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1.
Anal Bioanal Chem ; 413(29): 7251-7263, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34622322

RESUMO

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/isolamento & purificação , COVID-19/diagnóstico , Análise Custo-Benefício , Imunoensaio/economia , SARS-CoV-2/isolamento & purificação , Viremia/virologia , Anticorpos Antivirais/sangue , Antígenos Virais/sangue , COVID-19/virologia , Calorimetria , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , SARS-CoV-2/imunologia , Manejo de Espécimes , Espectrometria de Massas em Tandem , Viremia/sangue , Fluxo de Trabalho
2.
J Clin Invest ; 131(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34623326

RESUMO

Primary HIV-1 infection can be classified into six Fiebig stages based on virological and serological laboratory testing, whereas simian-HIV (SHIV) infection in nonhuman primates (NHPs) is defined in time post-infection, making it difficult to extrapolate NHP experiments to the clinics. We identified and extensively characterized the Fiebig-equivalent stages in NHPs challenged intrarectally or intravenously with SHIVAD8-EO. During the first month post-challenge, intrarectally challenged monkeys were up to 1 week delayed in progression through stages. However, regardless of the challenge route, stages I-II predominated before, and stages V-VI predominated after, peak viremia. Decrease in lymph node (LN) CD4+ T cell frequency and rise in CD8+ T cells occurred at stage V. LN virus-specific CD8+ T cell responses, dominated by degranulation and TNF, were first detected at stage V and increased at stage VI. A similar late elevation in follicular CXCR5+ CD8+ T cells occurred, consistent with higher plasma CXCL13 levels at these stages. LN SHIVAD8-EO RNA+ cells were present at stage II, but appeared to decline at stage VI when virions accumulated in follicles. Fiebig-equivalent staging of SHIVAD8-EO infection revealed concordance of immunological events between intrarectal and intravenous infection despite different infection progressions, and can inform comparisons of NHP studies with clinical data.


Assuntos
Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Administração Intravenosa , Administração Retal , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Progressão da Doença , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1 , Humanos , Macaca mulatta , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/classificação , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Fatores de Tempo , Carga Viral , Viremia/imunologia , Viremia/virologia
3.
Cells ; 10(10)2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34685766

RESUMO

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110-183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M's RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development.


Assuntos
Cromatina/metabolismo , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Transcrição Genética , Proteínas da Matriz Viral/metabolismo , Animais , Arginina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , DNA Viral/metabolismo , Modelos Animais de Doenças , Humanos , Lisina/metabolismo , Camundongos Endogâmicos BALB C , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Domínios Proteicos , RNA Viral/metabolismo , Células Vero , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Viremia/virologia
4.
Immunity ; 54(10): 2372-2384.e7, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34496223

RESUMO

Spontaneous control of HIV infection has been repeatedly linked to antiviral CD8+ T cells but is not always permanent. To address mechanisms of durable and aborted control of viremia, we evaluated immunologic and virologic parameters longitudinally among 34 HIV-infected subjects with differential outcomes. Despite sustained recognition of autologous virus, HIV-specific proliferative and cytolytic T cell effector functions became selectively and intrinsically impaired prior to aborted control. Longitudinal transcriptomic profiling of functionally impaired HIV-specific CD8+ T cells revealed altered expression of genes related to activation, cytokine-mediated signaling, and cell cycle regulation, including increased expression of the antiproliferative transcription factor KLF2 but not of genes associated with canonical exhaustion. Lymphoid HIV-specific CD8+ T cells also exhibited poor functionality during aborted control relative to durable control. Our results identify selective functional impairment of HIV-specific CD8+ T cells as prognostic of impending aborted HIV control, with implications for clinical monitoring and immunotherapeutic strategies.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Viremia/imunologia , Viremia/virologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva
5.
PLoS One ; 16(9): e0256865, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34473757

RESUMO

BACKGROUND: In the 21st century, understanding how population migration impacts human health is critical. Namibia has high migration rates and HIV prevalence, but little is known about how these intersect. We examined the association between migration and HIV-related outcomes using data from the 2017 Namibia Population-based HIV Impact Assessment (NAMPHIA). METHODS AND FINDINGS: The NAMPHIA survey selected a nationally representative sample of adults in 2017. All adults aged 15-64 years were invited to complete an interview and home-based HIV test. Recent infection (<130 days) was measured using HIV-1 LAg avidity combined with viral load (>1000 copies/mL) and antiretroviral analyte data. Awareness of HIV status and antiretroviral use were based on self-report and/or detectable antiretrovirals in blood. Viremia was defined as having a viral load ≥1000 copies/mL, including all participants in the denominator regardless of serostatus. We generated community viremia values as a weighted proportion at the EA level, excluding those classified as recently infected. Significant migrants were those who had lived outside their current region or away from home >one month in the past three years. Recent cross-community in-migrants were those who had moved to the community two years ago to determine the association of migration and timing with recent infection or viral load suppression (VLS). All proportions are weighted. Of eligible adults, we had HIV results and migration data on 9,625 (83.9%) of 11,474 women and 7,291 (73.0%) of 9,990 men. Most respondents (62.5%) reported significant migration. Of cross-community in-migrants, 15.3% were recent. HIV prevalence was 12.6% and did not differ by migration status. Population VLS was 77.4%. Recent cross-community in-migration was associated with recent HIV infection (aOR: 4.01, 95% CI 0.99-16.22) after adjusting for community viremia. Significant migration (aOR 0.73, 95% CI: 0.55-0.97) and recent cross-community in-migration (aOR 0.57, 95% CI: 0.35-0.92) were associated with lower VLS, primarily due to lack of awareness of HIV infection. The study was limited by lack of precise data on trajectory of migration. CONCLUSIONS: Despite a high population-level VLS, Namibia still has migrant populations that are not accessing effective treatment for HIV. Targeting migrants with effective prevention and testing programs in communities with viremia could enable further epidemic control.


Assuntos
Antirretrovirais/uso terapêutico , Emigração e Imigração , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , HIV-1/genética , HIV-1/imunologia , Viremia/epidemiologia , Adolescente , Adulto , Estudos Transversais , Características da Família , Feminino , Infecções por HIV/transmissão , Infecções por HIV/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Namíbia/epidemiologia , Prevalência , Autorrelato , Migrantes , Resultado do Tratamento , Carga Viral , Viremia/virologia , Adulto Jovem
6.
J Adv Res ; 32: 37-44, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34484824

RESUMO

Introduction: Dengue and Malaria are the most important mosquito-borne viral diseases affecting humans. Fever is transmitted between human hosts by infected female aedes mosquitoes. The modeling study of viral infections is very useful to show how the virus replicates in an infected individual and how the human antibody response acts to control that replication, which antibody playing a key role in controlling infection. Objectives: Optimal control of a novel variable-order nonlinear model of dengue virus is studied in the present work. Bang-bang control is suggested to minimize the viral infection as well as quick clearance of the virus from the host. Necessary conditions for the control problem are given. The variable-order derivatives are given in the sense of Caputo. Moreover, the parameters of the proposed model are dependent on the same variable-order fractional power. Two numerical schemes are constructed for solving the optimality systems. Comparative studies and numerical simulations are implemented. The variable-order fractional derivative can be describe the effects of long variable memory of time dependent systems than the integer order and fractional order derivatives. Methods: Both the nonstandard generalized fourth order Runge-Kutta and the nonstandard generalized Euler methods are presented. Results: We have successfully applied a kind of Pontryagin's maximum principle with bang-bang control and were able to reduce the viraemia level by adding the dose of DI particles. The nonstandard generalized fourth order Runge-Kutta method has the best results than nonstandard generalized Euler method. Conclusion: The combination of the variable-order fractional derivative and bang-bang control in the Dengue mathematical model improves the dynamics of the model. The nonstandard generalized Euler method and the nonstandard generalized fourth order Runge-Kutta method can be used to study the variable order fractional optimal control problem simply.


Assuntos
Vírus da Dengue , Dengue/virologia , Modelos Teóricos , Aedes , Animais , Feminino , Humanos , Viremia/virologia , Replicação Viral
7.
Sci Rep ; 11(1): 16377, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385528

RESUMO

Foot-and-mouth disease virus (FMDV) is one of the most important animal pathogens in the world. FMDV naturally infects swine, cattle, and other cloven-hoofed animals. FMD is not adequately controlled by vaccination. An alternative strategy is to develop swine that are genetically resistant to infection. Here, we generated FMDV-specific shRNA transgenic cells targeting either nonstructural protein 2B or polymerase 3D of FMDV. The shRNA-positive transgenic cells displayed significantly lower viral production than that of the control cells after infection with FMDV (P < 0.05). Twenty-three transgenic cloned swine (TGCS) and nine non-transgenic cloned swine (Non-TGCS) were produced by somatic cell nuclear transfer (SCNT). In the FMDV challenge study, one TGCS was completely protected, no clinical signs, no viremia and no viral RNA in the tissues, no non-structural antibody response, another one TGCS swine recovered after showing clinical signs for two days, whereas all of the normal control swine (NS) and Non-TGCS developed typical clinical signs, viremia and viral RNA was determined in the tissues, the non-structural antibody was determined, and one Non-TGCS swine died. The viral RNA load in the blood and tissues of the TGCS was reduced in both challenge doses. These results indicated that the TGCS displayed resistance to the FMDV infection. Immune cells, including CD3+, CD4+, CD8+, CD21+, and CD172+ cells, and the production of IFN-γ were analyzed, there were no significant differences observed between the TGCS and NS or Non-TGCS, suggesting that the FMDV resistance may be mainly derived from the RNAi-based antiviral pathway. Our work provides a foundation for a breeding approach to preventing infectious disease in swine.


Assuntos
Resistência à Doença/genética , Febre Aftosa/genética , Febre Aftosa/virologia , RNA Interferente Pequeno/genética , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Suínos/virologia , Animais , Animais Geneticamente Modificados/genética , Vírus da Febre Aftosa/patogenicidade , Interferência de RNA/fisiologia , RNA Viral/genética , Viremia/virologia
8.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424160

RESUMO

Senecavirus A (SVA) is a picornavirus that circulates in swine populations worldwide causing vesicular disease (VD) in affected animals. Here we developed a reverse genetics system for SVA based on the well-characterized wild-type SVA strain SD15-26 (wt SVA SD15-26). The full-length cDNA genome of SVA was cloned into a plasmid under a T7 RNA polymerase promoter. Following in vitro transcription, the genomic viral RNA was transfected into BHK-21 cells and rescue of infectious virus (rSVA SD15-26) was shown by inoculation of highly susceptible H1299 cells. In vitro characterization of the rSVA SD15-26 showed similar replication properties and protein expression levels as the wt SVA SD15-26. A pathogenesis study was conducted in 15-week-old finishing pigs to evaluate the pathogenicity and infection dynamics of the rSVA SD15-26 virus in comparison to the wt SVA SD15-26. Animals from both rSVA- and wt SVA SD15-26-inoculated groups presented characteristic SVA clinical signs (lethargy and lameness) followed by the development of vesicular lesions on the snout and/or feet. The clinical outcome of infection, including disease onset, severity and duration was similar in rSVA- and the wt SVA SD15-26-inoculated animals. All animals inoculated with rSVA or with wt SVA SD15-26 presented a short-term viremia, and animals from both groups shed similar amounts of virus in oral and nasal secretion, and faeces. Our data demonstrates that the rSVA SD5-26 clone is fully virulent and pathogenic in pigs, presenting comparable pathogenesis and infection dynamics to the wt SVA SD15-26 strain. The infectious clone generated here is a useful platform to study virulence determinants of SVA, and to dissect other aspects of SVA infection biology, pathogenesis and persistence.


Assuntos
Infecções por Picornaviridae , Picornaviridae/patogenicidade , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Cricetinae , Humanos , Infecções por Picornaviridae/veterinária , Infecções por Picornaviridae/virologia , Suínos , Viremia/virologia , Virulência
9.
Vet Microbiol ; 261: 109215, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34455356

RESUMO

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that has caused acute egg-drop syndrome in egg-laying ducks. DTMUV nonstructural protein 1 (NS1) contains three potential predicted N-linked glycosylation sites at residues 130, 175 and 207. In this study, we found that mutations at these sites affect the molecular weight of recombinant NS1, as assessed by western blot assays; however, the mutations do not affect their subcellular localization in the cytoplasm, as assessed by colocalization assays. Four recombinant viruses substituting the asparagine (N) residues at N130, N175, N207 or N130/N175/N207 of NS1 with alanine (A) residues were generated using rDTMUV-i, an infectious cDNA clone of the DTMUV CQW1 strain. Deglycosylation assays of the mutant virus NS1 were performed using endoglycosidases Endo H or PNGase F treatment in both mammalian and avian cells. The NS1-WT, NS1-N130A, NS1-N175A and NS1-N207A showed a shift in migration to 37 kDa after digestion with both endoglycosidases, which further confirmed that N130, N175 and N207 were the glycosylation sites of DTMUV NS1. Compared to the parental rDTMUV, the single mutants impaired viral multiplication in vitro, while the nonglycosylated virus rDTMUV-NS1-N130A/N175A/N207A showed a 5-fold to 178-fold decrease in viral titers and smaller plaque sizes. Notably, all mutant viruses were still highly virulent to duck embryos, but the embryos inoculated with rDTMUV-NS1-N130A/N175A/N207A started to die on the fourth day, which exhibited a prolonged time to death compared to that of rDTMUV. Moreover, rDTMUV-NS1-N130A/N175A/N207A was attenuated in vivo, showing no mortality and producing significantly lower viral titers in heart, spleen, kidney, brain and thymus as well as 2-fold to 3-fold lower viremia at 3 and 5 days post infection. Overall, our results indicated that N130, N175 and N207 are N-linked glycosylation sites of DTMUV NS1, which play crucial roles in viral multiplication, viremia and virulence in vitro and in vivo.


Assuntos
Infecções por Flavivirus/veterinária , Flavivirus/genética , Doenças das Aves Domésticas/virologia , Proteínas não Estruturais Virais/metabolismo , Viremia/veterinária , Virulência/genética , Replicação Viral/genética , Animais , Asparagina/genética , Asparagina/metabolismo , Patos , Infecções por Flavivirus/virologia , Glicosilação , Viremia/genética , Viremia/virologia
10.
J Clin Invest ; 131(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34196300

RESUMO

BACKGROUNDSARS-CoV-2 plasma viremia has been associated with severe disease and death in COVID-19 in small-scale cohort studies. The mechanisms behind this association remain elusive.METHODSWe evaluated the relationship between SARS-CoV-2 viremia, disease outcome, and inflammatory and proteomic profiles in a cohort of COVID-19 emergency department participants. SARS-CoV-2 viral load was measured using a quantitative reverse transcription PCR-based platform. Proteomic data were generated with Proximity Extension Assay using the Olink platform.RESULTSThis study included 300 participants with nucleic acid test-confirmed COVID-19. Plasma SARS-CoV-2 viremia levels at the time of presentation predicted adverse disease outcomes, with an adjusted OR of 10.6 (95% CI 4.4-25.5, P < 0.001) for severe disease (mechanical ventilation and/or 28-day mortality) and 3.9 (95% CI 1.5-10.1, P = 0.006) for 28-day mortality. Proteomic analyses revealed prominent proteomic pathways associated with SARS-CoV-2 viremia, including upregulation of SARS-CoV-2 entry factors (ACE2, CTSL, FURIN), heightened markers of tissue damage to the lungs, gastrointestinal tract, and endothelium/vasculature, and alterations in coagulation pathways.CONCLUSIONThese results highlight the cascade of vascular and tissue damage associated with SARS-CoV-2 plasma viremia that underlies its ability to predict COVID-19 disease outcomes.FUNDINGMark and Lisa Schwartz; the National Institutes of Health (U19AI082630); the American Lung Association; the Executive Committee on Research at Massachusetts General Hospital; the Chan Zuckerberg Initiative; Arthur, Sandra, and Sarah Irving for the David P. Ryan, MD, Endowed Chair in Cancer Research; an EMBO Long-Term Fellowship (ALTF 486-2018); a Cancer Research Institute/Bristol Myers Squibb Fellowship (CRI2993); the Harvard Catalyst/Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, NIH awards UL1TR001102 and UL1TR002541-01); and by the Harvard University Center for AIDS Research (National Institute of Allergy and Infectious Diseases, 5P30AI060354).


Assuntos
COVID-19/sangue , COVID-19/virologia , SARS-CoV-2 , Viremia/sangue , Viremia/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Coortes , Feminino , Interações entre Hospedeiro e Microrganismos , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Pandemias , Prognóstico , Proteoma/metabolismo , Proteômica , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Internalização do Vírus
11.
J Med Virol ; 93(12): 6788-6793, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34260081

RESUMO

This study aimed to report a case of mild novel coronavirus disease (COVID-19) in a pregnant woman with probable viremia, as reverse transcription-polymerase chain reaction (RT-PCR) testing of endometrial and placental swabs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was positive. A 26-year-old multigravida at 35 weeks 2 days of gestation, who had extensive thigh and abdominal cellulitis, tested SARS-CoV-2 positive by RT-PCR performed on samples from the endometrium and maternal side of the placenta. However, other samples (amniotic fluid, fetal side of the placenta, umbilical cord, maternal vagina, and neonatal nasopharynx) tested negative for SARS-CoV-2. This is one of the rare reports of probable SARS-CoV-2 viremia with the presence of SARS-CoV-2 in the endometrium and placenta, but not leading to vertical transmission and neonatal infection. Because knowledge about transplacental transmission and results is very limited, we conclude that more RT-PCR tests on placental and cord blood samples are needed in order to safely make definite conclusions.


Assuntos
COVID-19/virologia , Feto/virologia , Placenta/virologia , Complicações Infecciosas na Gravidez/virologia , SARS-CoV-2/genética , Viremia/virologia , Adulto , Feminino , Humanos , Transmissão Vertical de Doenças Infecciosas , Gravidez , Gestantes
12.
Viruses ; 13(5)2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065502

RESUMO

Circoviruses infect vertebrates where they can result in a wide range of disease signs or in asymptomatic infections. Using viral metagenomics we analyzed a pool of five sera from four healthy and one sick horse. Sequences from parvovirus-H, equus anellovirus, and distantly related to mammalian circoviruses were recognized. PCR identified the circovirus reads as originating from a pregnant mare with fever and hepatitis. That horse's serum was also positive by real time PCR for equine parvovirus H and negative for the flavivirus equine hepacivirus. The complete circular genome of equine circovirus 1 strain Charaf (EqCV1-Charaf) was completed using PCR and Sanger sequencing. EqCV1 replicase showed 73-74% identity to those of their closest relatives, pig circoviruses 1/2, and elk circovirus. The closest capsid proteins were from the same ungulate circoviruses with 62-63% identity. The overall nucleotide identity of 72% to its closest relative indicates that EqCV1 is a new species in the Circovirus genus, the first reported in genus Equus. Whether EqCV1 alone or in co-infections can result in disease and its prevalence in different equine populations will require further studies now facilitated using EqCV1's genome sequence.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus , Hepatite Viral Animal/virologia , Doenças dos Cavalos/virologia , Viremia/virologia , Animais , Circovirus/classificação , Circovirus/genética , Genoma Viral , Genômica/métodos , Hepatite Viral Animal/diagnóstico , Doenças dos Cavalos/diagnóstico , Cavalos , Filogenia , Viremia/diagnóstico
13.
AIDS Res Ther ; 18(1): 31, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088307

RESUMO

BACKGROUND: Brescia Province, northern Italy, was one of the worst epicenters of the COVID-19 pandemic. The division of infectious diseases of ASST (Azienda Socio Sanitaria Territoriale) Spedali Civili Hospital of Brescia had to face a great number of inpatients with severe COVID-19 infection and to ensure the continuum of care for almost 4000 outpatients with HIV infection actively followed by us. In a recent manuscript we described the impact of the pandemic on continuum of care in our HIV cohort expressed as number of missed visits, number of new HIV diagnosis, drop in ART (antiretroviral therapy) dispensation and number of hospitalized HIV patients due to SARS-CoV-2 infection. In this short communication, we completed the previous article with data of HIV plasmatic viremia of the same cohort before and during pandemic. METHODS: We considered all HIV-patients in stable ART for at least 6 months and with at least 1 available HIV viremia in the time window March 01-November 30, 2019, and another group of HIV patients with the same two requisites but in different time windows of the COVID-19 period (March 01-May 31, 2020, and June 01-November 30, 2020). For patients with positive viremia (PV) during COVID-19 period, we reported also the values of viral load (VL) just before and after PV. RESULTS: the percentage of patients with PV during COVID-19 period was lower than the previous year (2.8% vs 7%). Only 1% of our outpatients surely suffered from pandemic in term of loss of previous viral suppression. CONCLUSIONS: Our efforts to limit the impact of pandemic on our HIV outpatients were effective to ensure HIV continuum of care.


Assuntos
COVID-19/epidemiologia , Infecções por HIV/epidemiologia , Pandemias , Viremia/epidemiologia , COVID-19/virologia , Estudos de Coortes , Infecções por HIV/virologia , Humanos , Pacientes Internados , Itália/epidemiologia , Pacientes Ambulatoriais , Saúde Pública , SARS-CoV-2/isolamento & purificação , Carga Viral , Viremia/virologia
14.
mBio ; 12(3): e0056021, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34154408

RESUMO

Improved assays are critical to the successful implementation of novel HIV-1 cure strategies, given the limited ability of currently available assays to quantify true effects on the viral reservoir. As interventions based on immune clearance target infected cells producing viral antigens, irrespective of whether the viruses generated are infectious or not, we developed a novel assay to identify viral protein production at the single-cell level. The novel viral protein spot (VIP-SPOT) assay, based on the enzyme-linked ImmunoSpot (ELISpot) approach, quantifies the frequency of CD4+ T cells that produce HIV antigen upon stimulation. The performance of the VIP-SPOT assay was validated in samples from viremic (n = 18) and antiretroviral therapy (ART)-treated subjects (n = 35), and the results were compared with total and intact proviral DNA and plasma viremia. The size of the functional reservoir, measured by VIP-SPOT, correlates with total HIV-1 DNA and, more strongly, with intact proviruses. However, the frequency of HIV antigen-producing cells is 100-fold lower than that of intact proviruses, thus suggesting that most latently infected cells harboring full-length proviruses are not prone to reactivation. Furthermore, VIP-SPOT was useful for evaluating the efficacy of latency reversing agents (LRAs) in primary cells. VIP-SPOT is a novel tool for measuring the size of the functional HIV-1 reservoir in a rapid, sensitive, and precise manner. It might benefit the evaluation of cure strategies based on immune clearance, as these will specifically target this minor fraction of the viral reservoir, and might assist in the identification of novel therapeutic candidates that modulate viral latency. IMPORTANCE Current efforts aimed at finding a definitive cure for HIV-1 infection are hampered mainly by the persistence of a viral reservoir in latently infected cells. While complete viral eradication from the body remains elusive, finding a functional cure to enable control of viremia without the need for continuous treatment is a key goal. As the lower reservoir size increases the likelihood of controlling viremia, new therapeutic strategies aim to reduce the size of this viral reservoir. Evaluating the efficacy of these strategies requires a robust assay to measure the viral reservoir. Currently available options are subject to overestimation or underestimation of the productive reservoir. In order to overcome this limitation, we have developed a novel assay, viral protein spot (VIP-SPOT), to precisely quantify the frequency of infected cells that retain the ability to reactivate and produce viral proteins.


Assuntos
Linfócitos T CD4-Positivos/virologia , Reservatórios de Doenças/virologia , ELISPOT/métodos , HIV-1/fisiologia , Carga Viral/métodos , Proteínas Virais/análise , Antirretrovirais/uso terapêutico , DNA Viral/genética , ELISPOT/normas , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/química , Humanos , Provírus/genética , Estudos Retrospectivos , Análise de Célula Única/métodos , Viremia/virologia , Latência Viral
15.
J Virol ; 95(18): e0026821, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34190597

RESUMO

Preventing human immunodeficiency virus (HIV) infection in newborns by vertical transmission remains an important unmet medical need in resource-poor areas where antiretroviral therapy (ART) is not available and mothers and infants cannot be treated prepartum or during the breastfeeding period. In the present study, the protective efficacy of the potent HIV-neutralizing antibodies PGT121 and VRC07-523, both produced in plants, were assessed in a multiple-SHIV (simian-human immunodeficiency virus)-challenge breastfeeding macaque model. Newborn macaques received either six weekly subcutaneous injections with PGT121 alone or as a cocktail of PGT121-LS plus VRC07-523-LS injected three times every 2 weeks. Viral challenge with SHIVSF162P3 was twice weekly over 5.5 weeks using 11 exposures. Despite the transient presence of plasma viral RNA either immediately after the first challenge or as single-point blips, the antibodies prevented a productive infection in all babies with no sustained plasma viremia, compared to viral loads ranging from 103 to 5 × 108 virions/ml in four untreated controls. No virus was detected in peripheral blood mononuclear cells (PBMCs), and only 3 of 159 tissue samples were weakly positive in the treated babies. Newborn macaques proved to be immunocompetent, producing transient anti-Env antibodies and anti-drug antibody (ADA), which were maintained in the circulation after passive broadly neutralizing antibody clearance. ADA responses were directed to the IgG1 Fc CH2-CH3 domains, which has not been observed to date in adult monkeys passively treated with PGT121 or VRC01. In addition, high levels of VRC07-523 anti-idiotypic antibodies in the circulation of one newborn was concomitant with the rapid elimination of VRC07. Plant-expressed antibodies show promise as passive immunoprophylaxis in a breastfeeding model in newborns. IMPORTANCE Plant-produced human neutralizing antibody prophylaxis is highly effective in preventing infection in newborn monkeys during repeated oral exposure, modeling virus in breastmilk, and offers advantages in cost of production and safety. These findings raise the possibility that anti-Env antibodies may contribute to the control of viral replication in this newborn model and that the observed immune responsiveness may be driven by the long-lived presence of immune complexes.


Assuntos
Aleitamento Materno , Anticorpos Amplamente Neutralizantes/imunologia , HIV-1/fisiologia , Imunização Passiva/métodos , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Vírus da Imunodeficiência Símia/imunologia , Tabaco/imunologia , Animais , Animais Recém-Nascidos , Feminino , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Macaca mulatta , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Tabaco/virologia , Viremia/imunologia , Viremia/terapia , Viremia/virologia
16.
Nat Commun ; 12(1): 3727, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140517

RESUMO

Clonal expansion of HIV-infected cells contributes to the long-term persistence of the HIV reservoir in ART-suppressed individuals. However, the contribution from cell clones that harbor inducible proviruses to plasma viremia is poorly understood. Here, we describe a single-cell approach to simultaneously sequence the TCR, integration sites and proviral genomes from translation-competent reservoir cells, called STIP-Seq. By applying this approach to blood samples from eight participants, we show that the translation-competent reservoir mainly consists of proviruses with short deletions at the 5'-end of the genome, often involving the major splice donor site. TCR and integration site sequencing reveal that cell clones with predicted pathogen-specificity can harbor inducible proviruses integrated into cancer-related genes. Furthermore, we find several matches between proviruses retrieved with STIP-Seq and plasma viruses obtained during ART and upon treatment interruption, suggesting that STIP-Seq can capture clones that are responsible for low-level viremia or viral rebound.


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Provírus/genética , Análise de Célula Única/métodos , Viremia/virologia , Linfócitos T CD4-Positivos/virologia , DNA Viral/sangue , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Ionomicina/farmacologia , Masculino , Pessoa de Meia-Idade , Filogenia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Deleção de Sequência , Carga Viral/genética
17.
Sci Rep ; 11(1): 13134, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162948

RESUMO

COVID-19 has overloaded national health services worldwide. Thus, early identification of patients at risk of poor outcomes is critical. Our objective was to analyse SARS-CoV-2 RNA detection in serum as a severity biomarker in COVID-19. Retrospective observational study including 193 patients admitted for COVID-19. Detection of SARS-CoV-2 RNA in serum (viremia) was performed with samples collected at 48-72 h of admission by two techniques from Roche and Thermo Fischer Scientific (TFS). Main outcome variables were mortality and need for ICU admission during hospitalization for COVID-19. Viremia was detected in 50-60% of patients depending on technique. The correlation of Ct in serum between both techniques was good (intraclass correlation coefficient: 0.612; p < 0.001). Patients with viremia were older (p = 0.006), had poorer baseline oxygenation (PaO2/FiO2; p < 0.001), more severe lymphopenia (p < 0.001) and higher LDH (p < 0.001), IL-6 (p = 0.021), C-reactive protein (CRP; p = 0.022) and procalcitonin (p = 0.002) serum levels. We defined "relevant viremia" when detection Ct was < 34 with Roche and < 31 for TFS. These thresholds had 95% sensitivity and 35% specificity. Relevant viremia predicted death during hospitalization (OR 9.2 [3.8-22.6] for Roche, OR 10.3 [3.6-29.3] for TFS; p < 0.001). Cox regression models, adjusted by age, sex and Charlson index, identified increased LDH serum levels and relevant viremia (HR = 9.87 [4.13-23.57] for TFS viremia and HR = 7.09 [3.3-14.82] for Roche viremia) as the best markers to predict mortality. Viremia assessment at admission is the most useful biomarker for predicting mortality in COVID-19 patients. Viremia is highly reproducible with two different techniques (TFS and Roche), has a good consistency with other severity biomarkers for COVID-19 and better predictive accuracy.


Assuntos
COVID-19/sangue , RNA Viral/sangue , SARS-CoV-2/genética , Viremia/sangue , Idoso , Biomarcadores/sangue , COVID-19/mortalidade , COVID-19/virologia , Cuidados Críticos , Feminino , Hospitalização , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Gravidade do Paciente , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Fatores de Risco , Espanha , Viremia/virologia
18.
Sci Rep ; 11(1): 12354, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117329

RESUMO

Dengue virus (DENV) causes dengue fever (DF) and dengue hemorrhagic fever in humans. Some DF patients suddenly develop severe symptoms around the defervescent period. Although the pathogenic mechanism of the severe symptoms has not been fully elucidated, the viremia level in the early phase has been shown to correlate with the disease severity. One of the hypotheses is that a phenomenon called antibody-dependent enhancement (ADE) of infection leads to high level of viremia. To examine the plausibility of this hypothesis, we examined the relationship between in vitro ADE activity and in vivo viral load quantity in six patients with dengue diseases. Blood samples were collected at multiple time points between the acute and defervescent phases, and the balance between neutralizing and enhancing activities against the autologous and prototype viruses was examined. As the antibody levels against DENV were rapidly increased, ADE activity was decreased over time or partially maintained against some viruses at low serum dilution. In addition, positive correlations were observed between ADE activity representing in vitro progeny virus production and viremia levels in patient plasma samples. The measurement of ADE activity in dengue-seropositive samples may help to predict the level of viral load in the subsequent DENV infection.


Assuntos
Anticorpos Antivirais/imunologia , Dengue/virologia , Carga Viral , Viremia/virologia , Animais , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Dengue/sangue , Dengue/imunologia , Vírus da Dengue/imunologia , Humanos , Células K562 , Células Vero , Viremia/imunologia
19.
Sci Rep ; 11(1): 10946, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34040071

RESUMO

Avian leukosis virus subgroup J (ALV-J) causes oncogenic disease in chickens in China, resulting in great harm to poultry production, and remains widespread in China. Herein, we employed a cross-priming amplification (CPA) approach and a nucleic acid detection device to establish a visual rapid detection method for ALV-J. The sensitivity of CPA, polymerase chain reaction (PCR) and real-time PCR (RT-PCR) was compared, and the three methods were used to detect ALV-J in the cell cultures which inoculated with clinical plasma. The result showed when the amplification reaction was carried out at 60 °C for just 60 min, the sensitivity of CPA was 10 times higher than conventional PCR, with high specificity, which was comparable with RT-PCR, based on detection of 123 cell cultures which inoculated with clinical plasma, the coincidence rate with real-time PCR was 97.3% (71/73). CPA detection of ALV-J does not require an expensive PCR instrument; a simple water bath or incubator is sufficient for complete DNA amplification, and the closed nucleic acid detection device avoids aerosol pollution, making judgment of results more intuitive and objective. The CPA assay would be a promising simple, rapid and sensitive method for identification of ALV-J.


Assuntos
Vírus da Leucose Aviária/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Vírus da Leucose Aviária/classificação , Vírus da Leucose Aviária/genética , Biotinilação , Células Cultivadas , Galinhas/virologia , Primers do DNA , Eletroforese em Gel de Ágar , Fluoresceína-5-Isotiocianato/análise , Ouro , Nanopartículas Metálicas , Reação em Cadeia da Polimerase/métodos , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/virologia , RNA Viral/genética , Fitas Reagentes , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Temperatura , Infecções Tumorais por Vírus/diagnóstico , Infecções Tumorais por Vírus/veterinária , Infecções Tumorais por Vírus/virologia , Viremia/diagnóstico , Viremia/veterinária , Viremia/virologia
20.
J Med Virol ; 93(9): 5452-5457, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33969515

RESUMO

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is generally detected in nasopharyngeal swabs, viral RNA can be found in other samples including blood. Recently, associations between SARS-CoV-2 RNAaemia and disease severity and mortality have been reported in adults, while no reports are available in pediatric patients with coronavirus disease 2019 (COVID-19). The aim of this study was to evaluate the mortality, severity, clinical, and laboratory findings of SARS-CoV-2 RNA detection in blood in 96 pediatric patients with confirmed COVID-19. Among all patients, 6 (6%) had SARS-CoV-2 RNAaemia. Out of the six patients with SARS-CoV-2 RNAaemia, four (67%) had a severe form of the disease, and two out of the 6 patients with SARS-CoV-2 RNAaemia passed away (33%). Our results show that the symptoms more commonly found in the cases of COVID-19 in the study (fever, cough, tachypnea, and vomiting), were found at a higher percentage in the patients with SARS-CoV-2 RNAaemia. Creatine phosphokinase and magnesium tests showed significant differences between the positive and negative SARS-CoV-2 RNAaemia groups. Among all laboratory tests, magnesium and creatine phosphokinase could better predict SARS-CoV-2 RNAemia with area under the curve  levels of 0.808 and 0.748, respectively. In conclusion, 67% of individuals with SARS-CoV-2 RNAaemia showed a severe COVID-19 and one-third of the patients with SARS-CoV-2 RNAaemia passed away. Our findings suggest that magnesium and creatine phosphokinase might be considered as markers to estimate the SARS-CoV-2 RNAaemia.


Assuntos
COVID-19/patologia , Creatina Quinase/sangue , Magnésio/sangue , RNA Viral/sangue , SARS-CoV-2/patogenicidade , Viremia/patologia , Adolescente , Biomarcadores/sangue , COVID-19/diagnóstico , COVID-19/mortalidade , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Criança , Pré-Escolar , Tosse/diagnóstico , Tosse/mortalidade , Tosse/patologia , Tosse/virologia , Feminino , Febre/diagnóstico , Febre/mortalidade , Febre/patologia , Febre/virologia , Hospitais , Humanos , Lactente , Recém-Nascido , Irã (Geográfico) , Masculino , RNA Viral/genética , SARS-CoV-2/genética , Índice de Gravidade de Doença , Análise de Sobrevida , Taquipneia/diagnóstico , Taquipneia/mortalidade , Taquipneia/patologia , Taquipneia/virologia , Viremia/diagnóstico , Viremia/mortalidade , Viremia/virologia
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