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1.
Cell ; 184(1): 15-17, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33338422

RESUMEN

Complementary genome-wide CRISPR-Cas9 screens performed by multiple groups reveal new insights into SARS-CoV-2 biology including aspects of viral entry, translation, replication, egress, and the genes regulating these processes. Comparisons with other coronaviruses enhances our understanding of the cellular life cycle of this medically important family of emerging viruses.

2.
Cell ; 184(8): 2229-2238.e13, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33691138

RESUMEN

The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.


Asunto(s)
COVID-19/virología , Contención de Riesgos Biológicos/métodos , SARS-CoV-2 , Células A549 , Animales , Chlorocebus aethiops , Cricetinae , Prueba de Complementación Genética/métodos , Genoma Viral , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , ARN Viral , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Células Vero , Virulencia , Replicación Viral
3.
Cell ; 184(7): 1804-1820.e16, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33691139

RESUMEN

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.


Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Linfocitos T CD8-positivos , COVID-19 , Fragmentos Fc de Inmunoglobulinas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células CHO , COVID-19/inmunología , COVID-19/terapia , Chlorocebus aethiops , Cricetulus , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , SARS-CoV-2/inmunología , Células Vero , Carga Viral
4.
Cell ; 184(8): 2183-2200.e22, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33756110

RESUMEN

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Sitios de Unión de Anticuerpos , Células CHO , Chlorocebus aethiops , Cricetulus , Epítopos , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , SARS-CoV-2/inmunología , Células Vero
5.
Cell ; 182(3): 744-753.e4, 2020 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-32553273

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of human infections. One limitation to the evaluation of potential therapies and vaccines to inhibit SARS-CoV-2 infection and ameliorate disease is the lack of susceptible small animals in large numbers. Commercially available laboratory strains of mice are not readily infected by SARS-CoV-2 because of species-specific differences in their angiotensin-converting enzyme 2 (ACE2) receptors. Here, we transduced replication-defective adenoviruses encoding human ACE2 via intranasal administration into BALB/c mice and established receptor expression in lung tissues. hACE2-transduced mice were productively infected with SARS-CoV-2, and this resulted in high viral titers in the lung, lung pathology, and weight loss. Passive transfer of a neutralizing monoclonal antibody reduced viral burden in the lung and mitigated inflammation and weight loss. The development of an accessible mouse model of SARS-CoV-2 infection and pathogenesis will expedite the testing and deployment of therapeutics and vaccines.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Betacoronavirus/inmunología , Infecciones por Coronavirus/terapia , Modelos Animales de Enfermedad , Neumonía Viral/terapia , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/virología , Femenino , Células HEK293 , Humanos , Inmunización Pasiva/métodos , Pulmón/metabolismo , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Pandemias , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , SARS-CoV-2 , Transducción Genética , Células Vero , Carga Viral/inmunología
6.
Cell ; 183(1): 169-184.e13, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32931734

RESUMEN

The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.


Asunto(s)
Infecciones por Coronavirus/inmunología , Inmunogenicidad Vacunal , Neumonía Viral/inmunología , Vacunas Virales/inmunología , Adenoviridae/genética , Administración Intranasal , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , COVID-19 , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/prevención & control , Femenino , Células HEK293 , Humanos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Pandemias , Neumonía Viral/patología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células Vero , Vacunas Virales/administración & dosificación
7.
Nat Immunol ; 21(11): 1327-1335, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32839612

RESUMEN

Although animal models have been evaluated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, none have fully recapitulated the lung disease phenotypes seen in humans who have been hospitalized. Here, we evaluate transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice results in high levels of viral infection in lungs, with spread to other organs. A decline in pulmonary function occurs 4 days after peak viral titer and correlates with infiltration of monocytes, neutrophils and activated T cells. SARS-CoV-2-infected lung tissues show a massively upregulated innate immune response with signatures of nuclear factor-κB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection shares many features of severe COVID-19 infection and can be used to define the basis of lung disease and test immune and antiviral-based countermeasures.


Asunto(s)
Betacoronavirus/inmunología , Infecciones por Coronavirus/patología , Inmunidad Innata/inmunología , Peptidil-Dipeptidasa A/genética , Neumonía Viral/patología , Neumonía/patología , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Interferón Tipo I/inmunología , Interferón gamma/inmunología , Queratina-18/genética , Leucocitos/inmunología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Transgénicos , Monocitos/inmunología , FN-kappa B/inmunología , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Pandemias , Neumonía/genética , Neumonía/virología , Neumonía Viral/inmunología , Regiones Promotoras Genéticas/genética , SARS-CoV-2 , Linfocitos T/inmunología , Células Vero , Replicación Viral/inmunología
9.
Nature ; 591(7849): 293-299, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33494095

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.


Asunto(s)
COVID-19/virología , Furina/metabolismo , Mutación , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , COVID-19/patología , COVID-19/fisiopatología , Línea Celular , Chlorocebus aethiops , Cricetinae , Femenino , Humanos , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/fisiopatología , Enfermedades Pulmonares/virología , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteolisis , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Replicación Viral/genética
10.
PLoS Pathog ; 20(8): e1012436, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39196893

RESUMEN

Viruses capable of causing persistent infection have developed sophisticated mechanisms for evading host immunity, and understanding these processes can reveal novel features of the host immune system. One such virus, human pegivirus (HPgV), infects ~15% of the global human population, but little is known about its biology beyond the fact that it does not cause overt disease. We passaged a pegivirus isolate of feral brown rats (RPgV) in immunodeficient laboratory mice to develop a mouse-adapted virus (maPgV) that established persistent high-titer infection in a majority of wild-type laboratory mice. maRPgV viremia was detected in the blood of mice for >300 days without apparent disease, closely recapitulating the hallmarks of HPgV infection in humans. We found a pro-viral role for type-I interferon in chronic infection; a lack of PD-1-mediated tolerance to PgV infection; and multiple mechanisms by which PgV immunity can be achieved by an immunocompetent host. These data indicate that the PgV immune evasion strategy has aspects that are both common and unique among persistent viral infections. The creation of maPgV represents the first PgV infection model in wild-type mice, thus opening the entire toolkit of the mouse host to enable further investigation of this persistent RNA virus infections.


Asunto(s)
Infecciones por Flaviviridae , Flaviviridae , Animales , Ratones , Infecciones por Flaviviridae/virología , Infecciones por Flaviviridae/inmunología , Flaviviridae/genética , Flaviviridae/inmunología , Infección Persistente/inmunología , Infección Persistente/virología , Ratas , Evasión Inmune , Ratones Endogámicos C57BL , Humanos
11.
Emerg Infect Dis ; 30(4): 721-731, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38526136

RESUMEN

Genetically diverse simian arteriviruses (simarteriviruses) naturally infect geographically and phylogenetically diverse monkeys, and cross-species transmission and emergence are of considerable concern. Characterization of most simarteriviruses beyond sequence analysis has not been possible because the viruses fail to propagate in the laboratory. We attempted to isolate 4 simarteriviruses, Kibale red colobus virus 1, Pebjah virus, simian hemorrhagic fever virus, and Southwest baboon virus 1, by inoculating an immortalized grivet cell line (known to replicate simian hemorrhagic fever virus), primary macaque cells, macrophages derived from macaque induced pluripotent stem cells, and mice engrafted with macaque CD34+-enriched hematopoietic stem cells. The combined effort resulted in successful virus isolation; however, no single approach was successful for all 4 simarteriviruses. We describe several approaches that might be used to isolate additional simarteriviruses for phenotypic characterization. Our results will expedite laboratory studies of simarteriviruses to elucidate virus-host interactions, assess zoonotic risk, and develop medical countermeasures.


Asunto(s)
Arterivirus , Animales , Ratones , Arterivirus/genética , Macaca , Macrófagos , Línea Celular
12.
J Virol ; 97(10): e0093023, 2023 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-37792000

RESUMEN

IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).


Asunto(s)
Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Técnicas de Cultivo de Célula , Expresión Génica Ectópica , Virus Elevador de Lactato Deshidrogenasa , Receptores de Superficie Celular , Animales , Ratones , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Línea Celular/virología , Virus Elevador de Lactato Deshidrogenasa/genética , Virus Elevador de Lactato Deshidrogenasa/crecimiento & desarrollo , Virus Elevador de Lactato Deshidrogenasa/inmunología , Virus Elevador de Lactato Deshidrogenasa/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Factores de Tiempo
13.
Proc Natl Acad Sci U S A ; 117(51): 32648-32656, 2020 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-33268494

RESUMEN

Yellow fever (YF) is a mosquito-transmitted viral disease that causes tens of thousands of deaths each year despite the long-standing deployment of an effective vaccine. In its most severe form, YF manifests as a hemorrhagic fever that causes severe damage to visceral organs. Although coagulopathy is a defining feature of severe YF in humans, the mechanism by which it develops remains uncertain. Hepatocytes are a major target of yellow fever virus (YFV) infection, and the coagulopathy in severe YF has long been attributed to massive hepatocyte infection and destruction that results in a defect in clotting factor synthesis. However, when we analyzed blood from Brazilian patients with severe YF, we found high concentrations of plasma D-dimer, a fibrin split product, suggestive of a concurrent consumptive process. To define the relationship between coagulopathy and hepatocellular tropism, we compared infection and disease in Fah-/-, Rag2-/-, and Il2rɣ-/- mice engrafted with human hepatocytes (hFRG mice) and rhesus macaques using a highly pathogenic African YFV strain. YFV infection of macaques and hFRG mice caused substantial hepatocyte infection, liver damage, and coagulopathy as defined by virological, clinical, and pathological criteria. However, only macaques developed a consumptive coagulopathy whereas YFV-infected hFRG mice did not. Thus, infection of cell types other than hepatocytes likely contributes to the consumptive coagulopathy associated with severe YF in primates and humans. These findings expand our understanding of viral hemorrhagic disease and associated coagulopathy and suggest directions for clinical management of severe YF cases.


Asunto(s)
Coagulación Intravascular Diseminada/virología , Hepatopatías/virología , Tropismo Viral/fisiología , Fiebre Amarilla/fisiopatología , Virus de la Fiebre Amarilla/fisiología , Animales , Modelos Animales de Enfermedad , Coagulación Intravascular Diseminada/sangre , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Hepatocitos/trasplante , Hepatocitos/virología , Humanos , Hepatopatías/fisiopatología , Macaca mulatta , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fiebre Amarilla/complicaciones , Fiebre Amarilla/virología
14.
PLoS Pathog ; 13(10): e1006692, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29073258

RESUMEN

Human pegivirus (HPgV) protects HIV+ people from HIV-associated disease, but the mechanism of this protective effect remains poorly understood. We sequentially infected cynomolgus macaques with simian pegivirus (SPgV) and simian immunodeficiency virus (SIV) to model HIV+HPgV co-infection. SPgV had no effect on acute-phase SIV pathogenesis-as measured by SIV viral load, CD4+ T cell destruction, immune activation, or adaptive immune responses-suggesting that HPgV's protective effect is exerted primarily during the chronic phase of HIV infection. We also examined the immune response to SPgV in unprecedented detail, and found that this virus elicits virtually no activation of the immune system despite persistently high titers in the blood over long periods of time. Overall, this study expands our understanding of the pegiviruses-an understudied group of viruses with a high prevalence in the global human population-and suggests that the protective effect observed in HIV+HPgV co-infected people occurs primarily during the chronic phase of HIV infection.


Asunto(s)
Coinfección/virología , Infecciones por Flaviviridae/inmunología , Infecciones por Flaviviridae/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Animales , Coinfección/inmunología , Modelos Animales de Enfermedad , Virus GB-C , Macaca fascicularis , Virus de la Inmunodeficiencia de los Simios
15.
Clin Chem ; 65(5): 634-643, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30518664

RESUMEN

BACKGROUND: Historically, culture-based microbiology laboratory testing has relied on manual methods, and automated methods (such as those that have revolutionized clinical chemistry and hematology over the past several decades) were largely absent from the clinical microbiology laboratory. However, an increased demand for microbiology testing and standardization of sample-collection devices for microbiology culture, as well as a dwindling supply of microbiology technologists, has driven the adoption of automated methods for culture-based laboratory testing in clinical microbiology. CONTENT: We describe systems currently enabling total laboratory automation (TLA) for culture-based microbiology testing. We describe the general components of a microbiology automation system and the various functions of these instruments. We then introduce the 2 most widely used systems currently on the market: Becton Dickinson's Kiestra TLA and Copan's WASPLab. We discuss the impact of TLA on metrics such as turnaround time and recovery of microorganisms, providing a review of the current literature and perspectives from laboratory directors, managers, and technical staff. Finally, we provide an outlook for future advances in TLA for microbiology with a focus on artificial intelligence for automated culture interpretation. SUMMARY: TLA is playing an increasingly important role in clinical microbiology. Although challenges remain, TLA has great potential to affect laboratory efficiency, turnaround time, and the overall quality of culture-based microbiology testing.


Asunto(s)
Técnicas Microbiológicas/instrumentación , Automatización , Recuento de Colonia Microbiana , Eficiencia Organizacional , Humanos , Laboratorios/organización & administración , Pruebas de Sensibilidad Microbiana , Estudios de Tiempo y Movimiento
16.
Eur J Clin Microbiol Infect Dis ; 38(6): 1135-1141, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30806903

RESUMEN

In order to realize the full potential of total laboratory automation (TLA) in the clinical microbiology laboratory, workflows must be optimized to match each laboratory's capabilities, patient population, and staffing model. Using TLA-based digital photography to monitor urine cultures, we sought to improve culture result turn-around-time (TAT) by changing the time at which a culture is first photographed and thus available for analysis/work-up (Pre1) from 18 h (16,391 cultures) to 16 h (53,113 cultures) (with a total of 24-h culture incubation in both time periods); in both time periods, cultures were set up 24/7, and culture work-up occurred during the day shift only. With this change, we observed a significant decrease in time-to-final-result TAT for positive cultures (18 h-Pre1 median: 71.6 h; 16 h-Pre1 median: 61.0 h). This effect was most pronounced for Gram-negative organisms, with a median reduction in time-to-final-result for Escherichia coli cultures (51.8% of positive urine cultures) of 14.2 h (18 h-Pre1 median: 77.3 h; 16 h-Pre1 median: 63.1 h). This reduction in TAT was accompanied by a decrease in sensitivity at the Pre1 time point (18 h-Pre1 91.01%; 16 h-Pre1 88.06%), which we also found to vary by species: there was a reduction in sensitivity at the first culture reading of 1 to 2% for cultures with Gram-negative microorganisms, but for some Gram-positive microorganisms (e.g., Aerococcus urinae and non-aureus Staphylococcus species), there was a reduction in sensitivity at the Pre1 time-point of 3 to 7%. These results can guide workflow decisions for laboratories seeking to implement and/or optimize TLA systems, demonstrating a tradeoff between TAT and the sensitivity of preliminary urine culture results.


Asunto(s)
Automatización de Laboratorios , Orina/microbiología , Flujo de Trabajo , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Grampositivas/crecimiento & desarrollo , Bacterias Grampositivas/aislamiento & purificación , Humanos , Sensibilidad y Especificidad , Especificidad de la Especie , Factores de Tiempo
17.
J Clin Microbiol ; 56(9)2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29976590

RESUMEN

Ceftolozane-tazobactam (C/T) is a novel beta-lactam-beta-lactamase inhibitor combination antibiotic approved by the U.S. Food and Drug Administration in 2014 for the treatment of complicated intra-abdominal infections (in combination with metronidazole) and complicated urinary tract infections. In this study, we evaluated the performance of the C/T Etest, a gradient diffusion method. C/T Etest was compared to broth microdilution (BMD) for 51 Enterobacteriaceae challenge isolates and 39 Pseudomonas aeruginosa challenge isolates at three clinical sites. Essential agreement (EA) between the methods ranged from 47 to 49/51 (92.2 to 96.1%) for the Enterobacteriaceae, and categorical agreement (CA) ranged from 49 to 51/51 (96.1 to 100.0%). EA and CA for P. aeruginosa were 100% at all sites. The C/T Etest was also compared to BMD for susceptibility testing on 966 clinical isolates (793 Enterobacteriaceae, including 167 Klebsiella pneumoniae and 159 Escherichia coli isolates, in addition to 173 P. aeruginosa isolates) collected at four clinical sites. EA between Etest and BMD was 96.9% for Enterobacteriaceae isolates and 98.8% for P. aeruginosa isolates. Within the Enterobacteriaceae, isolates from each species examined had >96% CA. For the clinical isolates, no very major errors were identified but two major errors were found (one for K. pneumoniae and one for Providencia rettgeri). By BMD, 47.0% of Enterobacteriaceae and 46.2% of P. aeruginosa challenge strains were nonsusceptible to C/T by CLSI breakpoint criteria; 8.2% of clinical Enterobacteriaceae isolates and 12.1% of clinical P. aeruginosa isolates were nonsusceptible to C/T by CLSI breakpoint criteria. In conclusion, Etest is accurate and reproducible for C/T susceptibility testing of Enterobacteriaceae and P. aeruginosa.


Asunto(s)
Antibacterianos/farmacología , Cefalosporinas/farmacología , Pruebas Antimicrobianas de Difusión por Disco/métodos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Enterobacteriaceae/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Tazobactam/farmacología , Pruebas Antimicrobianas de Difusión por Disco/normas , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Humanos , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Reproducibilidad de los Resultados
18.
J Virol ; 91(4)2017 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-27974564

RESUMEN

Simian arteriviruses are a diverse clade of viruses infecting captive and wild nonhuman primates. We recently reported that Kibale red colobus virus 1 (KRCV-1) causes a mild and self-limiting disease in experimentally infected crab-eating macaques, while simian hemorrhagic fever virus (SHFV) causes lethal viral hemorrhagic fever. Here we characterize how these viruses evolved during replication in cell culture and in experimentally infected macaques. During passage in cell culture, 68 substitutions that were localized in open reading frames (ORFs) likely associated with host cell entry and exit became fixed in the KRCV-1 genome. However, we did not detect any strong signatures of selection during replication in macaques. We uncovered patterns of evolution that were distinct from those observed in surveys of wild red colobus monkeys, suggesting that these species may exert different adaptive challenges for KRCV-1. During SHFV infection, we detected signatures of selection on ORF 5a and on a small subset of sites in the genome. Overall, our data suggest that patterns of evolution differ markedly among simian arteriviruses and among host species. IMPORTANCE: Certain RNA viruses can cross species barriers and cause disease in new hosts. Simian arteriviruses are a diverse group of related viruses that infect captive and wild nonhuman primates, with associated disease severity ranging from apparently asymptomatic infections to severe, viral hemorrhagic fevers. We infected nonhuman primate cell cultures and then crab-eating macaques with either simian hemorrhagic fever virus (SHFV) or Kibale red colobus virus 1 (KRCV-1) and assessed within-host viral evolution. We found that KRCV-1 quickly acquired a large number of substitutions in its genome during replication in cell culture but that evolution in macaques was limited. In contrast, we detected selection focused on SHFV ORFs 5a and 5, which encode putative membrane proteins. These patterns suggest that in addition to diverse pathogenic phenotypes, these viruses may also exhibit distinct patterns of within-host evolution both in vitro and in vivo.


Asunto(s)
Infecciones por Arterivirus/veterinaria , Arterivirus/fisiología , Evolución Biológica , Interacciones Huésped-Patógeno , Enfermedades de los Monos/virología , Animales , Interacciones Huésped-Patógeno/genética , Macaca fascicularis , Enfermedades de los Monos/genética , Sistemas de Lectura Abierta , Polimorfismo de Nucleótido Simple , ARN Viral , Selección Genética , Internalización del Virus , Replicación Viral
19.
J Hepatol ; 77(6): 1711-1713, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-35981935
20.
J Virol ; 90(15): 6724-6737, 2016 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-27170760

RESUMEN

UNLABELLED: Nonhuman primates (NHPs) are a historically important source of zoonotic viruses and are a gold-standard model for research on many human pathogens. However, with the exception of simian immunodeficiency virus (SIV) (family Retroviridae), the blood-borne viruses harbored by these animals in the wild remain incompletely characterized. Here, we report the discovery and characterization of two novel simian pegiviruses (family Flaviviridae) and two novel simian arteriviruses (family Arteriviridae) in wild African green monkeys from Zambia (malbroucks [Chlorocebus cynosuros]) and South Africa (vervet monkeys [Chlorocebus pygerythrus]). We examine several aspects of infection, including viral load, genetic diversity, evolution, and geographic distribution, as well as host factors such as age, sex, and plasma cytokines. In combination with previous efforts to characterize blood-borne RNA viruses in wild primates across sub-Saharan Africa, these discoveries demonstrate that in addition to SIV, simian pegiviruses and simian arteriviruses are widespread and prevalent among many African cercopithecoid (i.e., Old World) monkeys. IMPORTANCE: Primates are an important source of viruses that infect humans and serve as an important laboratory model of human virus infection. Here, we discover two new viruses in African green monkeys from Zambia and South Africa. In combination with previous virus discovery efforts, this finding suggests that these virus types are widespread among African monkeys. Our analysis suggests that one of these virus types, the simian arteriviruses, may have the potential to jump between different primate species and cause disease. In contrast, the other virus type, the pegiviruses, are thought to reduce the disease caused by human immunodeficiency virus (HIV) in humans. However, we did not observe a similar protective effect in SIV-infected African monkeys coinfected with pegiviruses, possibly because SIV causes little to no disease in these hosts.


Asunto(s)
Infecciones por Arterivirus/epidemiología , Evolución Biológica , Infecciones por Flaviviridae/epidemiología , Variación Genética , Infecciones por Lentivirus/epidemiología , Carga Viral , África/epidemiología , Animales , Animales Salvajes , Arterivirus/genética , Arterivirus/patogenicidad , Infecciones por Arterivirus/genética , Infecciones por Arterivirus/virología , Flaviviridae/genética , Flaviviridae/patogenicidad , Infecciones por Flaviviridae/genética , Infecciones por Flaviviridae/virología , Genoma Viral , Haplorrinos , Humanos , Lentivirus/genética , Lentivirus/patogenicidad , Infecciones por Lentivirus/genética , Infecciones por Lentivirus/virología , Filogenia , Prevalencia
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