virologia , Monócitos/virologia , Animais , Permeabilidade Capilar , Citocinas/imunologia , Citocinas/metabolismo , Dengue/imunologia , Dengue/metabolismo , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/imunologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Microvasos/imunologia , Microvasos/metabolismo , Microvasos/virologia , Monócitos/imunologia , Monócitos/metabolismo , Fagocitose , Transdução de Sinais , Replicação Viral
17.
EBioMedicine ; 39: 332-347, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30527622

RESUMO

BACKGROUND: Activated platelets release cytokines/proteins including CXCL4 (PF4), CCL5 and fibrinopeptides, which regulate infection of several pathogenic viruses such as HIV, H1N1 and HCV in human. Since platelet activation is the hallmark of Dengue virus (DV) infection, we investigated the role of platelets in DV replication and also in a closely related Japanese Encephalitis virus (JEV). METHODS AND FINDINGS: Microscopy and PCR analysis revealed a 4-fold increase in DV replication in primary monocytes or monocytic THP-1 cells in vitro upon incubation with either DV-activated platelets or supernatant from DV-activated platelets. The mass spectrometry based proteomic data from extra-nuclear fraction of above THP-1 lysate showed the crucial association of PF4 with enhanced DV replication. Our cytokine analysis and immunoblot assay showed significant inhibition of IFN-α production in monocytes via p38MAPK-STAT2-IRF9 axis. Blocking PF4 through antibodies or its receptor CXCR3 through inhibitor i.e. AMG487, significantly rescued production of IFN-α resulting in potent inhibition of DV replication in monocytes. Further, flow cytometry and ELISA data showed the direct correlation between elevated plasma PF4 with increased viral NS1 in circulating monocytes in febrile DV patients at day-3 of fever than day-9. Similarly, PF4 also showed direct effects in promoting the JEV replication in monocytes and microglia cells in vitro. The in vitro results were also validated in mice, where AMG487 treatment significantly improved the survival of JEV infected animals. INTERPRETATION: Our study suggests that PF4-CXCR3-IFN axis is a potential target for developing treatment regimen against viral infections including JEV and DV.


Assuntos
Vírus da Dengue/fisiologia , Dengue/metabolismo , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Encefalite Japonesa/metabolismo , Fator Plaquetário 4/metabolismo , Acetamidas/farmacologia , Animais , Linhagem Celular , Dengue/sangue , Dengue/virologia , Modelos Animais de Doenças , Encefalite Japonesa/sangue , Encefalite Japonesa/virologia , Humanos , Interferons/metabolismo , Camundongos , Monócitos/virologia , Pirimidinonas/farmacologia , Células THP-1 , Células Vero , Replicação Viral
18.
Dig Dis Sci ; 64(5): 1226-1237, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30535782

RESUMO

BACKGROUND: Hepatitis C virus (HCV) has been classified as a strictly hepatotropic pathogen for a long time, and hepatocytes are target cells for HCV infection. More and more studies showed non-liver cells supported HCV entry and replication, such as macrophages. The mechanisms of HCV entry into macrophages are still not clear. AIMS: This study aims to determine the way of HCV entry into macrophages. METHODS: Cell culture-derived infectious HCV particles (HCVcc) were prepared using Huh7 cells transfected with HCV RNA. CD81-knockdown cells were obtained through siRNA transfection. HCV RNA levels were determined by RT-qPCR. Flow cytometry analyses were used to determine cell surface levels of CD11b, CD68, and CD81. ELISA and western blotting were performed to quantify the protein levels of IL-1ß, IL-6, and TNF-α. Phagocytic ability was determined by neutral red uptake assay. RESULTS: CD81 knockdown could not inhibit HCVcc entry into macrophages. The entry of HCV into macrophages could not be blocked by pooled IgG from chronic hepatitis C patient's sera. Macrophages derived from THP-1 cells displayed stronger phagocytic capacity, which also swallowed more HCV RNA. Treatment of macrophages with endocytic inhibitor, methyl-ß-cyclodextrin, decreased the internalization of HCV. HCV uptake by macrophages was related to the reorganization of F-actin cytoskeleton and PI3Ks activation. HCV infection significantly increased the expression of IL1ß and IL6 in macrophages and promoted apoptosis of macrophages. CONCLUSIONS: HCV entry into macrophages mainly depends on phagocytosis of macrophages.


Assuntos
Hepacivirus/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Monócitos/metabolismo , Monócitos/virologia , Fagocitose/fisiologia , Animais , Células Cultivadas , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/efeitos dos fármacos , Morfolinas/farmacologia , Fagocitose/efeitos dos fármacos , Células RAW 264.7 , Coelhos
19.
J Virol ; 93(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30404793

RESUMO

Varicella-zoster virus (VZV) is associated with viremia during primary infection that is presumed to stem from infection of circulating immune cells. While VZV has been shown to be capable of infecting a number of different subsets of circulating immune cells, such as T cells, dendritic cells, and NK cells, less is known about the interaction between VZV and monocytes. Here, we demonstrate that blood-derived human monocytes are permissive to VZV replication in vitro VZV-infected monocytes exhibited each temporal class of VZV gene expression, as evidenced by immunofluorescent staining. VZV virions were observed on the cell surface and viral nucleocapsids were observed in the nucleus of VZV-infected monocytes by scanning electron microscopy. In addition, VZV-infected monocytes were able to transfer infectious virus to human fibroblasts. Infected monocytes displayed impaired dextran-mediated endocytosis, and cell surface immunophenotyping revealed the downregulation of CD14, HLA-DR, CD11b, and the macrophage colony-stimulating factor (M-CSF) receptor. Analysis of the impact of VZV infection on M-CSF-stimulated monocyte-to-macrophage differentiation demonstrated the loss of cell viability, indicating that VZV-infected monocytes were unable to differentiate into viable macrophages. In contrast, macrophages differentiated from monocytes prior to exposure to VZV were highly permissive to infection. This study defines the permissiveness of these myeloid cell types to productive VZV infection and identifies the functional impairment of VZV-infected monocytes.IMPORTANCE Primary VZV infection results in the widespread dissemination of the virus throughout the host. Viral transportation is known to be directly influenced by susceptible immune cells in the circulation. Moreover, infection of immune cells by VZV results in attenuation of the antiviral mechanisms used to control infection and limit spread. Here, we provide evidence that human monocytes, which are highly abundant in the circulation, are permissive to productive VZV infection. Furthermore, monocyte-derived macrophages were also highly permissive to VZV infection, although VZV-infected monocytes were unable to differentiate into macrophages. Exploring the relationships between VZV and permissive immune cells, such as human monocytes and macrophages, elucidates novel immune evasion strategies and provides further insight into the control that VZV has over the immune system.


Assuntos
Diferenciação Celular , Fibroblastos/citologia , Macrófagos/citologia , Monócitos/citologia , Infecção pelo Vírus da Varicela-Zoster/patologia , Vírion , Replicação Viral , Antígenos Virais/metabolismo , Sobrevivência Celular , Células Cultivadas , Endocitose , Fibroblastos/metabolismo , Fibroblastos/virologia , Herpesvirus Humano 3/isolamento & purificação , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Monócitos/metabolismo , Monócitos/virologia , Infecção pelo Vírus da Varicela-Zoster/metabolismo , Infecção pelo Vírus da Varicela-Zoster/virologia
20.
Vet Microbiol ; 228: 134-142, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30593359

RESUMO

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that play an important role in inducing primary antigen-specific immune responses. Some viruses have evolved to specifically target DCs to circumvent the host immune responses for their persistence in the host. One example is porcine reproductive and respiratory syndrome virus (PRRSV) that causes a persistent infection in pigs through modulating DC-mediated antiviral response. To study the cellular protein responses in PRRSV-infected monocyte-derived dendritic cells (MoDCs), two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins in PRRSV-infected MoDCs and the control cells. A total of 252 cellular proteins in MoDCs that were significantly altered at different time periods post-infection were identified. Differentially expressed proteins that are involved in the endocytosis pathway, actin cytoskeleton network, antigen processing and presentation, JAK-STAT signaling pathway and PRRSV receptors were identified and further analyzed. Among them, the expression changes of STAT1, Mx1, PICALM and SLA-DR were further verified by Western blotting. The protein profiles associated with PRRSV infection of MoDCs should offer novel insights to further investigation of PRRSV-mediated antiviral evasion mechanism and its pathobiology in swine.


Assuntos
Biologia Computacional , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteoma , Animais , Cromatografia Líquida/veterinária , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Monócitos/metabolismo , Monócitos/virologia , Proteômica , Suínos , Espectrometria de Massas em Tandem/veterinária , Replicação Viral
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