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1.
Med Mol Morphol ; 57(2): 124-135, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38393367

RESUMO

In this study, we analyzed the morphological structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human cells. We identified the two types of viral particles present within the vacuoles of infected cells. Using transmission electron microscopy, we observed that SARS-CoV-2 particles exhibited both low- and high-electron-density structures, which was further confirmed through three-dimensional reconstruction using electron tomography. The budding of these particles was exclusively observed within these vacuoles. Intriguingly, viral particles with low-electron-density structures were confined to vacuoles, whereas those with high-electron-density structures were found in vacuoles and on the cell membrane surface of infected cells. Notably, high-electron-density particles found within vacuoles exhibited the same morphology as those outside the infected cells. This observation suggests that the two types of viral particles identified in this study had different maturation status. Our findings provide valuable insights into the molecular details of SARS-CoV-2 particles, contributing to our understanding of the virus.


Assuntos
COVID-19 , Tomografia com Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , SARS-CoV-2 , Vacúolos , Vírion , Humanos , SARS-CoV-2/ultraestrutura , SARS-CoV-2/fisiologia , Vacúolos/ultraestrutura , Vacúolos/virologia , Vírion/ultraestrutura , COVID-19/virologia , COVID-19/patologia , Imageamento Tridimensional , Chlorocebus aethiops , Células Vero
2.
Mol Microbiol ; 121(4): 671-678, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37700704

RESUMO

Bunyavirales constitute the largest order of enveloped RNA viruses, many members of which cause severe diseases in humans and domestic animals. In recent decades, innovative fluorescence-based methods have paved the way to visualize and track single fluorescent bunyaviral particles in fixed and live cells. This technological breakthrough has enabled imaging of the early stages of infection and the quantification of every step in the bunyavirus cell entry process. Here, we describe the latest procedures for rendering bunyaviral particles fluorescent and discuss the advantages and disadvantages of each approach in light of the most recent advances in fluorescence detection and monitoring of bunyavirus entry. In this mini-review, we also illustrate how fluorescent viral particles are a powerful tool for deciphering the cellular entry process of bunyaviruses, the vast majority of which have not yet been analyzed.


Assuntos
Orthobunyavirus , Vírus de RNA , Animais , Humanos , Fluorescência , Internalização do Vírus
3.
Microbiol Spectr ; 12(1): e0453422, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38051228

RESUMO

IMPORTANCE: The survival of the sinking prokaryotes and viruses in the deep-sea environment is crucial for deep-sea ecosystems and biogeochemical cycles. Through an in situ deep-sea long-term incubation device, our results showed that viral particles and infectivity had still not decayed completely after in situ incubation for 1 year. This suggests that, via infection and lysis, surface viruses with long-term infectious activity in situ deep-sea environments may influence deep-sea microbial populations in terms of activity, function, diversity, and community structure and ultimately affect deep-sea biogeochemical cycles, highlighting the need for additional research in this area.


Assuntos
Bacteriófagos , Vírus , Bacteriófagos/genética , Água do Mar , Ecossistema
4.
Int J Mol Sci ; 24(20)2023 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-37894953

RESUMO

Zinc, an essential trace element that serves as a cofactor for numerous cellular and viral proteins, plays a central role in the dynamics of HIV-1 infection. Among the viral proteins, the nucleocapsid NCp7, which contains two zinc finger motifs, is abundantly present viral particles and plays a crucial role in coating HIV-1 genomic RNA, thus concentrating zinc within virions. In this study, we investigated whether HIV-1 virus production impacts cellular zinc homeostasis and whether isotopic fractionation occurs between the growth medium, the producing cells, and the viral particles. We found that HIV-1 captures a significant proportion of cellular zinc in the neo-produced particles. Furthermore, as cells grow, they accumulate lighter zinc isotopes from the medium, resulting in a concentration of heavier isotopes in the media, and the viruses exhibit a similar isotopic fractionation to the producing cells. Moreover, we generated HIV-1 particles in HEK293T cells enriched with each of the five zinc isotopes to assess the potential effects on the structure and infectivity of the viruses. As no strong difference was observed between the HIV-1 particles produced in the various conditions, we have demonstrated that enriched isotopes can be accurately used in future studies to trace the fate of zinc in cells infected by HIV-1 particles. Comprehending the mechanisms underlying zinc absorption by HIV-1 viral particles offers the potential to provide insights for developing future treatments aimed at addressing this specific facet of the virus's life cycle.


Assuntos
HIV-1 , Humanos , HIV-1/metabolismo , Sequência de Aminoácidos , Células HEK293 , Proteínas Virais/metabolismo , Vírion/metabolismo , RNA/metabolismo , Zinco/metabolismo , Isótopos de Zinco/metabolismo , Isótopos/metabolismo , Dedos de Zinco
5.
Int J Mol Sci ; 24(10)2023 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-37239849

RESUMO

The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization.


Assuntos
Proteínas do Capsídeo , Dependovirus , Proteínas do Capsídeo/genética , Dependovirus/genética , Dependovirus/metabolismo , Cromatografia Líquida , Espectrometria de Massas , Proteínas Virais , Cromatografia de Fase Reversa , Dodecilsulfato de Sódio/química , Eletroforese Capilar/métodos
6.
Anal Bioanal Chem ; 415(18): 4209-4220, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37014373

RESUMO

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.


Assuntos
Capsídeo , Elétrons , Simulação por Computador , Síncrotrons , Raios X
7.
Int J Mol Sci ; 23(24)2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36555262

RESUMO

Self-replicating RNA viruses have become attractive delivery vehicles for therapeutic applications. They are easy to handle, can be rapidly produced in large quantities, and can be delivered as recombinant viral particles, naked or nanoparticle-encapsulated RNA, or plasmid DNA-based vectors. The self-replication of RNA in infected host cells provides the means for generating much higher transgene expression levels and the possibility to apply substantially reduced amounts of RNA to achieve similar expression levels or immune responses compared to conventional synthetic mRNA. Alphaviruses and flaviviruses, possessing a single-stranded RNA genome of positive polarity, as well as measles viruses and rhabdoviruses with a negative-stranded RNA genome, have frequently been utilized for therapeutic applications. Both naturally and engineered oncolytic self-replicating RNA viruses providing specific replication in tumor cells have been evaluated for cancer therapy. Therapeutic efficacy has been demonstrated in animal models. Furthermore, the safe application of oncolytic viruses has been confirmed in clinical trials. Multiple myeloma patients treated with an oncolytic measles virus (MV-NIS) resulted in increased T-cell responses against the measles virus and several tumor-associated antigen responses and complete remission in one patient. Furthermore, MV-CEA administration to patients with ovarian cancer resulted in a stable disease and more than doubled the median overall survival.


Assuntos
Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Neoplasias Ovarianas , Vírus de RNA , Humanos , Animais , Feminino , Vírus Oncolíticos/genética , RNA , Vírus de RNA/genética , Neoplasias Ovarianas/terapia , Vírus do Sarampo/genética , Terapia Viral Oncolítica/métodos , Neoplasias/terapia
8.
ACS Sens ; 7(11): 3560-3570, 2022 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-36382569

RESUMO

Current tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detect either the constituent nucleic acids/proteins of the viral particles or antibodies specific to the virus, but cannot provide information about viral neutralization by an antibody and the efficacy of an antibody. Such information is important about individuals' vulnerability to severe symptoms or their likelihood of showing no symptoms. We immobilized online SARS-CoV-2 spike (S1) protein and angiotensin-converting enzyme 2 (ACE2) into separate surface plasmon resonance (SPR) channels of a tris-nitrilotriacetic acid (tris-NTA) chip to simultaneously detect the anti-S1 antibody and viral particles in serum samples. In addition, with a high-molecular-weight-cutoff filter, we separated the neutralized viral particles from the free antibody molecules and used a sensing channel immobilized with Protein G to determine antibody-neutralized viral particles. The optimal density of probe molecules in each fluidic channel can be precisely controlled through the closure and opening of the specific ports. By utilizing the high surface density of ACE2, multiple assays can be carried out without regenerations. These three species can be determined with a short analysis time (<12 min per assay) and excellent sensor-to-sensor/cycle-to-cycle reproducibility (RSD < 5%). When coupled with an autosampler, continuous assays can be performed in an unattended manner at a single chip for up to 6 days. Such a sensor capable of assaying serum samples containing the three species at different levels provides additional insights into the disease status and immunity of persons being tested, which should be helpful for containing the SARS-CoV-2 spread during the era of incessant viral mutations.


Assuntos
COVID-19 , SARS-CoV-2 , Ressonância de Plasmônio de Superfície , Humanos , Enzima de Conversão de Angiotensina 2 , Anticorpos Antivirais , COVID-19/diagnóstico , Reprodutibilidade dos Testes , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus , Vírion/isolamento & purificação
9.
Viruses ; 14(9)2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-36146692

RESUMO

SARS-CoV-2 is a virus that belongs to the Betacoronavirus genus of the Coronaviridae family. Other coronaviruses, such as SARS-CoV and MERS-CoV, were associated with complications in pregnant women. Therefore, this study aimed to report the clinical history of five pregnant women infected with SARS-CoV-2 (four symptomatic and one asymptomatic who gave birth to a stillborn child) during the COVID-19 pandemic. They gave birth between August 2020 to January 2021, a period in which there was still no vaccination for COVID-19 in Brazil. In addition, their placental alterations were later investigated, focusing on macroscopic, histopathological, and ultrastructural aspects compared to a prepandemic sample. Three of five placentas presented SARS-CoV-2 RNA detected by RT-PCRq at least two to twenty weeks after primary pregnancy infection symptoms, and SARS-CoV-2 spike protein was detected in all placentas by immunoperoxidase assay. The macroscopic evaluation of the placentas presented congested vascular trunks, massive deposition of fibrin, areas of infarctions, and calcifications. Histopathological analysis showed fibrin deposition, inflammatory infiltrate, necrosis, and blood vessel thrombosis. Ultrastructural aspects of the infected placentas showed a similar pattern of alterations between the samples, with predominant characteristics of apoptosis and detection of virus-like particles. These findings contribute to a better understanding of the consequences of SARS-CoV-2 infection in placental tissue, vertical transmission.


Assuntos
COVID-19 , Complicações Infecciosas na Gravidez , Feminino , Fibrina , Humanos , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas , Pandemias , Placenta , Gravidez , RNA Viral , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus
10.
Front Vet Sci ; 9: 870009, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35615248

RESUMO

African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family that damages the immune system of pigs, resulting in the death or slaughter of millions of animals worldwide. Recent modern techniques in ASFV vaccination have highlighted the potential of viral replicon particles (RPs), which can efficiently express foreign proteins and induce robust cellular and humoral immune responses compared with the existing vaccines. In this study, we established a Semliki Forest virus (SFV) vector by producing replication-defective viral particles. This vector was used to deliver RPs expressing ASFV antigens. SFV-RPs expressing ASFV p32 (SFV-p32) and p54 (SFV-p54) were tested in baby hamster kidney (BHK-21) cells. Proteins expression was evaluated via western blotting and indirect immunofluorescence, while immunogenicity was evaluated in BALB/c mice. The resulting RPs exhibited high levels of protein expression and elicited robust humoral and cellular immune responses.

11.
Aquat Toxicol ; 245: 106104, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35176694

RESUMO

There have been significant impacts of the current COVID-19 pandemic on society including high health and economic costs. However, little is known about the potential ecological risks of this virus despite its presence in freshwater systems. In this study, we aimed to evaluate the exposure of Poecilia reticulata juveniles to two peptides derived from Spike protein of SARS-CoV-2, which was synthesized in the laboratory (named PSPD-2002 and PSPD-2003). For this, the animals were exposed for 35 days to the peptides at a concentration of 40 µg/L and different toxicity biomarkers were assessed. Our data indicated that the peptides were able to induce anxiety-like behavior in the open field test and increased acetylcholinesterase (AChE) activity. The biometric evaluation also revealed that the animals exposed to the peptides displayed alterations in the pattern of growth/development. Furthermore, the increased activity of superoxide dismutase (SOD) and catalase (CAT) enzymes were accompanied by increased levels of malondialdehyde (MDA), reactive oxygen species (ROS) and hydrogen peroxide (H2O2), which suggests a redox imbalance induced by SARS-CoV-2 spike protein peptides. Moreover, molecular docking analysis suggested a strong interaction of the peptides with the enzymes AChE, SOD and CAT, allowing us to infer that the observed effects are related to the direct action of the peptides on the functionality of these enzymes. Consequently, our study provided evidence that the presence of SARS-CoV-2 viral particles in the freshwater ecosystems offer a health risk to fish and other aquatic organisms.


Assuntos
COVID-19 , Poecilia , Poluentes Químicos da Água , Acetilcolinesterase/metabolismo , Animais , Catalase/metabolismo , Ecossistema , Humanos , Peróxido de Hidrogênio , Simulação de Acoplamento Molecular , Pandemias , Poecilia/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Superóxido Dismutase/metabolismo , Poluentes Químicos da Água/toxicidade
12.
Sci Total Environ ; 825: 153988, 2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35192827

RESUMO

The identification of SARS-CoV-2 particles in wastewater and freshwater ecosystems has raised concerns about its possible impacts on non-target aquatic organisms. In this particular, our knowledge of such impacts is still limited, and little attention has been given to this issue. Hence, in our study, we aimed to evaluate the possible induction of mutagenic (via micronucleus test) and genotoxic (via single cell gel electrophoresis assay, comet assay) effects in Poecilia reticulata adults exposed to fragments of the Spike protein of the new coronavirus at the level of 40 µg/L, denominated PSPD-2002. As a result, after 10 days of exposure, we have found that animals exposed to the peptides demonstrated an increase in the frequency of erythrocytic nuclear alteration (ENA) and all parameters assessed in the comet assay (length tail, %DNA in tail and Olive tail moment), suggesting that PSPD-2002 peptides were able to cause genomic instability and erythrocyte DNA damage. Besides, these effects were significantly correlated with the increase in lipid peroxidation processes [inferred by the high levels of malondialdehyde (MDA)] reported in the brain and liver of P. reticulata and with the reduction of the superoxide dismutase (SOD) and catalase (CAT) activity. Thus, our study constitutes a new insight and promising investigation into the toxicity associated with the dispersal of SARS-CoV-2 peptide fragments in freshwater environments.


Assuntos
COVID-19 , Poecilia , Poluentes Químicos da Água , Animais , Ensaio Cometa , Dano ao DNA , Ecossistema , Instabilidade Genômica , Humanos , Pandemias , Peptídeos , SARS-CoV-2 , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidade
13.
Front Bioeng Biotechnol ; 9: 753480, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34805110

RESUMO

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.

14.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638527

RESUMO

The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.


Assuntos
COVID-19/virologia , Clonagem Molecular/métodos , Genoma Viral , SARS-CoV-2/genética , Animais , Chlorocebus aethiops , Células HEK293 , Humanos , Mutagênese , Plasmídeos/genética , Recombinação Genética , Células Vero
15.
Respiration ; 100(12): 1196-1207, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34537778

RESUMO

BACKGROUND: Various forms of noninvasive respiratory support methods are used in the treatment of hypoxemic CO-VID-19 patients, but limited data are available about the corresponding respiratory droplet dispersion. OBJECTIVES: The aim of this study was to estimate the potential spread of infectious diseases for a broad selection of oxygen and respiratory support methods by revealing the therapy-induced aerodynamics and respiratory droplet dispersion. METHODS: The exhaled air-smoke plume from a 3D-printed upper airway geometry was visualized by recording light reflection during simulated spontaneous breathing, standard oxygen mask application, nasal high-flow therapy (NHFT), continuous positive airway pressure (CPAP), and bilevel positive airway pressure (BiPAP). The dispersion of 100 µm particles was estimated from the initial velocity of exhaled air and the theoretical terminal velocity. RESULTS: Estimated droplet dispersion was 16 cm for unassisted breathing, 10 cm for Venturi masks, 13 cm for the nebulizer, and 14 cm for the nonrebreathing mask. Estimated droplet spread increased up to 34 cm in NHFT, 57 cm in BiPAP, and 69 cm in CPAP. A nonsurgical face mask over the NHFT interface reduced estimated droplet dispersion. CONCLUSIONS: During NHFT and CPAP/BiPAP with vented masks, extensive jets with relatively high jet velocities were observed, indicating increased droplet spread and an increased risk of droplet-driven virus transmission. For the Venturi masks, a nonrebreathing mask, and a nebulizer, estimated jet velocities are comparable to unassisted breathing. Aerosols are transported unboundedly in all these unfiltered therapies. The adequate use of protective measures is of vital importance when using noninvasive unfiltered therapies in infectious respiratory diseases.


Assuntos
Movimentos do Ar , Expiração , Modelos Biológicos , Ventilação não Invasiva , Aerossóis e Gotículas Respiratórios , Humanos
16.
mSphere ; 6(5): e0053721, 2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34550005

RESUMO

Defective interfering (DI) genomes restrict viral replication and induce type I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity, and determine their long-term effects. To address this, we generated a recombinant canine distemper virus (rCDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques, and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate that these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues, which are a major site of CDV replication. Our established pipeline for detection, generation, and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome. IMPORTANCE Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes might be harnessed therapeutically. Negative-strand RNA virus research has provided a rich pool of natural DI genomes over many years, and they are probably the best understood in vitro. Here, we demonstrate the identification, synthesis, production, and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.


Assuntos
Vírus da Cinomose Canina/genética , Vírus da Cinomose Canina/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Vírus Defeituosos , Cães , Furões , Genoma Viral , Masculino , Guaxinins/virologia , Células Vero , Replicação Viral/genética , Replicação Viral/fisiologia
17.
Front Immunol ; 12: 661379, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34108964

RESUMO

Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemagglutinin-pseudotypes (IBV PV) using plasmid-directed transfection. To activate influenza B hemagglutinin, we have explored the use of proteases in increasing PV titers via their co-transfection during pseudotype virus production. When tested for their ability to transduce target cells, the influenza B pseudotypes produced exhibit tropism for different cell lines. The pseudotypes were evaluated as alternatives to live virus in microneutralization assays using reference sera standards, mouse and human sera collected during vaccine immunogenicity studies, surveillance sera from seals, and monoclonal antibodies (mAbs) against IBV. The influenza B pseudotype pMN was found to effectively detect neutralizing and cross-reactive responses in all assays and shows promise as an effective and versatile tool in influenza research.


Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunogenicidade da Vacina/imunologia , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Lentivirus/imunologia , Células A549 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Cães , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/classificação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza B/genética , Vírus da Influenza B/fisiologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Lentivirus/genética , Células Madin Darby de Rim Canino , Testes de Neutralização/métodos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Vacinação , Potência de Vacina
18.
Anal Bioanal Chem ; 413(29): 7265-7275, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34013402

RESUMO

COVID-19 is the most disturbing pandemic of the past hundred years. Its causative agent, the SARS-CoV-2 virus, has been the subject of an unprecedented investigation to characterize its molecular structure and intimate functioning. While markers for its detection have been proposed and several diagnostic methodologies developed, its propensity to evolve and evade diagnostic tools and the immune response is of great concern. The recent spread of new variants with increased infectivity requires even more attention. Here, we document how shotgun proteomics can be useful for rapidly monitoring the evolution of the SARS-CoV-2 virus. We evaluated the heterogeneity of purified SARS-CoV-2 virus obtained after culturing in the Vero E6 cell line. We found that cell culture induces significant changes that are translated at the protein level, such changes being detectable by tandem mass spectrometry. Production of viral particles requires careful quality control which can be easily performed by shotgun proteomics. Although considered relatively stable so far, the SARS-CoV-2 genome turns out to be prone to frequent variations. Therefore, the sequencing of SARS-CoV-2 variants from patients reporting only the consensus genome after its amplification would deserve more attention and could benefit from more in-depth analysis of low level but crystal-clear signals, as well as complementary and rapid analysis by shotgun proteomics.


Assuntos
Genoma Viral , Proteômica/métodos , SARS-CoV-2/isolamento & purificação , Sequência de Aminoácidos , Técnicas de Cultura de Células , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Espectrometria de Massas em Tandem/métodos , Proteínas Virais/química , Virulência
19.
Sci Total Environ ; 785: 147300, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33940414

RESUMO

The transmission of SARS-CoV-2 virus through aerosols has become an outstanding issue, where plenty of spread aspects are being analyzed. Portable Air Cleaners (PAC) with high-efficiency particulate air (HEPA) filters have been discussed as an adjunctive means for indoor environments coronavirus decontamination. This study evaluates, first, the air and surfaces SARS-COV-2 RNA contamination due to positive patients in households, and second, the efficiency of a PAC with HEPA filter to eliminate virus. A total of 29 air and surface samples were collected inside 9 households, by using an air portable collector with gelatin filters and swabs. SARS-CoV-2 RNA detection was performed using real-time reverse transcription polymerase chain reaction (RT-PCR). Overall, all the air samples collected before using PAC and 75% of swab samples were positive for SARS-CoV-2. After the PAC usage, all samples except one were negative, displaying a 80% device effectiveness. Portable HEPA cleaners usage allowed the removal of SARS CoV-2 and, therefore, they could be recommended for places with inadequate ventilation, considering the limitations and functionality of the device.


Assuntos
Filtros de Ar , COVID-19 , Ar Condicionado , Humanos , RNA Viral , SARS-CoV-2
20.
Curr Opin Colloid Interface Sci ; 52: 101417, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33642918

RESUMO

Since the outbreak of the COVID-19 pandemic, most countries have recommended their citizens to adopt social distance, hand hygiene, and face mask wearing. However, wearing face masks has not been well adopted by many citizens. While the reasons are complex, there is a general perception that the evidence to support face mask wearing is lacking, especially for the general public in a community setting. Face mask wearing can block or filter airborne virus-carrying particles through the working of colloid and interface science. This paper assesses current knowledge behind the design and functioning of face masks by reviewing the selection of materials, mask specifications, relevant laboratory tests, and respiratory virus transmission trials, with an overview of future development of reusable masks for the general public. This review highlights the effectiveness of face mask wearing in the prevention of COVID-19 infection.

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