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Betacoronavirus/ultraestructura , Núcleo Celular/ultraestructura , Leucocitos/ultraestructura , Neutrófilos/ultraestructura , Células Sanguíneas/virología , Recuento de Eritrocitos/métodos , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Recuento de Linfocitos , Subgrupos Linfocitarios/ultraestructura , Persona de Mediana Edad , SARS-CoV-2RESUMEN
Bovine leukemia virus (BLV) infects cattle worldwide and causes B-cell lymphoma in cattle. BLV has been identified in human breast and lung cancer and in blood, but the association of BLV and human cancer is controversial. In this study, we investigated the existence of BLV in 145 Japanese human blood cell lines and 54 human cancer cell lines, using a new highly sensitive PCR assay that can amplify even one copy of BLV using LTR primers different from those in previous studies on BLV provirus in breast cancer. All samples were found negative for BLV provirus, suggesting that BLV is unlikely to infect humans.
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Células Sanguíneas/virología , Línea Celular Tumoral/virología , Virus de la Leucemia Bovina/aislamiento & purificación , Zoonosis/diagnóstico , Adulto , Anciano , Animales , Células Sanguíneas/citología , Línea Celular , Línea Celular Tumoral/citología , Femenino , Humanos , Japón , Virus de la Leucemia Bovina/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Secuencias Repetidas Terminales , Adulto Joven , Zoonosis/virologíaRESUMEN
The collapse of iconic, keystone populations of sockeye (Oncorhynchus nerka) and Chinook (Oncorhynchus tshawytscha) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture.
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Arenavirus/aislamiento & purificación , Enfermedades de los Peces/virología , Nidovirales/aislamiento & purificación , Reoviridae/aislamiento & purificación , Salmón/virología , Virosis/veterinaria , Animales , Arenavirus/clasificación , Arenavirus/genética , Células Sanguíneas/virología , Hibridación in Situ , Metagenómica , Nidovirales/clasificación , Nidovirales/genética , Océano Pacífico , Reoviridae/clasificación , Reoviridae/genética , Análisis de Secuencia de ADN , Transcripción Genética , Virosis/virologíaRESUMEN
Understanding the cellular and anatomical sites of latent virus that contribute to human immunodeficiency virus (HIV) rebound is essential for eradication. In HIV-positive patients, CD4+ T lymphocytes comprise a well-defined functional latent reservoir, defined as cells containing transcriptionally silent genomes able to produce infectious virus once reactivated. However, the persistence of infectious latent virus in CD4+ T cells in compartments other than blood and lymph nodes is unclear. Macrophages (MÏ) are infected by HIV/simian immunodeficiency virus (SIV) and are likely to carry latent viral genomes during antiretroviral therapy (ART), contributing to the reservoir. Currently, the gold standard assay used to measure reservoirs containing replication-competent virus is the quantitative viral outgrowth assay (QVOA). Using an SIV-macaque model, the CD4+ T cell and MÏ functional latent reservoirs were measured in various tissues using cell-specific QVOAs. Our results showed that blood, spleen, and lung in the majority of suppressed animals contain latently infected MÏs. Surprisingly, the numbers of CD4+ T cells, monocytes, and MÏs carrying infectious genomes in blood and spleen were at comparable frequencies (â¼1 infected cell per million). We also demonstrate that ex vivo viruses produced in the MÏ QVOA are capable of infecting activated CD4+ T cells. These results strongly suggest that latently infected tissue MÏs can reestablish productive infection upon treatment interruption. This study provides the first comparison of CD4+ T cell and MÏ functional reservoirs in a macaque model. It is the first confirmation of the persistence of latent genomes in monocytes in blood and MÏs in the spleen and lung of SIV-infected ART-suppressed macaques. Our results demonstrate that transcriptionally silent genomes in MÏs can contribute to viral rebound after ART interruption and should be considered in future HIV cure strategies.IMPORTANCE This study suggests that CD4+ T cells found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. In addition, this study demonstrates that macrophages in tissues are another cellular reservoir for SIV and may contribute to viral rebound after treatment interruption. This new insight into the size and location of the SIV reservoir could have great implications for HIV-infected individuals and should be taken into consideration for the development of future HIV cure strategies.
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Antirretrovirales/administración & dosificación , Linfocitos T CD4-Positivos/virología , Macrófagos/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Latencia del Virus , Animales , Células Sanguíneas/virología , Células Cultivadas , Pulmón/virología , Macaca , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Bazo/virologíaRESUMEN
OBJECTIVE: The aim of this study was to explore the contribution of blood and colon myeloid cells to HIV persistence during antiretroviral therapy (ART). DESIGN: Leukapheresis was collected from HIV-infected individuals with undetectable plasma viral load during ART (HIVâ+âART; nâ=â15) and viremics untreated (HIV+; nâ=â6). Rectal sigmoid biopsies were collected from nâ=â8 HIV+ART. METHODS: Myeloid cells (total monocytes (Mo), CD16/CD16 Mo, CD1c dendritic cells) and CD4 T cells were isolated by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) from peripheral blood. Matched myeloid and CCR6CD4 T cells were isolated from blood and rectal biopsies by FACS. Levels of early (RU5 primers), late (Gag primers) and/or integrated HIV-DNA (Alu/HIV primers) were quantified by nested real-time PCR. Replication-competent HIV was amplified by co-culturing cells from HIV-positive individuals with CD3/CD28-activated CD4 T cells from uninfected donors. RESULTS: Early/late but not integrated HIV reverse transcripts were detected in blood myeloid subsets of four out of 10 HIV+ART; in contrast, integrated HIV-DNA was exclusively detected in CD4 T cells. In rectal biopsies, late HIV reverse transcripts were detected in myeloid cells and CCR6CD4 T cells from one out of eight and seven out of eight HIV+ART individuals, respectively. Replication-competent HIV was outgrown from CD4 T cells but not from myeloid of untreated/ART-treated HIV-positive individuals. CONCLUSION: In contrast to CD4 T cells, blood and colon myeloid cells carry detectable HIV only in a small fraction of HIV+ART individuals. This is consistent with the documented resistance of Mo to HIV infection and the rapid turnover of Mo-derived macrophages in the colon. Future assessment of multiple lymphoid and nonlymphoid tissues is required to include/exclude myeloid cells as relevant HIV reservoirs during ART.
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Antirretrovirales/uso terapéutico , Células Sanguíneas/virología , Colon Sigmoide/virología , Infecciones por VIH/tratamiento farmacológico , VIH-1/aislamiento & purificación , Células Mieloides/virología , Respuesta Virológica Sostenida , Adulto , Anciano , Femenino , Infecciones por VIH/virología , Humanos , Masculino , Persona de Mediana Edad , Carga Viral , Adulto JovenRESUMEN
Product safety assurance is crucial for the clinical use of manufactured cellular therapies. A rational approach for delivering products that fail release criteria (because of potentially false-positive sterility results) is important to avoid unwarranted wastage of highly personalized and costly therapies in critically ill patients where benefits may outweigh risk. Accurate and timely interpretation of microbial sterility assays represents a major challenge in cell therapies. We developed a systematic protocol for the assessment of positive microbial sterility test results using retrospective data from 2007 to 2016. This protocol was validated and applied prospectively between October 2016 and September 2017 to 13 products from which positive sterility results had been reported. Viable and nonviable environmental monitoring (EM) data were collected concurrently as part of a facility control assessment. Three of 13 (23%) positive sterility results were attributable to bone marrow collections that had been contaminated with skin flora during harvest; all were infused without pertinent infectious sequelae. Of the remaining 10, 1 was deemed a true positive and was discarded before infusion, whereas 9 were classified as false positives attributed to laboratory sampling and/or culturing processes. Three products deemed false positive were infused and 6 were withheld because of patient issues unrelated to microbial sterility results. No postinfusion-associated infectious complications were documented. Almost half of the positive EM findings were skin flora. Paired detection of an organism in both product and associated EM was identified in 1 case. Application of our validated protocol to positive product sterility test results allowed for systematic data compilation for regulatory evaluation and provided comprehensive information to clinical investigators to ensure timely and strategic management for product recipients.
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Células Sanguíneas , Tratamiento Basado en Trasplante de Células y Tejidos , Desinfección , Control de Calidad , Células Sanguíneas/microbiología , Células Sanguíneas/virología , Humanos , Estudios RetrospectivosRESUMEN
OBJECTIVE(S): To determine whether variation in cell-associated unspliced (CA-US) HIV RNA in HIV-infected individuals on antiretroviral therapy (ART) has a circadian basis. METHODS: Prospective observational study of HIV-infected individuals on ART. Blood was collected on three occasions and CA-US HIV RNA and mRNA of the circadian-locomotor-output-cycles-kaput (CLOCK)-associated genes quantified by real time PCR. CLOCK-associated proteins were over-expressed in a cell line stably transfected with an HIV long-terminal repeat (LTR) luciferase reporter. RESULTS: Using a mixed effects model, there was a significant increase in log-CA-US RNA at the third visit compared with the first visit (effect size of 0.619 with standard error (SE) of 0.098, Pâ<â0.001) and an independent effect of time of blood draw (effect size 0.051 (SE 0.025), Pâ=â0.040). The CLOCK-associated gene, brain-and-muscle-ARNT-like-1 (BMAL-1) had a significant relationship with log CA-US HIV RNA (effect size 8.508 (SE 3.777), Pâ=â0.028) and also with time (Pâ=â0.045). Over expression of BMAL-1 and CLOCK in a cell line stably transfected with an HIV-LTR luciferase reporter resulted in an increase in luciferase expression and this was reduced following mutation of the second E-box in the HIV-LTR. CONCLUSION: The basal level of HIV transcription on ART can vary significantly and is modulated by the circadian regulator BMAL-1, amongst other factors.
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Factores de Transcripción ARNTL/biosíntesis , Antirretrovirales/uso terapéutico , Células Sanguíneas/virología , Infecciones por VIH/virología , VIH/crecimiento & desarrollo , ARN Viral/análisis , Transcripción Genética , Factores de Transcripción ARNTL/genética , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Infecciones por VIH/tratamiento farmacológico , Interacciones Huésped-Patógeno , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Foot-and-mouth disease virus (FMDV) is a single-stranded positive RNA virus that belongs to the family Picornaviridae. FMDV infects cloven-hoofed animals, such as pigs, sheep, goats, cattle and diverse wildlife species, and remains a major threat to the livestock industry worldwide. In this study, a transcriptome analysis of whole blood from pigs infected with FMDV was performed using the paired-end Illumina sequencing technique to understand the interactions between the pathogen and its host cells. During infection with FMDV, a total of 120 differentially expressed genes (DEGs) were identified, including 110 up-regulated genes and 10 down-regulated genes. To further investigate the DEGs involved in interactions between the virus and its host, gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were conducted. GO annotation indicated that a number of DEGs were enriched in categories involved in host-virus interactions, such as response to stimulus, immune system process and regulation of biological process. KEGG enrichment analysis indicated that the DEGs were primarily involved in the ribosome signaling pathway and immune-related signaling pathways. Ten DEGs, including the immune-related genes BTK1, C1QB, TIMD4 and CXCL10, were selected and validated using quantitative PCR, which showed that the expression patterns of these genes are consistent with the results of the in silico expression analysis. In conclusion, this study presents the first transcriptome analysis of pig whole blood cells infected with FMDV, and the results obtained in this study improve our understanding of the interactions between FMDV and host cells as well as the diagnosis and control of FMD.
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Células Sanguíneas/metabolismo , Células Sanguíneas/virología , Virus de la Fiebre Aftosa/fisiología , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Marcadores Genéticos/genética , Anotación de Secuencia Molecular , PorcinosRESUMEN
In order to determine if an eradication strategy for HIV is effective, it will be important to measure persistent replication-competent virus, the current barrier to a cure. Various assays are available that measure persistent virus, each with advantages and disadvantages that must be balanced in order to select the best assay for the experimental aim. Assays of free virus do not measure the latent form of the virus but can be utilised in conjunction with other assays in order to better understand HIV persistence on ART. The quantitative viral outgrowth assay (QVOA) is the gold standard assay for measuring persistent replication-competent virus, but it, along with assays that vary the classical QVOA method, underestimates the frequency of latently infected cells in blood due to the presence of non-induced yet intact and replication-competent proviruses. Assays that quantify or sequence specific genomic regions of HIV overestimate the size of the reservoir as they are unable to distinguish between intact and defective virus. As an alternative, sequencing the full-length integrated genome can better distinguish replication-competent provirus, but these methods may be expensive and time-consuming. Novel assays, and the application of these assays to novel questions, will be key to the development of future curative therapies for HIV.
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Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Viremia/tratamiento farmacológico , Virología/métodos , Latencia del Virus , Fármacos Anti-VIH/farmacología , Células Sanguíneas/virología , Linfocitos T CD4-Positivos/virología , Líquido Cefalorraquídeo/virología , ADN Complementario/análisis , ADN Viral/análisis , Farmacorresistencia Viral , Predicción , Genoma Viral , Infecciones por VIH/sangre , Infecciones por VIH/líquido cefalorraquídeo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Células Mieloides/virología , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Carga Viral , Viremia/virología , Replicación ViralRESUMEN
The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.
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Vacunas Virales/administración & dosificación , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/fisiología , Aedes/virología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Células Sanguíneas/virología , Embrión de Mamíferos/virología , Femenino , Feto/virología , Humanos , Lípidos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , ARN Mensajero/genética , ARN Mensajero/inmunología , Organismos Libres de Patógenos Específicos , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.
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Apoptosis , Células Sanguíneas/citología , Crassostrea/virología , ADN Viral/metabolismo , Herpesviridae/fisiología , ARN Viral/metabolismo , Proteínas Virales/metabolismo , Animales , Células Sanguíneas/virología , Crassostrea/genética , Fragmentación del ADN , Regulación de la Expresión Génica , Herpesviridae/genética , Herpesviridae/metabolismo , Fosfatidilserinas/metabolismo , Factores de TiempoRESUMEN
Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.
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Cromosomas Humanos/virología , Células Germinativas/virología , Herpesvirus Humano 7/genética , Herpesvirus Humano 7/fisiología , Infecciones por Roseolovirus/virología , Telómero/virología , Integración Viral , Adulto , Células Sanguíneas/virología , Línea Celular , Femenino , Genoma Viral , Folículo Piloso/virología , Humanos , Hibridación Fluorescente in Situ , Masculino , Infecciones por Roseolovirus/transmisiónRESUMEN
BACKGROUND: Two new subclades of influenza A(H3N2) viruses became prominent during the 2014-2015 Northern Hemisphere influenza season. The HA glycoproteins of these viruses showed sequence changes previously associated with alterations in receptor-binding properties. To address how these changes influence virus propagation, viruses were isolated and propagated in conventional MDCK cells and MDCK-SIAT1 cells, cells with enhanced expression of the human receptor for the virus, and analysed at each passage. METHODS: Gene sequence analysis was undertaken as virus was passaged in conventional MDCK cells and MDCK-SIAT1 cells. Alterations in receptor recognition associated with passage of virus were examined by haemagglutination assays using red blood cells from guinea pigs, turkeys and humans. Microneutralisation assays were performed to determine how passage-acquired amino acid substitutions and polymorphisms affected virus antigenicity. RESULTS: Viruses were able to infect MDCK-SIAT1 cells more efficiently than conventional MDCK cells. Viruses of both the 3C.2a and 3C.3a subclades showed greater sequence change on passage in conventional MDCK cells than in MDCK-SIAT1 cells, with amino acid substitutions being seen in both HA and NA glycoproteins. However, virus passage in MDCK-SIAT1 cells at low inoculum dilutions showed reducing infectivity on continued passage. CONCLUSIONS: Current H3N2 viruses should be cultured in the MDCK-SIAT1 cell line to maintain faithful replication of the virus, and at an appropriate multiplicity of infection to retain infectivity.
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Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/inmunología , Gripe Humana/virología , Pruebas de Aglutinación , Sustitución de Aminoácidos , Animales , Células Sanguíneas/inmunología , Células Sanguíneas/virología , Perros , Cobayas , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H3N2 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Células de Riñón Canino Madin Darby , Pase Seriado , PavosRESUMEN
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection of pregnant females causes fetal death and increased piglet mortality, but there is substantial variation in the extent of reproductive pathology between individual dams. This study used RNA-sequencing to characterize the whole blood transcriptional response to type 2 PRRSV in pregnant gilts during the first week of infection (at 0, 2, and 6 days post-inoculation), and attempted to identify gene expression signatures associated with a low or high level of fetal mortality rates (LFM and HFM; n = 8/group) at necropsy, 21 days post-inoculation. The initial response to infection measured at 2 days post-inoculation saw an upregulation of genes involved in innate immunity, such as interferon-stimulated antiviral genes and inflammatory markers, and apoptosis. A concomitant decrease in expression of protein synthesis and T lymphocyte markers was observed. By day 6 the pattern had reversed, with a drop in innate immune signaling and an increase in the expression of genes involved in cell division and T cell signaling. Differentially expressed genes (DEGs) associated with extremes of litter mortality rate were identified at all three time-points. Among the 15 DEGs upregulated in LFM gilts on all three days were several genes involved in platelet function, including integrins ITGA2B and ITGB3, and the chemokine PF4 (CXCL4). LFM gilts exhibited a higher baseline expression of interferon-stimulated and pro-inflammatory genes prior to infection, and of T cell markers two days post-infection, indicative of a more rapid progression of the immune response to PRRSV. This study has increased our knowledge of the early response to PRRSV in the blood of pregnant gilts, and could ultimately lead to the development of a biomarker panel that can be used to predict PRRSV-associated reproductive pathology.
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Células Sanguíneas/virología , Expresión Génica/genética , Síndrome Respiratorio y de la Reproducción Porcina/genética , Síndrome Respiratorio y de la Reproducción Porcina/mortalidad , Reproducción/genética , Porcinos/virología , Animales , Células Sanguíneas/inmunología , Femenino , Muerte Fetal , Mortalidad Fetal , Inmunidad Innata/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Embarazo , Complicaciones Infecciosas del Embarazo/genética , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/mortalidad , Reproducción/inmunología , Porcinos/inmunología , Linfocitos T/inmunologíaRESUMEN
This study examined the prevalence of three human herpesviruses (HHV), namely HHV-4 (Epstein-Barr virus/EBV), HHV-6b and HHV-7 in leucoreduced blood products obtained from the Sainte-Justine Hospital blood bank. A total of 100 specimens, including 34 red blood cell concentrates, 33 platelet bags and 33 plasma units, were collected and screened by a sensitive PCR assay using virus-specific primers. Positive units were then retested by quantitative PCR. Of the 100 specimens, one platelet unit tested positive for EBV.
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Bancos de Sangre/estadística & datos numéricos , Herpesvirus Humano 4/aislamiento & purificación , Plasma/virología , Bancos de Sangre/normas , Células Sanguíneas/virología , HumanosRESUMEN
Epidemics caused by cyprinid herpesvirus 2 (CyHV-2) in domestic cyprinid species have been reported in both European and Asian countries. Although the mechanisms remain unknown, acute CyHV-2 infections generally result in high mortality, and the surviving carps become chronic carriers displaying no external clinical signs. In this study, in situ hybridization analysis showed that CyHV-2 tended to infect peripheral blood cells during either acute or chronic infections in silver crucian carp, Carassius auratus gibelio (Bloch). Laboratory challenge experiments coupled with real-time PCR quantification assays further indicated that steady-state levels of the viral genomic copy number in fish serum exhibited a typical 'one-step' growth curve post-viral challenge. Transcriptional expression of open reading frames (ORF) 121, which was selected due to its highest transcriptional levels in almost all tested tissues, was monitored to represent the replication kinetics of CyHV-2 in peripheral blood cells. Similar kinetic curve of active viral gene transcription in blood cells was obtained as that of serum viral load, indicating that CyHV-2 replicated in peripheral blood cells as well as in other well-characterized tissues. This study should pave the way for designing non-invasive and cost-effective serum diagnostic methods for quick detection of CyHV-2 infection.
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Células Sanguíneas/virología , Carpas/virología , Enfermedades de los Peces/virología , Infecciones por Herpesviridae/veterinaria , Herpesviridae/fisiología , Animales , ADN Viral/sangre , Enfermedades de los Peces/diagnóstico , Regulación Viral de la Expresión Génica , Herpesviridae/genética , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/virología , Replicación ViralRESUMEN
OBJECTIVES: The objective of this study was to address the dynamics of archived resistant quasispecies in cell-associated HIV-1 DNA over time in heavily ART-experienced patients with currently suppressed plasma HIV-1 RNA. METHODS: Longitudinal ultra-deep sequencing (UDS) analysis of reverse transcriptase, protease and V3 Env regions was performed on blood-cell-associated HIV-1 DNA samples. Drug-resistance-associated mutations (DRAMs) and tropism were interpreted using the ANRS and Geno2Pheno algorithms. We analysed frozen blood cells from patients enrolled in the INNOVE and ANRS 123 ETOILE studies who achieved sustained viral suppression after salvage optimized ART (SOT). RESULTS: Samples were available at baseline and 6 and ≥12 months after SOT initiation in 10 patients. V3 loop sequences displayed wide intra-individual dynamics over time. Viral variants harbouring DRAMs exhibited three non-exclusive scenarios. First, when SOT exerted the same selective pressure as previous failing regimens, some viral quasispecies still harboured the same DRAMs at the same level as at the time of virological failure. Thus, as DRAMs were mostly associated with the same viral variant, variants with a complete resistance pattern remained archived. Second, some viral variants harbouring DRAMs were no longer detected over time when SOT consisted of new antiretroviral classes or had resistance profiles distinct from those of previous failing regimens. Third, variants with new DRAMs associated with SOT emerged in blood cells during follow-up despite sustained virological control. CONCLUSIONS: Using longitudinal UDS analysis and focusing on DRAMs and tropism as markers, we demonstrated that, despite sustained virological control, archived HIV-1 DNA quasispecies continued to evolve.
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Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Tropismo Viral , Células Sanguíneas/virología , ADN Viral/química , ADN Viral/genética , ADN Viral/aislamiento & purificación , Genotipo , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , VIH-1/genética , VIH-1/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estudios Longitudinales , Mutación , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure.
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Fármacos Anti-VIH/farmacocinética , Citidina Trifosfato/análogos & derivados , Didesoxinucleótidos/farmacocinética , Lamivudine/análogos & derivados , Leucocitos Mononucleares/metabolismo , Modelos Estadísticos , Semen/metabolismo , Nucleótidos de Timina/farmacocinética , Zidovudina/análogos & derivados , Adulto , Fármacos Anti-VIH/farmacología , Disponibilidad Biológica , Transporte Biológico , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Células Sanguíneas/patología , Células Sanguíneas/virología , Simulación por Computador , Citidina Trifosfato/farmacocinética , Citidina Trifosfato/farmacología , Didesoxinucleótidos/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Lamivudine/farmacocinética , Lamivudine/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/virología , Masculino , Semen/citología , Semen/efectos de los fármacos , Semen/virología , Nucleótidos de Timina/farmacología , Factores de Tiempo , Zidovudina/farmacocinética , Zidovudina/farmacologíaAsunto(s)
Descontaminación/métodos , Coloración y Etiquetado/métodos , Inactivación de Virus , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/patología , Células Sanguíneas/virología , Recolección de Muestras de Sangre/normas , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Células de la Médula Ósea/virología , Pruebas Hematológicas , Fiebre Hemorrágica Ebola/sangre , Fiebre Hemorrágica Ebola/virología , Calor/uso terapéutico , Humanos , Hipoclorito de Sodio/uso terapéutico , Carga ViralRESUMEN
BACKGROUND: Treatment with interferon-alpha (IFN-α) and ribavirin successfully clears hepatitis C virus (HCV) infection in 50% of patients infected with genotype 1. Addition of NS3-4A protease inhibitors (PIs) increases response rates but results in additional side effects and significant economic costs. Here, we hypothesised that in vitro responsiveness of peripheral blood mononuclear cells (PBMCs) to IFN-α stimulation would identify patients who achieved sustained virological response (SVR) on dual therapy alone and thus not require addition of PIs. METHODS: PBMCs were isolated from HCV infected patients (n = 42), infected with either HCV genotype 1 or genotype 3, before commencing therapy and stimulated in vitro with IFN-α. Expression of the IFN stimulated genes (ISGs) PKR, OAS and MxA was measured and correlated with subsequent treatment response and IL28B genotype. RESULTS: Genotype 1 infected patients who achieved SVR had significantly higher pre-treatment expression of PKR (p = 0.0148), OAS (p = 0.0019) and MxA (p = 0.0019) in IFN-α stimulated PBMCs, compared to genotype 1 infected patients who did not achieve SVR or patients infected with genotype 3, whose in vitro ISG expression did not correlate with clinical responsiveness. IL28B genotype (rs12979860) did not correlate with endogenous or IFN-α stimulated ISG responsiveness. CONCLUSIONS: In vitro responsiveness of PBMCs to IFN-α from genotype 1 infected patients predicts clinical responsiveness to dual therapy, independently of IL28B genotype. These results indicate that this sub-group of HCV infected patients could be identified pre-treatment and successfully treated without PIs, thus reducing adverse side effects and emergence of PI resistant virus while making significant economic savings.