RESUMO
Cytotoxic CD4 T lymphocytes (CD4-CTL) are important in antiviral immunity. For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection. How viral infections induce CD4-CTL responses remains incompletely understood. We demonstrate here that not only ECTV but also vaccinia virus and lymphocytic choriomeningitis virus induce CD4-CTL, though the response to ECTV is stronger. Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled. We also show that major histocompatibility complex class II molecules on CD11c+ cells are required for CD4-CTL differentiation and for mousepox resistance. Transcriptional analysis indicated that antiviral CD4-CTL and noncytolytic T helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells. Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment, and Runx3, required for CD8 T cell development and effector function. However, at the protein level, CD4-CTL had higher levels of the three transcription factors, suggesting that further posttranscriptional regulation is required for CD4-CTL differentiation. Finally, CRISPR/Cas9-mediated deletion of Runx3 in CD4 T cells inhibited CD4-CTL but not classical Th1 cell differentiation in response to ECTV infection. These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of posttranscriptionally regulated Runx3 in this process. IMPORTANCE While it is well established that cytotoxic CD4 T cells (CD4-CTLs) directly contribute to viral clearance, it remains unclear how CD4-CTL are induced. We now show that CD4-CTLs require sustained antigen presentation and are induced by CD11c-expressing antigen-presenting cells. Moreover, we show that CD4-CTLs are derived from the terminal differentiation of classical T helper 1 (Th1) subset of CD4 cells. Compared to Th1 cells, CD4-CTLs upregulate protein levels of the transcription factors ThPOK, Runx3, and GATA-3 posttranscriptionally. Deletion of Runx3 in differentiated CD4 T cells prevents induction of CD4-CTLs but not classical Th1 cells. These results advance our knowledge of how CD4-CTLs are induced during viral infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Ectromelia Infecciosa/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Viroses/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD11/análise , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Citotoxicidade Imunológica , Vírus da Ectromelia/fisiologia , Ectromelia Infecciosa/virologia , Antígenos de Histocompatibilidade Classe II/análise , Fígado/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/metabolismo , Células Th1/metabolismo , Transcriptoma , Replicação ViralRESUMO
Ectromelia virus (ECTV), the causative agent of mousepox, has threatened laboratory mouse colonies worldwide for almost a century. Mousepox has been valuable for the understanding of poxvirus pathogenesis and immune evasion. Here, we have monitored in parallel the pathogenesis of nine ECTVs in BALB/cJ mice and report the full-length genome sequence of eight novel ECTV isolates or strains, including the first ECTV isolated from a field mouse, ECTV-MouKre. This approach allowed us to identify several genes, absent in strains attenuated through serial passages in culture, that may play a role in virulence and a set of putative genes that may be involved in enhancing viral growth in vitro. We identified a putative strong inhibitor of the host inflammatory response in ECTV-MouKre, an isolate that did not cause local foot swelling and developed a moderate virulence. Most of the ECTVs, except ECTV-Hampstead, encode a truncated version of the P4c protein that impairs the recruitment of virions into the A-type inclusion bodies, and our data suggest that P4c may play a role in viral dissemination and transmission. This is the first comprehensive report that sheds light into the phylogenetic and geographic relationship of the worldwide outbreak dynamics for the ECTV species.
Assuntos
Vírus da Ectromelia/genética , Vírus da Ectromelia/patogenicidade , Ectromelia Infecciosa/patologia , Ectromelia Infecciosa/virologia , Genômica , Filogenia , Animais , Modelos Animais de Doenças , Vírus da Ectromelia/classificação , Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Feminino , Evasão da Resposta Imune , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Filogeografia , Proteínas Virais/genética , VirulênciaRESUMO
Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.
Assuntos
Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais , Animais , Citocinas/imunologia , Resistência à Doença , Ectromelia Infecciosa/virologia , Feminino , Hepatócitos/imunologia , Hepatócitos/virologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/virologia , Receptor de Interferon alfa e beta/genéticaRESUMO
Smallpox and monkeypox pose severe threats to human health. Other orthopoxviruses are comparably virulent in their natural hosts, including ectromelia, the cause of mousepox. Disease severity is linked to an array of immunomodulatory proteins including the B22 family, which has homologs in all pathogenic orthopoxviruses but not attenuated vaccine strains. We demonstrate that the ectromelia B22 member, C15, is necessary and sufficient for selective inhibition of CD4+ but not CD8+ T cell activation by immunogenic peptide and superantigen. Inhibition is achieved not by down-regulation of surface MHC- II or co-stimulatory protein surface expression but rather by interference with antigen presentation. The appreciable outcome is interference with CD4+ T cell synapse formation as determined by imaging studies and lipid raft disruption. Consequently, CD4+ T cell activating stimulus shifts to uninfected antigen-presenting cells that have received antigen from infected cells. This work provides insight into the immunomodulatory strategies of orthopoxviruses by elucidating a mechanism for specific targeting of CD4+ T cell activation, reflecting the importance of this cell type in control of the virus.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Proteínas Virais/imunologia , Animais , Ectromelia Infecciosa/metabolismo , Ectromelia Infecciosa/virologia , Feminino , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Virais/metabolismo , VirulênciaRESUMO
NK cells play an important role in antiviral resistance. The integrin α2, which dimerizes with integrin ß1, distinguishes NK cells from innate lymphoid cells 1 and other leukocytes. Despite its use as an NK cell marker, little is known about the role of α2ß1 in NK cell biology. In this study, we show that in mice α2ß1 deficiency does not alter the balance of NK cell/ innate lymphoid cell 1 generation and slightly decreases the number of NK cells in the bone marrow and spleen without affecting NK cell maturation. NK cells deficient in α2ß1 had no impairment at entering or distributing within the draining lymph node of ectromelia virus (ECTV)-infected mice or at becoming effectors but proliferated poorly in response to ECTV and did not increase in numbers following infection with mouse CMV (MCMV). Still, α2ß1-deficient NK cells efficiently protected from lethal mousepox and controlled MCMV titers in the spleen. Thus, α2ß1 is required for optimal NK cell proliferation but is dispensable for protection against ECTV and MCMV, two well-established models of viral infection in which NK cells are known to be important.
Assuntos
Ectromelia Infecciosa/imunologia , Infecções por Herpesviridae/imunologia , Integrina alfa2beta1/metabolismo , Células Matadoras Naturais/imunologia , Animais , Contagem de Células , Proliferação de Células , Modelos Animais de Doenças , Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/sangue , Ectromelia Infecciosa/virologia , Feminino , Infecções por Herpesviridae/sangue , Infecções por Herpesviridae/virologia , Humanos , Imunidade Inata , Integrina alfa2beta1/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Muromegalovirus/imunologia , Replicação Viral/imunologiaRESUMO
Cells sensing infection produce Type I interferons (IFN-I) to stimulate Interferon Stimulated Genes (ISGs) that confer resistance to viruses. During lympho-hematogenous spread of the mouse pathogen ectromelia virus (ECTV), the adaptor STING and the transcription factor IRF7 are required for IFN-I and ISG induction and resistance to ECTV. However, it is unknown which cells sense ECTV and which pathogen recognition receptor (PRR) upstream of STING is required for IFN-I and ISG induction. We found that cyclic-GMP-AMP (cGAMP) synthase (cGAS), a DNA-sensing PRR, is required in bone marrow-derived (BMD) but not in other cells for IFN-I and ISG induction and for resistance to lethal mousepox. Also, local administration of cGAMP, the product of cGAS that activates STING, rescues cGAS but not IRF7 or IFN-I receptor deficient mice from mousepox. Thus, sensing of infection by BMD cells via cGAS and IRF7 is critical for resistance to a lethal viral disease in a natural host.
Assuntos
Medula Óssea/virologia , Vírus da Ectromelia/patogenicidade , Ectromelia Infecciosa/virologia , Nucleotídeos Cíclicos/metabolismo , Animais , Medula Óssea/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/metabolismo , Camundongos Transgênicos , Nucleotidiltransferases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4+ T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4+ T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4+ T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.
Assuntos
Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Linfócitos T CD4-Positivos/imunologia , Vírus da Ectromelia , Ectromelia Infecciosa/imunologia , Animais , Citocinas/imunologia , Ectromelia Infecciosa/virologia , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Especificidade da EspécieRESUMO
Etanercept is a soluble form of the tumor necrosis factor receptor 2 (TNFR2) that inhibits pathological tumor necrosis factor (TNF) responses in rheumatoid arthritis and other inflammatory diseases. However, besides TNF, etanercept also blocks lymphotoxin-α (LTα), which has no clear therapeutic value and might aggravate some of the adverse effects associated with etanercept. Poxviruses encode soluble TNFR2 homologs, termed viral TNF decoy receptors (vTNFRs), that display unique specificity properties. For instance, cytokine response modifier D (CrmD) inhibits mouse and human TNF and mouse LTα, but it is inactive against human LTα. Here, we analyzed the molecular basis of these immunomodulatory activities in the ectromelia virus-encoded CrmD. We found that the overall molecular mechanism to bind TNF and LTα from mouse and human origin is fairly conserved in CrmD and dominated by a groove under its 50s loop. However, other ligand-specific binding determinants optimize CrmD for the inhibition of mouse ligands, especially mouse TNF. Moreover, we show that the inability of CrmD to inhibit human LTα is caused by a Glu-Phe-Glu motif in its 90s loop. Importantly, transfer of this motif to etanercept diminished its anti-LTα activity in >60-fold while weakening its TNF-inhibitory capacity in 3-fold. This new etanercept variant could potentially be used in the clinic as a safer alternative to conventional etanercept. This work is the most detailed study of the vTNFR-ligand interactions to date and illustrates that a better knowledge of vTNFRs can provide valuable information to improve current anti-TNF therapies.
Assuntos
Vírus da Ectromelia/imunologia , Fatores Imunológicos/imunologia , Linfotoxina-alfa/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Receptores Chamariz do Fator de Necrose Tumoral/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Vírus da Ectromelia/química , Ectromelia Infecciosa/virologia , Humanos , Fatores Imunológicos/química , Camundongos , Modelos Moleculares , Domínios Proteicos , Fator de Necrose Tumoral alfa/imunologia , Proteínas Virais/químicaRESUMO
Ectromelia virus (ECTV) is an orthopoxvirus that productively replicates in dendritic cells (DCs), but its influence on the microtubule (MT) cytoskeleton in DCs is not known. Here, we show that ECTV infection of primary murine granulocyte-macrophage colony stimulating factor-derived bone marrow cells (GM-BM) downregulates numerous genes engaged in MT cytoskeleton organization and dynamics. In infected cells, the MT cytoskeleton undergoes dramatic rearrangement and relaxation, accompanied by disappearance of the microtubule organizing centre (MTOC) and increased acetylation and stabilization of MTs, which are exploited by progeny virions for intracellular transport. This indicates a strong ability of ECTV to subvert the MT cytoskeleton of highly specialized immune cells.
Assuntos
Citoesqueleto/metabolismo , Células Dendríticas/metabolismo , Vírus da Ectromelia/fisiologia , Ectromelia Infecciosa/metabolismo , Macrófagos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Linhagem Celular , Ectromelia Infecciosa/virologia , Interações Hospedeiro-Patógeno , Camundongos , Camundongos Endogâmicos BALB C , Microtúbulos/metabolismoRESUMO
Activation of the DNA-dependent innate immune pathway plays a pivotal role in the host defense against poxvirus. Cyclic GMP-AMP synthase (cGAS) is a key cytosolic DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which triggers stimulator of interferon genes (STING), leading to type I Interferons (IFNs) production and an antiviral response. Ectromelia virus (ECTV) has emerged as a valuable model for investigating the host-Orthopoxvirus relationship. However, the role of cGas-Sting pathway in response to ECTV is not clearly understood. Here, we showed that murine cells (L929 and RAW264.7) mount type I IFN responses to ECTV that are dependent upon cGas, Sting, TANK binding kinase 1 (Tbk1), and interferon regulatory factor 3 (Irf3) signaling. Disruption of cGas or Sting expression in mouse macrophages blocked the type I IFN production and facilitated ECTV replication. Consistently, mice deficient in cGas or Sting exhibited lower type I IFN levels and higher viral loads, and are more susceptible to mousepox. Collectively, our study indicates that the cGas-Sting pathway is critical for sensing of ECTV infection, inducing the type I IFN production, and controlling ECTV replication.
Assuntos
Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Ectromelia Infecciosa/metabolismo , Imunidade Inata , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Animais , Chlorocebus aethiops , Ectromelia Infecciosa/virologia , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/biossíntese , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células NIH 3T3 , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Células RAW 264.7 , Células Vero , Replicação ViralRESUMO
The ectromelia virus (ECTV) is a mouse specific Orthopoxvirus that causes lethal infection in some mouse strains. ECTV infection of these mouse strains has been used as a valuable model for understanding the interplay between Orthopoxvirus species and their hosts, including variola virus in humans. Although poxviruses encode numerous proteins required for DNA and RNA synthesis, and are less dependent on host functions than other DNA viruses, a detailed understanding of the host factors required for the replication of poxviruses is lacking. Heat shock protein 70 (Hsp70) isoforms have been reported to serve various roles in the replication cycle of numerous viruses. In the present study, microarray and reverse transcriptionquantitative polymerase chain reaction analysis were conducted to investigate the host gene expression profiles following ECTV infection in mice and cell cultures. The results indicated that one Hsp70 isoform, Hsp70 member 1B (Hspa1b), was highly upregulated during ECTV infection in vitro and in vivo. Subsequently, overexpression of Hspa1b protein and small interfering RNAmediated gene silencing of Hspa1b revealed that Hspa1b is required for efficient replication of ECTV. Furthermore, the results demonstrated that ECTV replication may be significantly suppressed by two chemical Hspa1b inhibitors: Quercetin and VER155008. In conclusion, the present study clearly demonstrated that ECTV infection upregulates the expression of Hspa1b in order to promote its replication. The dependence on Hsp70 may be used as a novel therapeutic target for the treatment of Orthopoxvirus infection.
Assuntos
Vírus da Ectromelia/fisiologia , Ectromelia Infecciosa/genética , Ectromelia Infecciosa/virologia , Proteínas de Choque Térmico HSP70/genética , Interações Hospedeiro-Patógeno , Camundongos/virologia , Replicação Viral , Animais , Replicação do DNA , Masculino , Camundongos Endogâmicos BALB C , Regulação para CimaRESUMO
Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when A36R is deleted.
Assuntos
Actinas/metabolismo , Vírus da Ectromelia/fisiologia , Ectromelia Infecciosa/metabolismo , Ectromelia Infecciosa/virologia , Liberação de Vírus , Animais , Transporte Biológico , Linhagem Celular , Chlorocebus aethiops , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Mutação , Ligação Proteica , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Ectromelia virus (ECTV) is the causative agent of mousepox, which has devastating effects in laboratory-mouse colonies and causes economic loss in biomedical research. More importantly, ECTV has been extensively used as an excellent model for studies of the pathogenesis and immunobiology of human smallpox. A rapid and sensitive SYBR Green I-based real-time PCR assay was developed and used for the detection and quantitation of orthopoxvirus by using ECTV in this study. Primers targeted to the highly conserved region of major core protein P4b gene of orthopoxvirus were designed and the standard plasmid was constructed. This assay was able to detect a minimum of 10 copies of standard DNA and 5 TCID50 units of ECTV. In addition, no cross-reactions were observed with two DNA viruses, such as herpes simplex virus and swine pseudorabies virus, and one RNA virus, vesicular stomatitis virus. Furthermore, intra- and inter-assay variability data showed that this method had a highly reproducibility and reliability. Moreover, the current assay was faster and had a higher sensitivity for detection of ECTV genomic DNA in cell cultured and clinical test samples. Therefore, the high sensitivity and reproducibility of this SYBR Green real-time PCR approach is a more effective method than the conventional PCR for ECTV diagnosis and quantitation.
Assuntos
Vírus da Ectromelia/isolamento & purificação , Compostos Orgânicos/química , Orthopoxvirus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Benzotiazóis , Chlorocebus aethiops , Diaminas , Ectromelia Infecciosa/virologia , Limite de Detecção , Masculino , Camundongos Endogâmicos C57BL , Quinolinas , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células VeroRESUMO
Taterapox virus (TATV), which was isolated from an African gerbil (Tatera kempi) in 1975, is the most closely related virus to variola; however, only the original report has examined its virology. We have evaluated the tropism of TATV in vivo in small animals. We found that TATV does not infect Graphiurus kelleni, a species of African dormouse, but does induce seroconversion in the Mongolian gerbil (Meriones unguiculatus) and in mice; however, in wild-type mice and gerbils, the virus produces an unapparent infection. Following intranasal and footpad inoculations with 1 × 106 plaque forming units (PFU) of TATV, immunocompromised stat1-/- mice showed signs of disease but did not die; however, SCID mice were susceptible to intranasal and footpad infections with 100% mortality observed by Day 35 and Day 54, respectively. We show that death is unlikely to be a result of the virus mutating to have increased virulence and that SCID mice are capable of transmitting TATV to C57BL/6 and C57BL/6 stat1-/- animals; however, transmission did not occur from TATV inoculated wild-type or stat1-/- mice. Comparisons with ectromelia (the etiological agent of mousepox) suggest that TATV behaves differently both at the site of inoculation and in the immune response that it triggers.
Assuntos
Orthopoxvirus/fisiologia , Infecções por Poxviridae/virologia , Tropismo Viral , Animais , Antivirais/uso terapêutico , Modelos Animais de Doenças , Vírus da Ectromelia/genética , Vírus da Ectromelia/fisiologia , Ectromelia Infecciosa/virologia , Especificidade de Hospedeiro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Orthopoxvirus/genética , Orthopoxvirus/imunologia , Orthopoxvirus/isolamento & purificação , Infecções por Poxviridae/tratamento farmacológico , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/transmissão , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genéticaRESUMO
Dendritic cells (DCs) are effector cells linking the innate immune system with the adaptive immune response. Many viruses eliminate DCs to prevent host response, induce immunosuppression and to maintain chronic infection. In this study, we examined apoptotic response of dendritic cells during in vitro and in vivo infection with ectromelia virus (ECTV), the causative agent of mousepox. ECTV-infected bone marrow dendritic cells (BMDCs) from BALB/c mice underwent apoptosis through mitochondrial pathway at 48 h post infection, up-regulated FasL and decreased expression of anti-apoptotic Bcl-2 and pro-apoptotic Fas. Similar pattern of Bcl-2, Fas and FasL expression was observed for DCs early during in vivo infection of BALB/c mice. Both BMDCs and DCs from BALB/c mice showed no maturation upon ECTV infection. We conclude that ECTV-infected DCs from BALB/c mouse strain help the virus to spread and to maintain infection.
Assuntos
Apoptose , Células Dendríticas/imunologia , Vírus da Ectromelia/fisiologia , Vírus da Ectromelia/patogenicidade , Ectromelia Infecciosa/imunologia , Imunidade Adaptativa , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3 , Chlorocebus aethiops , Células Dendríticas/patologia , Células Dendríticas/fisiologia , Células Dendríticas/virologia , Modelos Animais de Doenças , Ectromelia Infecciosa/virologia , Proteína Ligante Fas/metabolismo , Regulação da Expressão Gênica , Imunidade Inata , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Regulação para Cima , Células VeroRESUMO
Orthopoxviruses continue to pose a significant threat to the population as potential agents of bioterrorism. An intentional release of natural or engineered variola virus (VARV) or monkeypox viruses would cause mortality and morbidity in the target population. To address this, antivirals have been developed and evaluated in animal models of smallpox and monkeypox. One such antiviral, brincidofovir (BCV, previously CMX001), has demonstrated high levels of efficacy against orthopoxviruses in animal models and is currently under clinical evaluation for prevention and treatment of diseases caused by cytomegaloviruses and adenoviruses. In this study we use the mousepox model of smallpox to evaluate the relationship between the magnitude of the infectious virus dose and an efficacious BCV therapy outcome when treatment is initiated concomitant with detection of ectromelia virus viral DNA (vDNA) in mouse buccal swabs. We found that vDNA could be detected in buccal swabs of some, but not all infected mice over a range of challenge doses by day 3 or 4 postexposure, when initiation of BCV treatment was efficacious, suggesting that detection of vDNA in buccal swabs could be used as a trigger to initiate BCV treatment of an entire potentially exposed population. However, buccal swabs of some mice did not become positive until 5 days postexposure, when initiation of BCV therapy failed to protect mice that received high doses of virus. And finally, the data suggest that the therapeutic window for efficacious BCV treatment decreases as the virus infectious dose increases. Extrapolating these findings to VARV, the data suggest that treatment should be initiated as soon as possible after exposure and not rely on a diagnostic tool such as the measurement of vDNA in buccal cavity swabs; however, consideration should be given to the fact that the behavior/disease-course of VARV in humans is different from that of ectromelia virus in the mouse.
Assuntos
Antivirais/uso terapêutico , Citosina/análogos & derivados , DNA Viral/efeitos dos fármacos , Vírus da Ectromelia/efeitos dos fármacos , Ectromelia Infecciosa/tratamento farmacológico , Mucosa Bucal/virologia , Organofosfonatos/uso terapêutico , Animais , Antivirais/administração & dosagem , Citosina/administração & dosagem , Citosina/uso terapêutico , DNA Viral/isolamento & purificação , Modelos Animais de Doenças , Ectromelia Infecciosa/virologia , Camundongos , Organofosfonatos/administração & dosagem , Orthopoxvirus/efeitos dos fármacos , Varíola/tratamento farmacológico , Varíola/virologiaRESUMO
Mousepox is caused by the orthopoxvirus ectromelia virus (ECTV), and is thought to be transmitted via skin abrasions. We studied the ECTV virulence factor N1 following subcutaneous infection of mousepox-susceptible BALB/c mice. In this model, ECTV lacking N1L gene was attenuated more than 1000-fold compared with wild-type virus and replication was profoundly reduced as early as four days after infection. However, in contrast to data from an intranasal model, N1 protein was not required for virus dissemination. Further, neither T cell nor cytokine responses were enhanced in the absence of N1. Together with the early timing of reduced virus titres, this suggests that in a cutaneous model, N1 exerts its function at the level of infected cells or in the inhibition of the very earliest effectors of innate immunity.
Assuntos
Vírus da Ectromelia/fisiologia , Ectromelia Infecciosa/virologia , Proteínas Virais/genética , Animais , Interações Hospedeiro-Patógeno , Camundongos , Carga Viral , Proteínas Virais/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Replicação ViralRESUMO
Most poxviruses encode a homolog of a ~200,000-kDa membrane protein originally identified in variola virus. We investigated the importance of the ectromelia virus (ECTV) homolog C15 in a natural infection model. In cultured mouse cells, the replication of a mutant virus with stop codons near the N-terminus (ECTV-C15Stop) was indistinguishable from a control virus (ECTV-C15Rev). However, for a range of doses injected into the footpads of BALB/c mice there was less mortality with the mutant. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less ECTV-C15Stop in popliteal and inguinal lymph nodes, spleen and liver indicating decreased virus spread and replication. The latter results were supported by immunohistochemical analyses. Decreased spread was evidently due to immune modulatory activity of C15, rather than to an intrinsic viral function, as the survival of infected mice depended on CD4+ and CD8+ T cells.
Assuntos
Vírus da Ectromelia/metabolismo , Vírus da Ectromelia/patogenicidade , Ectromelia Infecciosa/metabolismo , Ectromelia Infecciosa/virologia , Proteínas de Membrana/metabolismo , Proteínas Virais/metabolismo , Animais , Modelos Animais de Doenças , Vírus da Ectromelia/genética , Ectromelia Infecciosa/genética , Ectromelia Infecciosa/patologia , Feminino , Humanos , Fígado/patologia , Fígado/virologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Baço/patologia , Baço/virologia , Vírus da Varíola/genética , Vírus da Varíola/metabolismo , Proteínas Virais/genética , VirulênciaRESUMO
Ectromelia virus (ECTV), the causative agent of mousepox, has emerged as a valuable model for investigating the host-Orthopoxvirus relationship as it relates to pathogenesis and the immune response. ECTV is a mouse-specific virus and causes high mortality in susceptible mice strains, including BALB/c and C3H, whereas C57BL/6 and 129 strains are resistant to the disease. To understand the host genetic factors in different mouse strains during the ECTV infection, we carried out a microarray analysis of spleen tissues derived from BALB/c and C57BL/6 mice, respectively, at 3 and 10 days after ECTV infection. Differential Expression of Genes (DEGs) analyses revealed distinct differences in the gene profiles of susceptible and resistant mice. The susceptible BALB/c mice generated more DEGs than the resistant C57BL/6 mice. Additionally, gene ontology and KEGG pathway analysis showed the DEGs of susceptible mice were involved in innate immunity, apoptosis, metabolism, and cancer-related pathways, while the DEGs of resistant mice were largely involved in MAPK signaling and leukocyte transendothelial migration. Furthermore, the BALB/c mice showed a strong induction of interferon-induced genes, which, however, were weaker in the C57BL/6 mice. Collectively, the differential transcriptome profiles of susceptible and resistant mouse strains with ECTV infection will be crucial for further uncovering the molecular mechanisms of the host-Orthopoxvirus interaction.
Assuntos
Resistência à Doença/genética , Ectromelia Infecciosa/genética , Interações Hospedeiro-Parasita/genética , Transcriptoma/genética , Animais , Suscetibilidade a Doenças/virologia , Vírus da Ectromelia/patogenicidade , Ectromelia Infecciosa/patologia , Ectromelia Infecciosa/virologia , Regulação da Expressão Gênica , Imunidade Inata/genética , Interferons/genética , Camundongos , Baço/metabolismo , Baço/virologiaRESUMO
In 2007, the United States- Food and Drug Administration (FDA) issued guidance concerning animal models for testing the efficacy of medical countermeasures against variola virus (VARV), the etiologic agent for smallpox. Ectromelia virus (ECTV) is naturally-occurring and responsible for severe mortality and morbidity as a result of mousepox disease in the murine model, displaying similarities to variola infection in humans. Due to the increased need of acceptable surrogate animal models for poxvirus disease, we have characterized ECTV infection in the BALB/c mouse. Mice were inoculated intranasally with a high lethal dose (125 PFU) of ECTV, resulting in complete mortality 10 days after infection. Decreases in weight and temperature from baseline were observed eight to nine days following infection. Viral titers via quantitative polymerase chain reaction (qPCR) and plaque assay were first observed in the blood at 4.5 days post-infection and in tissue (spleen and liver) at 3.5 days post-infection. Adverse clinical signs of disease were first observed four and five days post-infection, with severe signs occurring on day 7. Pathological changes consistent with ECTV infection were first observed five days after infection. Examination of data obtained from these parameters suggests the ECTV BALB/c model is suitable for potential use in medical countermeasures (MCMs) development and efficacy testing.