Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 247
Filtrar
1.
Sci Rep ; 12(1): 1060, 2022 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-35058513

RESUMO

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.

2.
Microbiol Spectr ; : e0056121, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34851162

RESUMO

Vaccines against severe acute respiratory syndrome coronavirus-2 have been introduced. To investigate the relationship between vaccine-induced humoral immunity and patient age, we measured antibody levels and neutralization in vaccinated sera. Sera from 13 to 17 days after the second dose of the BNT162b2 vaccine were collected from health care workers at the University of Toyama (n = 740). Antibody levels were measured by the anti-receptor binding domain antibody test (anti-RBD test), and neutralization against wild-type (WT), α- and ß-variant pseudotyped viruses were assayed using a high-throughput chemiluminescent reduction neutralizing test (htCRNT; positivity cutoff, 50% neutralization at serum dilution 1:100). Basic clinical characteristics were obtained from questionnaires. Antibodies were confirmed in all participants in both the anti-RBD test (median, 2,112 U/ml; interquartile range [IQR], 1,275 to 3,390 U/ml) and the htCRNT against WT (median % inhibition, >99.9; IQR, >99.9 to >99.9). For randomly selected sera (n = 61), 100.0% had positive htCRNT values against the α- and ß-derived variants. Among those who answered the questionnaire (n = 237), the values of the anti-RBD test were negatively correlated with age in females (P < 0.01). An age-dependent decline in neutralization was observed against the variants but not against the wild-type virus (wild type, P = 0.09; α, P < 0.01; ß, P < 0.01). The neutralizing activity induced by BNT162b2 was obtained not only against the wild-type virus, but also against the variants; however, there was an age-dependent decrease in the latter. Age-related heterogeneity of vaccine-acquired immunity is a concern in preventive strategies in the era dominated by variants. IMPORTANCE Since mRNA vaccines utilize wild-type SARS-CoV-2 spike protein as an antigen, there are potential concerns about acquiring immunity to variants of this virus. The neutralizing activity in BNT162b2-vaccinated individuals was higher against the wild-type virus than against its variants; this effect was more apparent in older age groups. This finding suggests that one of the weaknesses of the mRNA vaccine is the high risk of variant infection in the elderly population. Because the elderly are at a higher risk of SARS-CoV-2 infection, the age-dependent decline of neutralization against viral variants should be considered while planning vaccination programs that include boosters.

3.
Microbiol Spectr ; : e0056021, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34851163

RESUMO

Serological tests are beneficial for recognizing the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). To identify protective immunity, optimization of the chemiluminescent reduction neutralizing test (CRNT) is critical. Whether commercial antibody tests have comparable accuracy is unknown. Serum samples were obtained from COVID-19 patients (n = 74), SARS-CoV-2 PCR-negative (n = 179), and suspected healthy individuals (n = 229) before SARS-CoV-2 variants had been detected locally. The convalescent phase was defined as the period after day 10 from disease onset or the episode of close contact. The CRNT using pseudotyped viruses displaying the wild-type (WT) spike protein and a commercial anti-receptor-binding domain (RBD) antibody test were assayed. Serology for the B.1.1.7 and B.1.351 variants was also assayed. Both tests concurred for symptomatic COVID-19 patients in the convalescent phase. They clearly differentiated between patients and suspected healthy individuals (sensitivity: 95.8% and 100%, respectively; specificity: 99.1% and 100%, respectively). Anti-RBD antibody test results correlated with neutralizing titers (r = 0.31, 95% confidence interval [CI] 0.22-0.38). Compared with the WT, lower CRNT values were observed for the variants. Of the samples with ≥100 U/mL by the anti-RBD antibody test, 77.8% and 88.9% showed ≥50% neutralization against the B.1.1.7 and the B.1.351 variants, respectively. Exceeding 100 U/mL in the anti-RBD antibody test was associated with neutralization of variants (P < 0.01). The CRNT and commercial anti-RBD antibody test effectively classified convalescent COVID-19 patients. Strong positive results with the anti-RBD antibody test can reflect neutralizing activity against emerging variants. IMPORTANCE This study provides a diagnostic evidence of test validity, which can lead to vaccine efficacy and proof of recovery after COVID-19. It is not easy to know neutralization against SARS-CoV-2 in the clinical laboratory because of technical and biohazard issues. The correlation of the quantitative anti-receptor-binding domain antibody test, which is widely available, with neutralizing test indicates that we can know indirectly the state of acquisition of functional immunity against wild and variant-type viruses in the clinical laboratory.

4.
Heliyon ; 7(10): e08250, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34761137

RESUMO

Norovirus infection is a major cause of acute gastroenteritis, although some infected individuals are asymptomatic. GII.4 is the predominant genotype worldwide and, since 2000, has been the most prevalent in patients in Thailand with acute gastroenteritis. We screened stool samples for norovirus in 786 patients with acute gastroenteritis who were admitted to a hospital in Bangkok from 2017 to early 2019 and detected it in 136 specimens (17.3%). Eight and 124 specimens were positive for the GI and GII genogroups, respectively, and the remaining 4 specimens were double-positive. Nine genotypes (GI.3, GI.5, GII.2, GII.3, GII.4, GII.6, GII.8, GII.13, and GII.17) were identified from 140 strains, and 72 strains (51.4%) were GII.4. We had previously conducted a one-year survey of norovirus infection in residents of a community in Bangkok from May 2018 to April 2019 and found that a substantial portion of the residents were infected asymptomatically. The 9 genotypes identified in the patients were also commonly identified in the community residents. To investigate the relationship between noroviruses identified in the acute gastroenteritis patients and those identified in the community residents, phylogenetic tree analysis was conducted. Of the 9 genotypes, 8 showed similarities in both their genomic sequences and their deduced amino acid sequences. In addition, strain replacement of GI.3 was observed in both the patients and the community residents within the overlapping period. These results suggested that norovirus spreads efficiently to the community by simultaneously causing symptomatic and asymptomatic infections.

5.
J Exp Med ; 218(12)2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34623376

RESUMO

Broadly protective vaccines against SARS-related coronaviruses that may cause future outbreaks are urgently needed. The SARS-CoV-2 spike receptor-binding domain (RBD) comprises two regions, the core-RBD and the receptor-binding motif (RBM); the former is structurally conserved between SARS-CoV-2 and SARS-CoV. Here, in order to elicit humoral responses to the more conserved core-RBD, we introduced N-linked glycans onto RBM surfaces of the SARS-CoV-2 RBD and used them as immunogens in a mouse model. We found that glycan addition elicited higher proportions of the core-RBD-specific germinal center (GC) B cells and antibody responses, thereby manifesting significant neutralizing activity for SARS-CoV, SARS-CoV-2, and the bat WIV1-CoV. These results have implications for the design of SARS-like virus vaccines.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , Polissacarídeos/imunologia , Vírus da SARS/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Motivos de Aminoácidos , Animais , COVID-19/genética , COVID-19/prevenção & controle , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polissacarídeos/genética , Domínios Proteicos , Vírus da SARS/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
6.
J Gen Virol ; 102(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34661519

RESUMO

Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.


Assuntos
Citocinas/metabolismo , Vírus da Hepatite B/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transativadores/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral , Linhagem Celular , Farmacorresistência Viral , Vírus da Hepatite B/genética , Humanos , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Interferons/farmacologia , Transativadores/química , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais Reguladoras e Acessórias/química
7.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34625475

RESUMO

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and life. A useful pathological animal model accurately reflecting human pathology is needed to overcome the COVID-19 crisis. In the present study, COVID-19 cynomolgus monkey models including monkeys with underlying diseases causing severe pathogenicity such as metabolic disease and elderly monkeys were examined. Cynomolgus macaques with various clinical conditions were intranasally and/or intratracheally inoculated with SARS-CoV-2. Infection with SARS-CoV-2 was found in mucosal swab samples, and a higher level and longer period of viral RNA was detected in elderly monkeys than in young monkeys. Pneumonia was confirmed in all of the monkeys by computed tomography images. When monkeys were readministrated SARS-CoV-2 at 56 d or later after initial infection all of the animals showed inflammatory responses without virus detection in swab samples. Surprisingly, in elderly monkeys reinfection showed transient severe pneumonia with increased levels of various serum cytokines and chemokines compared with those in primary infection. The results of this study indicated that the COVID-19 cynomolgus monkey model reflects the pathophysiology of humans and would be useful for elucidating the pathophysiology and developing therapeutic agents and vaccines.


Assuntos
COVID-19/imunologia , Modelos Animais de Doenças , Macaca fascicularis/imunologia , Doenças dos Primatas/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/virologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Pulmão/diagnóstico por imagem , Pulmão/imunologia , Pulmão/virologia , Macaca fascicularis/virologia , Masculino , Doenças dos Primatas/virologia , SARS-CoV-2/fisiologia , Tomografia Computadorizada por Raios X/métodos , Eliminação de Partículas Virais/imunologia , Eliminação de Partículas Virais/fisiologia
8.
Immunity ; 54(10): 2385-2398.e10, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34508662

RESUMO

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.


Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Reações Cruzadas/imunologia , SARS-CoV-2/imunologia , Animais , Betacoronavirus/imunologia , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/uso terapêutico , COVID-19/prevenção & controle , COVID-19/terapia , COVID-19/virologia , Cricetinae , Humanos , Switching de Imunoglobulina , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/imunologia , Camundongos , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
9.
Viruses ; 13(7)2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34372516

RESUMO

Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases.

10.
Viruses ; 13(6)2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205062

RESUMO

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe disease in humans with case fatality rates of approximately 30%. There are few treatment options for SFTSV infection. SFTSV RNA synthesis is conducted using a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are, therefore, potential antiviral targets. A library of small molecule compounds was processed using a high-throughput screening (HTS) based on an SFTSV minigenome assay (MGA) in a 96-well microplate format to identify potential lead inhibitors of SFTSV RNA synthesis. The assay confirmed inhibitory activities of previously reported SFTSV inhibitors, favipiravir and ribavirin. A small-scale screening using MGA identified four candidate inhibitors that inhibited SFTSV minigenome activity by more than 80% while exhibiting less than 20% cell cytotoxicity with selectivity index (SI) values of more than 100. These included mycophenolate mofetil, methotrexate, clofarabine, and bleomycin. Overall, these data demonstrate that the SFTSV MGA is useful for anti-SFTSV drug development research.

11.
Cell Host Microbe ; 29(7): 1124-1136.e11, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34171266

RESUMO

Many SARS-CoV-2 variants with naturally acquired mutations have emerged. These mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains largely unexplored. Here, we demonstrate that two recently emerging mutations in the receptor-binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429 and B.1.617) and Y453F (in B.1.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce affinity toward the host entry receptor ACE2. Notably, the L452R mutation increases spike stability, viral infectivity, viral fusogenicity, and thereby promotes viral replication. These data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.


Assuntos
COVID-19/virologia , Imunidade Celular , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2 , COVID-19/epidemiologia , Genoma Viral , Humanos , Mutação , Filogenia , Ligação Proteica , Proteínas Virais/genética , Replicação Viral
12.
Nat Commun ; 12(1): 3726, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140500

RESUMO

High-throughput, high-accuracy detection of emerging viruses allows for the control of disease outbreaks. Currently, reverse transcription-polymerase chain reaction (RT-PCR) is currently the most-widely used technology to diagnose the presence of SARS-CoV-2. However, RT-PCR requires the extraction of viral RNA from clinical specimens to obtain high sensitivity. Here, we report a method for detecting novel coronaviruses with high sensitivity by using nanopores together with artificial intelligence, a relatively simple procedure that does not require RNA extraction. Our final platform, which we call the artificially intelligent nanopore, consists of machine learning software on a server, a portable high-speed and high-precision current measuring instrument, and scalable, cost-effective semiconducting nanopore modules. We show that artificially intelligent nanopores are successful in accurately identifying four types of coronaviruses similar in size, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2. Detection of SARS-CoV-2 in saliva specimen is achieved with a sensitivity of 90% and specificity of 96% with a 5-minute measurement.


Assuntos
Inteligência Artificial , Teste de Ácido Nucleico para COVID-19/métodos , Aprendizado de Máquina , Nanoporos , Teste de Ácido Nucleico para COVID-19/instrumentação , Coronavirus Humano 229E/genética , Desenho de Equipamento/economia , Humanos , Limite de Detecção , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Nanopartículas/química , Reação em Cadeia da Polimerase , SARS-CoV-2/genética , Saliva/virologia , Sensibilidade e Especificidade , Software
13.
Nat Commun ; 12(1): 3802, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155214

RESUMO

SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/farmacologia , COVID-19/tratamento farmacológico , Mutação , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/metabolismo , COVID-19/virologia , Células Cultivadas , Cricetinae , Cristalografia por Raios X , Modelos Animais de Doenças , Humanos , Masculino , Simulação de Dinâmica Molecular , Ligação Proteica , Engenharia de Proteínas/métodos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo
14.
Cell ; 184(13): 3452-3466.e18, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34139176

RESUMO

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , COVID-19/imunologia , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Ligação Proteica/imunologia , Domínios Proteicos/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Células Vero
15.
Proc Natl Acad Sci U S A ; 118(22)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34035171

RESUMO

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8+ T cells. The expression of MHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses.

17.
Hepatol Commun ; 5(4): 634-649, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33860122

RESUMO

Compared with each monoinfection, coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is well known to increase the risks of developing liver cirrhosis and hepatocellular carcinoma. However, the mechanism by which HBV/HCV coinfection is established in hepatocytes is not well understood. Common cell culture models for coinfection are required to examine viral propagation. In this study, we aimed to establish a cell line permissive for both HBV and HCV infection. We first prepared a HepG2 cell line expressing sodium taurocholate cotransporting polypeptide, an HBV receptor, and then selected a cell line highly permissive for HBV infection, G2/NT18-B. After transduction with a lentivirus-encoding microRNA-122, the cell line harboring the highest level of replicon RNA was selected and then treated with anti-HCV compounds to eliminate the replicon RNA. The resulting cured cell line was transduced with a plasmid-encoding CD81. The cell line permissive for HCV infection was cloned and then designated the G2BC-C2 cell line, which exhibited permissiveness for HBV and HCV propagation. JAK inhibitor I potentiated the HCV superinfection of HBV-infected cells, and fluorescence-activated cell-sorting analysis indicated that HBV/HCV double-positive cells accounted for approximately 30% of the coinfected cells. Among several host genes tested, cyclooxygenase-2 showed synergistic induction by coinfection compared with each monoinfection. Conclusion: These data indicate that our in vitro HBV/HCV coinfection system provides an easy-to-use platform for the study of host and viral responses against coinfection and the development of antiviral agents targeting HBV and HCV.

18.
Cell Rep ; 35(3): 109014, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33838744

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (∼5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.


Assuntos
COVID-19/genética , Genética Reversa , SARS-CoV-2/genética , Animais , Cricetinae , Células HEK293 , Humanos
19.
Sci Rep ; 11(1): 5934, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33723294

RESUMO

The aim of this study is to understand adaptive immunity to SARS-CoV-2 through the analysis of B cell epitope and neutralizing activity in coronavirus disease 2019 (COVID-19) patients. We obtained serum from forty-three COVID-19 patients from patients in the intensive care unit of Osaka University Hospital (n = 12) and in Osaka City Juso Hospital (n = 31). Most individuals revealed neutralizing activity against SARS-CoV-2 assessed by a pseudotype virus-neutralizing assay. The antibody production against the spike glycoprotein (S protein) or receptor-binding domain (RBD) of SARS-CoV-2 was elevated, with large individual differences, as assessed by ELISA. We observed the correlation between neutralizing antibody titer and IgG, but not IgM, antibody titer of COVID-19 patients. In the analysis of the predicted the linear B cell epitopes, hot spots in the N-terminal domain of the S protein were observed in the serum from patients in the intensive care unit of Osaka University Hospital. Overall, the analysis of antibody production and B cell epitopes of the S protein from patient serum may provide a novel target for the vaccine development against SARS-CoV-2.


Assuntos
COVID-19/epidemiologia , COVID-19/imunologia , Epitopos de Linfócito B/imunologia , Imunidade Humoral , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Interações Hospedeiro-Patógeno , Humanos , Masculino , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
20.
Cancer Med ; 10(5): 1501-1514, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33600074

RESUMO

Autophagy removes damaged organelles to inhibit malignant transformation during tumor initiation. Once a cancer matures, it uses the autophagic pathway as an energy source. Optineurin (OPTN) is an autophagy adaptor protein that recruits microtubule-associated protein 1 light chain 3, an autophagosome marker, to the autophagosome. Despite studies of the relation between cancer progression and autophagy adaptor proteins, there are no reports to our knowledge of a correlation between hepatocellular carcinoma (HCC) and OPTN. We aimed here to investigate the effects of OPTN expression on HCC progression through autophagy. Immunohistochemistry was used to measure the OPTN expression in the tissues of 141 Japanese patients with HCC. The effects of OPTN expression on HCC progression and mitophagy were assessed using an OPTN knockout (KO) cell line in vitro. We used this KO cell line to establish and exploit a mouse model of HCC to determine the effects of OPTN expression on tumor progression. Immunohistochemical analysis showed that patients with elevated expression of OPTN experienced shorter overall survival (OS) and recurrence-free survival (RFS). OPTN KO cells proliferated relatively slower versus wild-type (WT) cells in vitro. Western blot analysis showed that mitophagy was suppressed in OPTN KO cells, and ATP synthesis and beta-oxidation were reduced. The mouse model of HCC showed that OPTN KO cells formed smaller tumors versus WT cells less 10 weeks after implantation. Overall, the present findings suggest that OPTN is a key mediator of mitophagy that contributes to HCC progression through mitochondrial energy production.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitofagia/fisiologia , Proteínas de Neoplasias/metabolismo , Trifosfato de Adenosina/biossíntese , Idoso , Animais , Autofagossomos/metabolismo , Autofagia/fisiologia , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Transformação Celular Neoplásica , Modelos Animais de Doenças , Progressão da Doença , Intervalo Livre de Doença , Feminino , Técnicas de Inativação de Genes , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Mitocôndrias Hepáticas/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...