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1.
Sci Rep ; 11(1): 18085, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508172

RESUMO

Effective vaccines are slowing the COVID-19 pandemic, but SARS-CoV-2 will likely remain an issue in the future making it important to have therapeutics to treat patients. There are few options for treating patients with COVID-19. We show probenecid potently blocks SARS-CoV-2 replication in mammalian cells and virus replication in a hamster model. Furthermore, we demonstrate that plasma concentrations up to 50-fold higher than the protein binding adjusted IC90 value are achievable for 24 h following a single oral dose. These data support the potential clinical utility of probenecid to control SARS-CoV-2 infection in humans.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Probenecid/farmacologia , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Células Epiteliais/virologia , Humanos , Pulmão/virologia , Células Vero
2.
Signal Transduct Target Ther ; 6(1): 337, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489403

RESUMO

SARS-CoV-2 has been reported to show a capacity for invading the brains of humans and model animals. However, it remains unclear whether and how SARS-CoV-2 crosses the blood-brain barrier (BBB). Herein, SARS-CoV-2 RNA was occasionally detected in the vascular wall and perivascular space, as well as in brain microvascular endothelial cells (BMECs) in the infected K18-hACE2 transgenic mice. Moreover, the permeability of the infected vessel was increased. Furthermore, disintegrity of BBB was discovered in the infected hamsters by administration of Evans blue. Interestingly, the expression of claudin5, ZO-1, occludin and the ultrastructure of tight junctions (TJs) showed unchanged, whereas, the basement membrane was disrupted in the infected animals. Using an in vitro BBB model that comprises primary BMECs with astrocytes, SARS-CoV-2 was found to infect and cross through the BMECs. Consistent with in vivo experiments, the expression of MMP9 was increased and collagen IV was decreased while the markers for TJs were not altered in the SARS-CoV-2-infected BMECs. Besides, inflammatory responses including vasculitis, glial activation, and upregulated inflammatory factors occurred after SARS-CoV-2 infection. Overall, our results provide evidence supporting that SARS-CoV-2 can cross the BBB in a transcellular pathway accompanied with basement membrane disrupted without obvious alteration of TJs.


Assuntos
Membrana Basal/metabolismo , Barreira Hematoencefálica/metabolismo , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Junções Íntimas/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Membrana Basal/patologia , Membrana Basal/virologia , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/virologia , COVID-19/genética , COVID-19/patologia , Chlorocebus aethiops , Modelos Animais de Doenças , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , SARS-CoV-2/genética , Junções Íntimas/genética , Junções Íntimas/patologia , Junções Íntimas/virologia , Células Vero
3.
Signal Transduct Target Ther ; 6(1): 340, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504054

RESUMO

As COVID-19 continues to spread rapidly worldwide and variants continue to emerge, the development and deployment of safe and effective vaccines are urgently needed. Here, we developed an mRNA vaccine based on the trimeric receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein fused to ferritin-formed nanoparticles (TF-RBD). Compared to the trimeric form of the RBD mRNA vaccine (T-RBD), TF-RBD delivered intramuscularly elicited robust and durable humoral immunity as well as a Th1-biased cellular response. After further challenge with live SARS-CoV-2, immunization with a two-shot low-dose regimen of TF-RBD provided adequate protection in hACE2-transduced mice. In addition, the mRNA template of TF-RBD was easily and quickly engineered into a variant vaccine to address SARS-CoV-2 mutations. The TF-RBD multivalent vaccine produced broad-spectrum neutralizing antibodies against Alpha (B.1.1.7) and Beta (B.1.351) variants. This mRNA vaccine based on the encoded self-assembled nanoparticle-based trimer RBD provides a reference for the design of mRNA vaccines targeting SARS-CoV-2.


Assuntos
Vacinas contra COVID-19 , COVID-19/prevenção & controle , Nanopartículas , SARS-CoV-2/imunologia , Vacinas Sintéticas , Animais , COVID-19/imunologia , COVID-19/patologia , Vacinas contra COVID-19/química , Vacinas contra COVID-19/farmacologia , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Nanopartículas/química , Nanopartículas/uso terapêutico , Células Th1/imunologia , Células Th1/patologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Células Vero
4.
Artigo em Inglês | MEDLINE | ID: mdl-34501610

RESUMO

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is mainly transmitted through respiratory droplets from positive subjects to susceptible hosts or by direct contact with an infected individual. Our study focuses on the in vitro minimal time of viral absorption as well as the minimal quantity of virus able to establish a persistent infection in Vero E6 cells. We observed that 1 min of in vitro virus exposure is sufficient to generate a cytopathic effect in cells after 7 days of infection, even at a multiplicity of infection (MOI) value of 0.01. Being aware that our findings have been obtained using an in vitro cellular model, we demonstrated that short-time exposures and low viral concentrations are able to cause infection, thus opening questions about the risk of SARS-CoV-2 transmissibility even following short contact times.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Chlorocebus aethiops , Efeito Citopatogênico Viral , Humanos , Células Vero
5.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502139

RESUMO

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.


Assuntos
COVID-19/transmissão , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , SARS-CoV-2/patogenicidade , Animais , Sítios de Ligação/genética , COVID-19/imunologia , COVID-19/virologia , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/isolamento & purificação , Mutação , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Células Vero
6.
Vaccine ; 39(36): 5214-5223, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34334254

RESUMO

Smallpox, a disease caused by the variola virus, is one of the most dangerous diseases and had killed numerous people before it was eradicated in 1980. However, smallpox has emerged as the most threatening bio-terrorism agent; as the first- and second-generation smallpox vaccines have been controversial and have caused severe adverse reactions, new demands for safe smallpox vaccines have been raised and some attenuated smallpox vaccines have been developed. We have developed a cell culture-based highly attenuated third-generation smallpox vaccine candidate KVAC103 strain by 103 serial passages of the Lancy-Vaxina strain derived from the Lister in Vero cells. Several clones were selected, taking into consideration their shape, size, and growth rate in mammalian cells. The clones were then inoculated intracerebrally in suckling mice to test for neurovirulence by observing survival. Protective immune responses in adult mice were examined by measuring the levels of neutralization antibodies and IFN-γ expression. Among several clones, clone 7 was considered the best alternative candidate because there was no mortality in suckling mice against a lethal challenge. In addition, enhanced neutralizing antibodies and T-cell mediated IFN-γ production were observed in clone 7-immunized mice. Clone 7 was named "KVAC103" and was used for the skin toxicity test and full-genome analysis. KVAC103-inoculated rabbits showed reduced skin lesions compared to those inoculated with the Lister strain, Lancy-Vaxina. A whole genome analysis of KVAC103 revealed two major deleted regions that might contribute to the reduced virulence of KVAC103 compared to the Lister strain. Phylogenetic inference supported the close relationship with the Lister strain. Collectively, our data demonstrate that KVAC103 holds promise for use as a third-generation smallpox vaccine strain due to its enhanced safety and efficacy.


Assuntos
Vacina Antivariólica , Varíola , Vírus da Varíola , Animais , Anticorpos Antivirais , Chlorocebus aethiops , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Coelhos , Varíola/prevenção & controle , Vacinas Atenuadas , Vírus Vaccinia/genética , Células Vero
7.
Cells ; 10(8)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34440816

RESUMO

The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.


Assuntos
COVID-19/fisiopatologia , SARS-CoV-2/fisiologia , Vesículas Secretórias/ultraestrutura , Replicação Viral , Animais , Chlorocebus aethiops , Microscopia Eletrônica de Transmissão , Células Vero , Vírion/fisiologia
8.
Molecules ; 26(16)2021 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-34443390

RESUMO

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is producing a large number of infections and deaths globally, the development of supportive and auxiliary treatments is attracting increasing attention. Here, we evaluated SARS-CoV-2-inactivation activity of the polyphenol-rich tea leaf extract TY-1 containing concentrated theaflavins and other virucidal catechins. The TY-1 was mixed with SARS-CoV-2 solution, and its virucidal activity was evaluated. To evaluate the inhibition activity of TY-1 in SARS-CoV-2 infection, TY-1 was co-added with SARS-CoV-2 into cell culture media. After 1 h of incubation, the cell culture medium was replaced, and the cells were further incubated in the absence of TY-1. The viral titers were then evaluated. To evaluate the impacts of TY-1 on viral proteins and genome, TY-1-treated SARS-CoV-2 structural proteins and viral RNA were analyzed using western blotting and real-time RT-PCR, respectively. TY-1 showed time- and concentration-dependent virucidal activity. TY-1 inhibited SARS-CoV-2 infection of cells. The results of western blotting and real-time RT-PCR suggested that TY-1 induced structural change in the S2 subunit of the S protein and viral genome destruction, respectively. Our findings provided basic insights in vitro into the possible value of TY-1 as a virucidal agent, which could enhance the current SARS-CoV-2 control measures.


Assuntos
COVID-19/virologia , Polifenóis/farmacologia , SARS-CoV-2/efeitos dos fármacos , Chá/química , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Biflavonoides/química , Biflavonoides/farmacologia , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Camellia sinensis/metabolismo , Catequina/química , Catequina/farmacologia , Linhagem Celular , Chlorocebus aethiops , Genoma Viral/efeitos dos fármacos , Humanos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Polifenóis/isolamento & purificação , SARS-CoV-2/metabolismo , Células Vero , Carga Viral/efeitos dos fármacos
9.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445605

RESUMO

Coronavirus disease (COVID-19) is a contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This case report presents a patient who had difficulty eradicating the corona virus due to being treated with Rituximab, which depletes B lymphocyte cells and therefore disables the production of neutralizing antibodies. The combined use of external anti-viral agents like convalescent plasma, IVIG and Remdesivir successfully helped the patient's immune system to eradicate the virus without B-cell population recovery. In vitro studies showed that convalescent plasma is the main agent that helped in eradicating the virus.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , COVID-19/tratamento farmacológico , COVID-19/imunologia , COVID-19/terapia , SARS-CoV-2/imunologia , Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Animais , Anticorpos Neutralizantes/uso terapêutico , Antivirais/uso terapêutico , COVID-19/diagnóstico por imagem , Chlorocebus aethiops , Humanos , Imunização Passiva , Hospedeiro Imunocomprometido , Rituximab/uso terapêutico , Linfócitos T/imunologia , Células Vero
10.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34413211

RESUMO

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.


Assuntos
Antivirais/farmacologia , Fatores Imunológicos/farmacologia , Lactoferrina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , COVID-19/tratamento farmacológico , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Células CACO-2 , Linhagem Celular Tumoral , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Descoberta de Drogas , Reposicionamento de Medicamentos/métodos , Células Epiteliais , Heparitina Sulfato/antagonistas & inibidores , Heparitina Sulfato/imunologia , Heparitina Sulfato/metabolismo , Hepatócitos , Ensaios de Triagem em Larga Escala , Humanos , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/patogenicidade , Células Vero
11.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34417349

RESUMO

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.


Assuntos
Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Anticorpos Antivirais/farmacologia , Sítios de Ligação , COVID-19/genética , COVID-19/virologia , Chlorocebus aethiops , Convalescença , Expressão Gênica , Humanos , Evasão da Resposta Imune , Soros Imunes/química , Modelos Moleculares , Mutação , Testes de Neutralização , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero
12.
PLoS Pathog ; 17(8): e1009800, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34437657

RESUMO

Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.


Assuntos
Interferon beta/metabolismo , SARS-CoV-2/imunologia , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Chlorocebus aethiops , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Humanos , Interferon beta/genética , Interferon beta/farmacologia , SARS-CoV-2/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Células Vero , Proteínas Virais/genética
13.
J Exp Med ; 218(10)2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34357402

RESUMO

IFN-I and IFN-III immunity in the nasal mucosa is poorly characterized during SARS-CoV-2 infection. We analyze the nasal IFN-I/III signature, namely the expression of ISGF-3-dependent IFN-stimulated genes, in mildly symptomatic COVID-19 patients and show its correlation with serum IFN-α2 levels, which peak at symptom onset and return to baseline from day 10 onward. Moreover, the nasal IFN-I/III signature correlates with the nasopharyngeal viral load and is associated with the presence of infectious viruses. By contrast, we observe low nasal IFN-I/III scores despite high nasal viral loads in a subset of critically ill COVID-19 patients, which correlates with the presence of autoantibodies (auto-Abs) against IFN-I in both blood and nasopharyngeal mucosa. In addition, functional assays in a reconstituted human airway epithelium model of SARS-CoV-2 infection confirm the role of such auto-Abs in abrogating the antiviral effects of IFN-I, but not those of IFN-III. Thus, IFN-I auto-Abs may compromise not only systemic but also local antiviral IFN-I immunity at the early stages of SARS-CoV-2 infection.


Assuntos
Autoanticorpos/imunologia , COVID-19/imunologia , Interferon Tipo I/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Animais , Antivirais/imunologia , Antivirais/farmacologia , Autoanticorpos/sangue , COVID-19/sangue , COVID-19/virologia , Chlorocebus aethiops , Feminino , Humanos , Interferon Tipo I/farmacologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Cavidade Nasal/imunologia , Cavidade Nasal/virologia , Estudos Prospectivos , SARS-CoV-2/fisiologia , Células Vero , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia
14.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361724

RESUMO

Orchids are rich treasure troves of various important phytomolecules. Among the various medicinal orchids, Ansellia africana stands out prominently in the preparing of various herbal medicines due to its high therapeutic importance. The nodal explants of A. africana were sampled from asymbiotically germinated seedlings on basal Murashige and Skoog (MS) medium and were micropropagated in MS medium supplemented with 3% sucrose and 10 µM meta topolin (mT) + 5 µM naphthalene acetic acid (NAA) +15 µM indole butyric acid (IBA) + 30 µM phloroglucinol (PG). In the present study, the essential oil was extracted by hydrodistillation and the oleoresins by the solvent extraction method from the micropropagated A. africana. The essential oil and the oleoresins were analysed by Gas Chromatography (GC) and GC/MS (Mass spectrometry). A total of 84 compounds were identified. The most predominant components among them were linoleic acid (18.42%), l-ascorbyl 2,6-dipalmitate (11.50%), linolenic acid (10.98%) and p-cresol (9.99%) in the essential oil; and eicosane (26.34%), n-butyl acetate (21.13%), heptadecane (16.48%) and 2-pentanone, 4-hydroxy-4-methyl (11.13%) were detected in the acetone extract; heptadecane (9.40%), heneicosane (9.45%), eicosane (6.40%), n-butyl acetate (14.34%) and styrene (22.20%) were identified and quantified in the ethyl acetate extract. The cytotoxic activity of essential oil and oleoresins of micropropagated A. africana was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) assay on Vero cells compared to the standard drug doxorubicin chloride. The present research contains primary information about the therapeutic utility of the essential oil and oleoresins of A. africana with a promising future research potential of qualitative and quantitative improvement through synchronised use of biotechnological techniques.


Assuntos
Citotoxinas/isolamento & purificação , Óleos Voláteis/isolamento & purificação , Orchidaceae/química , Extratos Vegetais/isolamento & purificação , Plântula/química , Acrilatos/isolamento & purificação , Alcanos/isolamento & purificação , Animais , Ácido Ascórbico/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Cresóis/isolamento & purificação , Meios de Cultura/química , Meios de Cultura/farmacologia , Citotoxinas/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Hidroponia/métodos , Ácido Linoleico/isolamento & purificação , Extração Líquido-Líquido/métodos , Óleos Voláteis/farmacologia , Orchidaceae/metabolismo , Palmitatos/isolamento & purificação , Pentanóis/isolamento & purificação , Pentanonas/isolamento & purificação , Extratos Vegetais/farmacologia , Plantas Medicinais , Plântula/metabolismo , África do Sul , Estireno/isolamento & purificação , Células Vero , Ácido alfa-Linoleico/isolamento & purificação
15.
PLoS Pathog ; 17(8): e1009758, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34379705

RESUMO

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.


Assuntos
COVID-19 , Doenças Transmissíveis Emergentes , Modelos Animais de Doenças , Células 3T3 , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , COVID-19/imunologia , COVID-19/patologia , COVID-19/fisiopatologia , Chlorocebus aethiops , Dependovirus/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução Genética , Células Vero
16.
PLoS Pathog ; 17(8): e1009865, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424943

RESUMO

While evidence exists supporting the potential for aerosol transmission of SARS-CoV-2, the infectious dose by inhalation remains unknown. In the present study, the probability of infection following inhalation of SARS-CoV-2 was dose-dependent in a nonhuman primate model of inhalational COVID-19. The median infectious dose, assessed by seroconversion, was 52 TCID50 (95% CI: 23-363 TCID50), and was significantly lower than the median dose for fever (256 TCID50, 95% CI: 102-603 TCID50), resulting in a group of animals that developed an immune response post-exposure but did not develop fever or other clinical signs of infection. In a subset of these animals, virus was detected in nasopharyngeal and/or oropharyngeal swabs, suggesting that infected animals without signs of disease are able to shed virus and may be infectious, which is consistent with reports of asymptomatic spread in human cases of COVID-19. These results suggest that differences in exposure dose may be a factor influencing disease presentation in humans, and reinforce the importance of public health measures that limit exposure dose, such as social distancing, masking, and increased ventilation. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, and, ultimately, mitigation strategies. Additionally, these data will be useful to inform dose selection in future studies examining the efficacy of therapeutics and vaccines against inhalational COVID-19, and as a baseline in healthy, young adult animals for assessment of the importance of other factors, such as age, comorbidities, and viral variant, on the infectious dose and disease presentation.


Assuntos
COVID-19/transmissão , COVID-19/virologia , Macaca fascicularis , Soroconversão , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Febre/virologia , Exposição por Inalação , Masculino , Células Vero , Carga Viral
17.
Signal Transduct Target Ther ; 6(1): 315, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433803

RESUMO

The evolution of coronaviruses, such as SARS-CoV-2, makes broad-spectrum coronavirus preventional or therapeutical strategies highly sought after. Here we report a human angiotensin-converting enzyme 2 (ACE2)-targeting monoclonal antibody, 3E8, blocked the S1-subunits and pseudo-typed virus constructs from multiple coronaviruses including SARS-CoV-2, SARS-CoV-2 mutant variants (SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617.1, and P.1), SARS-CoV and HCoV-NL63, without markedly affecting the physiological activities of ACE2 or causing severe toxicity in ACE2 "knock-in" mice. 3E8 also blocked live SARS-CoV-2 infection in vitro and in a prophylactic mouse model of COVID-19. Cryo-EM and "alanine walk" studies revealed the key binding residues on ACE2 interacting with the CDR3 domain of 3E8 heavy chain. Although full evaluation of safety in non-human primates is necessary before clinical development of 3E8, we provided a potentially potent and "broad-spectrum" management strategy against all coronaviruses that utilize ACE2 as entry receptors and disclosed an anti-coronavirus epitope on human ACE2.


Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Anticorpos Monoclonais Murinos/farmacologia , Antivirais/farmacologia , COVID-19/tratamento farmacológico , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Antivirais/imunologia , Chlorocebus aethiops , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células Vero
18.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34406116

RESUMO

African swine fever is a devastating disease of domestic swine and wild boar caused by a large double-stranded DNA virus that encodes for more than 150 open reading frames. There is no licensed vaccine for the disease and the most promising current candidates are modified live viruses that have been attenuated by deletion of virulence factors. Like many viruses African swine fever virus significantly alters the host cell machinery to benefit its replication and viral genes that modify host pathways represent promising targets for development of gene deleted vaccines. Autophagy is an important cellular pathway that is involved in cellular homeostasis, innate and adaptive immunity and therefore is manipulated by a number of different viruses. Autophagy is regulated by a complex protein cascade and here we show that African swine fever virus can block formation of autophagosomes, a critical functional step of the autophagy pathway through at least two different mechanisms. Interestingly this does not require the A179L gene that has been shown to interact with Beclin-1, an important autophagy regulator.


Assuntos
Vírus da Febre Suína Africana/patogenicidade , Febre Suína Africana/virologia , Proteínas Virais/metabolismo , Animais , Autofagia , Chlorocebus aethiops , Suínos , Células Vero , Virulência
19.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34410903

RESUMO

An infectious agent's pathogenic and transmission potential is heavily influenced by early events during the asymptomatic or subclinical phase of disease. During this phase, the presence of infectious agent may be relatively low. An important example of this is Zika virus (ZIKV), which can cross the placenta and infect the foetus, even in mothers with subclinical infections. These subclinical infections represent roughly 80 % of all human infections. Initial ZIKV pathogenesis studies were performed in type I interferon receptor (IFNAR) knockout mice. Blunting the interferon response resulted in robust infectivity, and increased the utility of mice to model ZIKV infections. However, due to the removal of the interferon response, the use of these models impedes full characterization of immune responses to ZIKV-related pathologies. Moreover, IFNAR-deficient models represent severe disease whereas less is known regarding subclinical infections. Investigation of the anti-viral immune response elicited at the maternal-foetal interface is critical to fully understand mechanisms involved in foetal infection, foetal development, and disease processes recognized to occur during subclinical maternal infections. Thus, immunocompetent experimental models that recapitulate natural infections are needed. We have established subclinical intravaginal ZIKV infections in mice and guinea pigs. We found that these infections resulted in: the presence of both ZIKV RNA transcripts and infectious virus in maternal and placental tissues, establishment of foetal infections and ZIKV-mediated CXCL10 expression. These models will aid in discerning the mechanisms of subclinical ZIKV mother-to-offspring transmission, and by extension can be used to investigate other maternal infections that impact foetal development.


Assuntos
Feto , Placenta , Complicações Infecciosas na Gravidez , Infecção por Zika virus/virologia , Zika virus , Animais , Chlorocebus aethiops , Feminino , Feto/imunologia , Feto/virologia , Cobaias , Humanos , Transmissão Vertical de Doenças Infecciosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placenta/imunologia , Placenta/virologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , Células Vero , Zika virus/imunologia , Zika virus/patogenicidade
20.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34410905

RESUMO

The capsid protein (C) of dengue virus is required for viral infectivity as it packages viral RNA genome into infectious particles. C exists as a homodimer that forms via hydrophobic interactions between the α2 and α4 helices of monomers. To identify C region(s) important for virus particle production, a complementation system was employed in which single-round infectious particles are generated by trans-encapsidation of a viral C-deleted genome by recombinant C expressed in mosquito cells. Mutants harbouring a complete α3 deletion, or a dual Ile65-/Trp69-to-Ala substitution in the α3 helix, exhibited reduced production of infectious virus. Unexpectedly, higher proportions of oligomeric C were detected in cells expressing both mutated forms as compared with the wild-type counterpart, indicating that the α3 helix, through its internal hydrophobic residues, may down-modulate oligomerization of C during particle formation. Compared with wild-type C, the double Ile65-/Trp69 to Ala mutations appeared to hamper viral infectivity but not C and genomic RNA incorporation into the pseudo-infectious virus particles, suggesting that increased C oligomerization may impair DENV replication at the cell entry step.


Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , Vírus da Dengue/metabolismo , Dengue/virologia , Aedes , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Chlorocebus aethiops , Humanos , Células Vero , Montagem de Vírus , Replicação Viral
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