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1.
Viruses ; 12(12)2020 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-33353185

RESUMEN

Human parvovirus B19 (B19V) causes various human diseases, ranging from childhood benign infection to arthropathies, severe anemia and fetal hydrops, depending on the health state and hematological status of the patient. To counteract B19V blood-borne contamination, evaluation of B19 DNA in plasma pools and viral inactivation/removal steps are performed, but nucleic acid testing does not correctly reflect B19V infectivity. There is currently no appropriate cellular model for detection of infectious units of B19V. We describe here an improved cell-based method for detecting B19V infectious units by evaluating its host transcription. We evaluated the ability of various cell lines to support B19V infection. Of all tested, UT7/Epo cell line, UT7/Epo-STI, showed the greatest sensitivity to B19 infection combined with ease of performance. We generated stable clones by limiting dilution on the UT7/Epo-STI cell line with graduated permissiveness for B19V and demonstrated a direct correlation between infectivity and S/G2/M cell cycle stage. Two of the clones tested, B12 and E2, reached sensitivity levels higher than those of UT7/Epo-S1 and CD36+ erythroid progenitor cells. These findings highlight the importance of cell cycle status for sensitivity to B19V, and we propose a promising new straightforward cell-based method for quantifying B19V infectious units.


Asunto(s)
Ciclo Celular , Infecciones por Parvoviridae/diagnóstico , Infecciones por Parvoviridae/virología , Parvovirus B19 Humano/fisiología , Biomarcadores , Línea Celular , Células Eritroides/metabolismo , Células Eritroides/virología , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/virología , Expresión Génica , Regulación Viral de la Expresión Génica , Humanos , Técnicas de Diagnóstico Molecular , Infecciones por Parvoviridae/metabolismo , Sensibilidad y Especificidad , Tropismo Viral
2.
mBio ; 10(6)2019 11 26.
Artículo en Inglés | MEDLINE | ID: mdl-31772057

RESUMEN

CD71+ erythroid cells (CECs) have a wide range of immunomodulatory properties. Here, we show that CECs are expanded in the peripheral blood of HIV patients, with a positive correlation between their frequency and the plasma viral load. CECs from HIV patients and human cord blood/placenta exacerbate HIV-1 infection/replication when cocultured with CD4+ T cells, and that preexposure of CD4+ T cells to CECs enhances their permissibility to HIV infection. However, mature red blood cells (RBCs) do not enhance HIV replication when cocultured with CD4+ T cells. We also found CECs express substantial levels of the NOX2 gene and via a mitochondrial reactive oxygen species (ROS)-dependent mechanism possibly upregulate NF-κB in CD4+ T cells once cocultured, which affects the cell cycle machinery to facilitate HIV-1 replication. The complement receptor-1 (CD35) and the Duffy antigen receptor for chemokines (DARC) as potential HIV target molecules are expressed significantly higher on CECs compared to mature red blood cells. Blocking CD35 or DARC substantially abolishes HIV-1 transmission by RBCs to uninfected CD4+ T cells but not by CECs. In contrast, we observed CECs bind to HIV-1 via CD235a and subsequently transfer the virus to uninfected CD4+ T cells, which can be partially blocked by the anti-CD235a antibody. More importantly, we found that CECs from HIV-infected individuals in the presence of antiretroviral therapy harbor infective viral particles, which mediate HIV-1 trans-infection of CD4+ T cells. Therefore, our findings provide a novel insight into the role of CECs in HIV pathogenesis as potential contributing cells in viral persistence and transmission.IMPORTANCE Immature red blood cells (erythroid precursors or CD71+ erythroid cells) have a wide range of immunomodulatory properties. In this study, we found that these erythroid precursors are abundant in the human cord blood/placental tissues, in the blood of HIV-infected and anemic individuals. We observed that these cells exacerbate HIV-1 replication/infection in target cells and even make HIV target cells more permissible to HIV infection. In addition, we found that HIV gets a free ride by binding on the surface of these cells and thus can travel to different parts of the body. In agreement, we noticed a positive correlation between the plasma viral load and the frequency of these cells in HIV patients. More importantly, we observed that infective HIV particles reside inside these erythroid precursors but not mature red blood cells. Therefore, these cells by harboring HIV can play an important role in HIV pathogenesis.


Asunto(s)
Células Eritroides/virología , Infecciones por VIH/virología , VIH-1/fisiología , Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Células Eritroides/inmunología , Femenino , Sangre Fetal/inmunología , Sangre Fetal/virología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/genética , Humanos , Embarazo , Receptores de Transferrina/genética , Receptores de Transferrina/inmunología , Replicación Viral
3.
Vet Microbiol ; 201: 126-135, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28284598

RESUMEN

Porcine reproductive and respiratory syndrome viruses (PRRSV) present a wide phenotypic and genetic diversity. Experimental infections have demonstrated viral replication, including highly pathogenic strains (HP-PRRSV), in primary lymphoid organs such as the thymus. However, studies of the bone marrow are scarce but necessary to help elucidate the immunobiology of PRRSV strains of differing virulence. In this study, whereas viral RNA was detected within the bone marrow of animals experimentally infected with both low virulent Lelystad (LV) and 215-06 PRRSV-1 strains and with the highly virulent SU1-bel strain, PRRSV positive cells were only occasionally detected in one SU1-bel infected animal. PRRSV RNA levels were associated to circulating virus with the highest levels detected in LV-infected pigs. At 3 dpi, a decrease in the proportion of haematopoietic tissue and number of erythroid cells in all infected groups was associated with an increase in TUNEL or cleaved caspase 3 labelling and higher counts of myeloid cells compared to control. The expression of IL-1α and IL-6 was elevated at the beginning of the infection in all infected animals. The expression of TNF-α was increased at the end of the study in all infected groups with respect to control. Different PRRSV-1 strains induced, presummably by indirect mechanisms and independently of viral load and strain virulence, moderate and sustained hypoplasia of erythroid cells and myeloid cell hyperplasia at early stages of infection. These changes were paralleled by a peak in the local expression of IL-1α, IL-6 and TNF-α in all infected groups.


Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina/patología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Animales , Médula Ósea/patología , Médula Ósea/virología , Citocinas/inmunología , Células Eritroides/patología , Células Eritroides/virología , Hiperplasia/patología , Hiperplasia/veterinaria , Hiperplasia/virología , Masculino , Células Mieloides/patología , Células Mieloides/virología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Porcinos , Carga Viral , Virulencia , Replicación Viral
4.
PLoS One ; 8(3): e58757, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23516552

RESUMEN

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34(+) cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: ß-globin promoter (ßp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the ß-globin locus control region (HS2-ßbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-ßp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34(+) cells in vitro. Transgene expression from the B19p6 or the HS2-ßp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-ßp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-ßp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as ß-thalassemia and sickle cell disease.


Asunto(s)
Linaje de la Célula , Dependovirus/genética , Células Eritroides/metabolismo , Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Transducción Genética/métodos , Trasplante Heterólogo , Animales , Dependovirus/clasificación , Células Eritroides/citología , Células Eritroides/virología , Femenino , Expresión Génica , Células HEK293 , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/virología , Humanos , Células K562 , Ratones , Mutación , Regiones Promotoras Genéticas/genética , Serotipificación , Transcripción Genética , Transgenes/genética
5.
Virology ; 349(2): 254-63, 2006 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-16643977

RESUMEN

Human parvovirus B19 (B19) is a well-known pathogenic agent which causes apoptosis in erythrocyte lineage cells. Here, we provide the first evidence that mitochondrial autophagy is specifically found in the B19-infected cells. The protein expression ratio for LC3-II/LC3-I increased significantly in infected cells, indicating possible involvement of cellular autophagy in the infection process. Immunofluorescence confocal microscopy analyses revealed that B19 infection induced an intracellular autophagosome as judged by endogenous LC3 staining. Moreover, inhibition of autophagy by 3-MA significantly facilitated B19-infection-mediated cell death. These results suggest a novel mechanism by which B19-infected cells survive by cellular autophagy.


Asunto(s)
Autofagia/fisiología , Células Eritroides/virología , Parvovirus B19 Humano/fisiología , Adenina/análogos & derivados , Adenina/farmacología , Western Blotting , Línea Celular Tumoral , Supervivencia Celular , Citoplasma/química , Citoplasma/ultraestructura , Inhibidores Enzimáticos/farmacología , Células Eritroides/ultraestructura , Citometría de Flujo , Humanos , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/análisis , Fagosomas/química , Fagosomas/ultraestructura , Proteínas no Estructurales Virales/análisis
6.
Blood ; 106(10): 3449-56, 2005 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-16076874

RESUMEN

Human parvovirus B19 (B19) infects human erythroid cells expressing P antigen. However, some cell lines that were positive for P antigen failed to bind B19, whereas some cell lines had an ability to bind B19 despite undetectable expression of P antigen. We here demonstrate that B19 specifically binds with Ku80 autoantigen on the cell surface. Furthermore, transfection of HeLa cells with the gene of Ku80 enabled the binding of B19 and allowed its entry into cells. Moreover, reduction of cell-surface expression of Ku80 in KU812Ep6 cells, which was a high-sensitive cell line for B19 infection, by short interfering RNA for Ku80 resulted in the marked inhibition of B19 binding in KU812Ep6 cells. Although Ku80 originally has been described as a nuclear protein, human bone marrow erythroid cells with glycophorin A or CD36, B cells with CD20, or T cells with CD3 were all positive for cell-surface expression of Ku80. B19 infection of KU812Ep6 cells and bone marrow cells was inhibited in the presence of anti-Ku80 antibody. Our data suggest that Ku80 functions as a novel coreceptor for B19 infection, and this finding may provide an explanation for the pathologic immunity associated with B19 infection.


Asunto(s)
Antígenos Nucleares/inmunología , Proteínas de Unión al ADN/inmunología , Células Eritroides/inmunología , Sistema del Grupo Sanguíneo P , Infecciones por Parvoviridae/inmunología , Parvovirus B19 Humano/inmunología , Receptores Virales/inmunología , Anticuerpos/inmunología , Anticuerpos/farmacología , Antígenos CD/inmunología , Células de la Médula Ósea , Membrana Celular/inmunología , Células Eritroides/virología , Regulación de la Expresión Génica/inmunología , Células HeLa , Humanos , Autoantígeno Ku , Sistema del Grupo Sanguíneo P/sangre , Sistema del Grupo Sanguíneo P/inmunología , Infecciones por Parvoviridae/sangre , Células U937
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