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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(5): 1718-1725, 2020 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-33067980

RESUMO

OBJECTIVE: To investigate the adenovirus-mediated expression of human clotting factor IX (hFIX) gene in mouse adipose-derived stem cells(ADSC). METHODS: The mouse ADSC were isolated and cultured in vitro, the morphology of cells was observed and its growth viability was detected by using CCK-8. Cell surface markers CD29,CD90,CD45 were identified by flow cytometry, and its diferentiation ability was identified by adipogenic and osteogenic induction. Morphological changes was observed and the growth curve should be drawn after transfecting ADSC with adenovirus containing hFIX gene. The expression of hFIX gene was detected by RT-PCR. The expression of hFIX protein in ADSC or in culture supernatant was detected by Western blot. hFIX protein in the supernatant was measured by ELISA, and the clotting factor activity of hFIX in culture supernatant was measured by one-stage method. RESULTS: The in vitro cultured mouse ADSC displayed microspherical shape and strong refractive property. Anchoring growth was lasted for 4-6 hours after planting into culture flask. After cultured for 72 hours, the cells showed long spindle-shaped fibrous and swirling arrangement. The overall growth trend of the third generation ADSC cultured in vitro was S-shaped. The formation of lipid droplets could be observed in the induced cells with Oil red O staining by inverted microscope. After alizarin red staining, the orange-red calcified bone nodes were observed in the induced cells under inverted phase contrast microscope. CD29 (99.91%) and CD90 (99.02%) highly expressed in the third generation of ADSC, but CD45 (0.94%) almost not expressed. RT-PCR showed the hFIX gene could expressed in mouse ADSC. Western blot showed that hFIX protein expressed in both ADSC and culture supernatant. FIX:Ag in cell supernatant was 21.33±3.93 ng/(106 cells.24 h) on the first day, 12.63±0.86 ng/(106 cells.24 h) on the third day and 12.63±2.36 ng/(106 cells.24 h) on the ninth day. FIX:C in culture supernatant was 8.5%. CONCLUSION: Adenovirus-carried hFIX gene can effectively transfect ADSC. ADSC modified by hFIX gene can secrete hFIX protein with coagulation activity.


Assuntos
Adenoviridae , Fator IX , Adenoviridae/genética , Adipogenia , Animais , Fator IX/genética , Humanos , Camundongos , Osteogênese , Células-Tronco
3.
Curr Opin HIV AIDS ; 15(6): 351-358, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32969973

RESUMO

PURPOSE OF REVIEW: Coronavirus disease-19 (COVID-19) is a highly transmittable and pathogenic pneumonia-causing disease, which is caused by severe acute respiratory syndrome coronavirus-2, resulting in millions of deaths globally. Severe acute respiratory syndrome coronavirus-2 may coexist with human populations for a long time. Therefore, high-effective COVID-19 vaccines are an urgent need. RECENT FINDINGS: Vaccines help in the development of long-lasting humoral or cellular immunity, or both, by exposing individuals to antigens that induce an immunological response and memory prior to infections with live pathogens. New vaccine technologies, such as viral vectors and nucleic acid-based vaccines, which represent highly versatile technologies, may allow for faster vaccine manufacture and scale up production. SUMMARY: We summarized the recent progress made in relation to COVID-19 vaccine development using several promising technologies, with particular emphasis on advancements that are currently at the clinical trial stage.


Assuntos
Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Adenoviridae/genética , Infecções por Coronavirus/imunologia , Humanos , Vacinação , Vacinas Atenuadas/imunologia , Vacinas de DNA/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas Sintéticas/imunologia
4.
Cell ; 183(1): 169-184.e13, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32931734

RESUMO

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


Assuntos
Infecções por Coronavirus/imunologia , Imunogenicidade da Vacina , Pneumonia Viral/imunologia , Vacinas Virais/imunologia , Adenoviridae/genética , Administração Intranasal , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Feminino , Células HEK293 , Humanos , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Pandemias , Pneumonia Viral/patologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero , Vacinas Virais/administração & dosagem
6.
Eur Rev Med Pharmacol Sci ; 24(17): 9196-9201, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32965014

RESUMO

OBJECTIVE: The aim of this study is to find the distributions of pathogens in 164 suspected COVID-19 patients from the outpatient clinic of Shenjing Hospital of China Medical University from 24th January, 2020, to 29th February of 2020. PATIENTS AND METHODS: 164 COVID-19 suspected patients were from the Shengjing Hospital of China Medical University. Oropharyngeal swab specimens were acquired by respiratory doctors under standardized conditions. Specific nucleic acids of SARS-CoV-2, influenza A and B, respiratory syncytial virus A and B, adenovirus, parainfluenza virus, along with pneumonic mycoplasma were detected by real-time fluorescence PCR. Symptomatic, epidemiologic, laboratory and radiological data of the patients were obtained from the electronic medical record system of our hospital. RESULTS: Among the 164 patients, 3 were positive for SARS-CoV-2, 15 were positive for other respiratory viruses and 16 were positive for pneumonic mycoplasma. Of the positive patients above, 1 patient was co-infected with SARS-CoV-2 and adenovirus, and 1 was co-infected with influenza B and pneumonic mycoplasma. The 3 SARS-CoV-2 infected patients were clinically diagnosed as COVID-19 because they meet the diagnostic criteria listed in "Chinese Clinical Guidance for COVID-19 Pneumonia diagnosis and treatment", including epidemic history, symptom and pathogenic detection, as well as abnormalities of the laboratory and radiological data. However, the clinical characteristics of COVID-19 patients were non-specific compared to those of the patients infected with other respiratory viruses. CONCLUSIONS: The endemic common respiratory pathogens are more prevalent than SARS-CoV-2 in the SARS-CoV-2 non-epidemic areas of this research. Detection of the pathogen is the unique means for definite COVID-19 diagnosis.


Assuntos
Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Adenoviridae/genética , Adenoviridae/isolamento & purificação , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , China/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Influenzavirus B/genética , Influenzavirus B/isolamento & purificação , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tórax/diagnóstico por imagem , Tomografia Computadorizada por Raios X
7.
Nat Commun ; 11(1): 4207, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826924

RESUMO

The rapid spread of coronavirus SARS-CoV-2 greatly threatens global public health but no prophylactic vaccine is available. Here, we report the generation of a replication-incompetent recombinant serotype 5 adenovirus, Ad5-S-nb2, carrying a codon-optimized gene encoding Spike protein (S). In mice and rhesus macaques, intramuscular injection with Ad5-S-nb2 elicits systemic S-specific antibody and cell-mediated immune (CMI) responses. Intranasal inoculation elicits both systemic and pulmonary antibody responses but weaker CMI response. At 30 days after a single vaccination with Ad5-S-nb2 either intramuscularly or intranasally, macaques are protected against SARS-CoV-2 challenge. A subsequent challenge reveals that macaques vaccinated with a 10-fold lower vaccine dosage (1 × 1010 viral particles) are also protected, demonstrating the effectiveness of Ad5-S-nb2 and the possibility of offering more vaccine dosages within a shorter timeframe. Thus, Ad5-S-nb2 is a promising candidate vaccine and warrants further clinical evaluation.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas Virais/administração & dosagem , Adenoviridae/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Coronavirus/imunologia , Relação Dose-Resposta Imunológica , Feminino , Células HEK293 , Humanos , Imunidade Celular , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Viral/imunologia , Sistema Respiratório/patologia , Sistema Respiratório/virologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Sintéticas/administração & dosagem
8.
BMC Infect Dis ; 20(1): 633, 2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-32847534

RESUMO

BACKGROUND: Cases of refractory Mycoplasma pneumoniae pneumonia have been increasing recently; however, whether viral coinfection or macrolide-resistant M. infection contribute to the development of refractory M. pneumoniae pneumonia remains unclear. This study aimed to investigate the impacts of viral coinfection and macrolide-resistant M. pneumoniae infection on M. pneumoniae pneumonia in hospitalized children and build a model to predict a severe disease course. METHODS: Nasopharyngeal swabs or sputum specimens were collected from patients with community-acquired pneumonia meeting our protocol who were admitted to Shanghai Children's Medical Center from December 1, 2016, to May 31, 2019. The specimens were tested with the FilmArray Respiratory Panel, a multiplex polymerase chain reaction assay that detects 16 viruses, Bordetella pertussis, M. pneumoniae, and Chlamydophila pneumoniae. Univariate and multivariate logistic regression models were used to identify the risk factors for adenovirus coinfection and macrolide-resistant mycoplasma infection. RESULTS: Among the 107 M. pneumoniae pneumonia patients, the coinfection rate was 56.07%, and 60 (60/107, 56.07%) patients were infected by drug-resistant M. pneumoniae. Adenovirus was the most prevalent coinfecting organism, accounting for 22.43% (24/107). The classification tree confirmed that viral coinfection was more common in patients younger than 3 years old. Adenovirus coinfection and drug-resistant M. pneumoniae infection occurred more commonly in patients with refractory M. pneumoniae pneumonia (P = 0.019; P = 0.001). A prediction model including wheezing, lung consolidation and extrapulmonary complications was used to predict adenovirus coinfection. The area under the receiver operating characteristic curve of the prediction model was 0.795 (95% CI 0.679-0.893, P < 0.001). A prolonged fever duration after the application of macrolides for 48 h was found more commonly in patients infected by drug-resistant M. pneumoniae (P = 0.002). A fever duration longer than 7 days was an independent risk factor for drug-resistant Mycoplasma infection (OR = 3.500, 95% CI = 1.310-9.353, P = 0.012). CONCLUSIONS: The occurrence of refractory M. pneumoniae pneumonia is associated with adenovirus coinfection and infection by drug-resistant M. pneumoniae. A prediction model combining wheezing, extrapulmonary complications and lung consolidation can be used to predict adenovirus coinfection in children with M. pneumoniae pneumonia. A prolonged fever duration indicates drug-resistant M. pneumoniae infection, and a reasonable change in antibiotics is necessary.


Assuntos
Infecções por Adenoviridae/epidemiologia , Adenoviridae/genética , Antibacterianos/uso terapêutico , Coinfecção/epidemiologia , Farmacorresistência Bacteriana , Macrolídeos/uso terapêutico , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/tratamento farmacológico , Pneumonia por Mycoplasma/epidemiologia , Infecções por Adenoviridae/virologia , Adolescente , Criança , Pré-Escolar , China/epidemiologia , Coinfecção/virologia , Infecções Comunitárias Adquiridas/tratamento farmacológico , Feminino , Hospitalização , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase Multiplex , Mycoplasma pneumoniae/efeitos dos fármacos , Pneumonia por Mycoplasma/virologia , Prevalência , Prognóstico , Resultado do Tratamento
10.
PLoS One ; 15(8): e0237098, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745124

RESUMO

The EGFR-targeting cancer therapies are commonly facing drug resistance, mostly due to mutations. Gene therapy with artificial microRNA targeting EGFR conserved sequence may avoid such problem. In this study, we constructed a recombinant adenovirus expressing EGFR-targeting artificial microRNA and active revCASP3 (Ad-EC), under the control of tumor-specific SLPI promoter, and evaluated its inhibitory effect on HEP-2 cancer cells both in vitro and in vivo. MTT assay showed that cell growth inhibition rate at 72h was 44.0% in Ad-EC group at MOI 50, while the rate was 7.7% in the control virus Ad-GFP group and 3.6% in Cetuximab (500 µg/ml) group respectively. Flow cytometry analysis revealed the late apoptotic cells rate was 36.1% in Ad-EC group, significantly higher than 6.5% of Ad-GFP group (p < 0.001). When Ad-EC (MOI 50) was combined with CDDP (0.25 µg/ml), late apoptotic cells rate increased to 61.2%, significantly higher than each monotherapy group (P < 0.001). The real-time xCELLigence system recorded an effective cell growth inhibition in Ad-EC and CDDP groups, and more enhanced effect in Ad-EC plus CDDP group. Western blot revealed that Ad-EC could inhibit the activation of AKT pathway and ERK1/2 pathway, while Cetuximab had the AKT pathway over-activated. In vivo experiments with HEP-2 xenograft in nude mice confirmed the tumor inhibition in Ad-EC, CDDP and Ad-EC plus CDDP groups compared with PBS group (P < 0.01). Collectively, these data support the effective inhibition of cancer cells by this novel gene therapy strategy.


Assuntos
Caspase 3/metabolismo , Receptores ErbB/genética , MicroRNAs/genética , Neoplasias Experimentais/terapia , Terapêutica com RNAi/métodos , Adenoviridae/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células , Cetuximab/administração & dosagem , Cetuximab/uso terapêutico , Cisplatino/administração & dosagem , Cisplatino/uso terapêutico , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo
11.
Life Sci ; 256: 117985, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32562692

RESUMO

AIMS: To assess the combination therapy of anti-CD20 mabs and adenovirus-mediated interleukin-10 (IL-10) gene delivery on the prevention of type 1 diabetes (T1D) in non-obese diabetes (NOD) mice. MAIN METHODS: In present study, we simultaneously blocked the B cell interactions and recovered the Th cell subset proportion by using through anti-CD20 Mab and adenovirus-mediated gene delivery of IL-10, respectively. After 9 consecutive days of combination therapy, various measurements, including hematoxylin-eosin staining (HE), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling assay (TUNEL), immunohistochemistry, ELISA, PCR and western blot were applied to further assess the efficacy. KEY FINDINGS: The results suggested that the combination intervention reduced the T1D-associated morbidity of NOD mice, promote insulin secretion, control blood glucose and ease pancreatitis. Moreover, the combination therapy might play a protective role in pancreatic ß cells by suppressing the expression of TNF-α and Fas, blocking the Caspase-8 and Caspase-3 apoptotic pathways and activating the Bcl-2 anti-apoptotic pathway. Finally, the combination intervention may up-regulate the gene expression of CK-19 and PDX-1 and further accelerate the differentiation and proliferation of pancreatic ß cells. SIGNIFICANCE: Therefore, the combination intervention with anti-CD20 mabs and the IL-10 gene plays a role in the prevention of T1D to some extent in NOD mice.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígenos CD20/administração & dosagem , Apoptose/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Interleucina-10/administração & dosagem , Pancreatite/tratamento farmacológico , Adenoviridae/genética , Animais , Anticorpos Monoclonais/genética , Antígenos CD20/genética , Apoptose/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Quimioterapia Combinada , Feminino , Interleucina-10/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pancreatite/genética , Pancreatite/patologia
12.
Nucleic Acids Res ; 48(11): 6294-6309, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402057

RESUMO

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.


Assuntos
Pareamento de Bases , Éxons , Íntrons , Processamento de RNA , Adenoviridae/genética , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Éxons/genética , Inativação Gênica , Íntrons/genética , Camundongos , Células-Tronco Embrionárias Murinas , Mutação , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento de RNA/genética , Spliceossomos/metabolismo , Transcriptoma/genética , Globinas beta/genética
13.
Int J Legal Med ; 134(4): 1271-1274, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32458044

RESUMO

In the setting of the coronavirus disease 2019 (COVID-19) pandemic, only few data regarding lung pathology induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is available, especially without medical intervention interfering with the natural evolution of the disease. We present here the first case of forensic autopsy of a COVID-19 fatality occurring in a young woman, in the community. Diagnosis was made at necropsy and lung histology showed diffuse alveolar damage, edema, and interstitial pneumonia with a geographically heterogeneous pattern, mostly affecting the central part of the lungs. This death related to COVID-19 pathology highlights the heterogeneity and severity of central lung lesions after natural evolution of the disease.


Assuntos
Betacoronavirus , Infecções por Coronavirus/patologia , Pulmão/patologia , Pneumonia Viral/patologia , Adenoviridae/genética , Adenoviridae/isolamento & purificação , Adulto , Autopsia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Bocavirus/genética , Bocavirus/isolamento & purificação , Proteína C-Reativa/análise , Coronavirus/genética , Coronavirus/isolamento & purificação , Feminino , Humanos , Influenzavirus A/genética , Influenzavirus A/isolamento & purificação , Influenzavirus B/genética , Influenzavirus B/isolamento & purificação , Macrófagos/patologia , Megacariócitos/patologia , Metapneumovirus/genética , Metapneumovirus/isolamento & purificação , Neutrófilos/patologia , Obesidade Mórbida , Pandemias , Pró-Calcitonina/sangue , Reação em Cadeia da Polimerase em Tempo Real , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhinovirus/genética , Rhinovirus/isolamento & purificação , Suíça , Linfócitos T/patologia
14.
PLoS One ; 15(4): e0232092, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352995

RESUMO

Human adenovirus (HAdV-7) is a highly contagious pathogen that causes severe respiratory illnesses. However, the epidemic patterns and genetic variability of HAdV-7 circulating in mainland China have not been well elucidated. In this study, we used Chinese HAdV sentinel surveillance data obtained from 2012-2015 to investigate the clinical features of 122 HAdV-7-positive cases and performed amplification and sequence determination of three capsid genes (penton base, hexon, and fiber) from 69 isolated viruses covering from seven provinces of China. Additionally, we compared with data from representative sequences of 21 strains covering seven more provinces in China and 32 international HAdV-7 strains obtained from GenBank database to determine the phylogenetic, sequence variations, and molecular evolution of HAdV-7. The results indicated that HAdV-7 infection occurred throughout the year, and a high proportion of severe cases (27 cases, 22.1%) exhibited infantile pneumonia. Moreover, phylogenetic analysis showed that all HAdV-7 strains could be divided into two major evolutionary branches, including subtype 1 and subtype 2, and subtype 3 was also formed according to analysis of the penton base gene. Subtypes 1 and 2 co-circulated in China before 2008, and HAdV-7 strains currently circulating in China belonged to subtype 2, which was also the predominant strain circulating worldwide in recent years. Further sequence variation analysis indicated that three genes of HAdV-7 were relatively stable across time and geographic space, particularly for viruses within subtypes, which shared almost the same variation sites. Owing to continuous outbreaks caused by HAdV-7, resulting in increased illness severity and fatality rates in China, the establishment of a national HAdV surveillance system is urgently needed for the development of effective preventive and infection-control interventions for adenovirus respiratory infections in China.


Assuntos
Infecções por Adenovirus Humanos/genética , Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , Adenoviridae/genética , Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/virologia , China/epidemiologia , Surtos de Doenças , Evolução Molecular , Variação Genética/genética , Humanos , Filogenia , Infecções Respiratórias/epidemiologia , Análise de Sequência de DNA/métodos
15.
Nat Commun ; 11(1): 1997, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332742

RESUMO

Persistent viruses cause chronic disease, and threaten the lives of immunosuppressed individuals. Here, we elucidate a mechanism supporting the persistence of human adenovirus (AdV), a virus that can kill immunosuppressed patients. Cell biological analyses, genetics and chemical interference demonstrate that one of five AdV membrane proteins, the E3-19K glycoprotein specifically triggers the unfolded protein response (UPR) sensor IRE1α in the endoplasmic reticulum (ER), but not other UPR sensors, such as protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The E3-19K lumenal domain activates the IRE1α nuclease, which initiates mRNA splicing of X-box binding protein-1 (XBP1). XBP1s binds to the viral E1A-enhancer/promoter sequence, and boosts E1A transcription, E3-19K levels and lytic infection. Inhibition of IRE1α nuclease interrupts the five components feedforward loop, E1A, E3-19K, IRE1α, XBP1s, E1A enhancer/promoter. This loop sustains persistent infection in the presence of the immune activator interferon, and lytic infection in the absence of interferon.


Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/patogenicidade , Proteínas E3 de Adenovirus/metabolismo , Endorribonucleases/metabolismo , Regulação Viral da Expressão Gênica/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Células A549 , Adenoviridae/genética , Adenoviridae/imunologia , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas E1A de Adenovirus/genética , Doença Crônica , Retículo Endoplasmático/metabolismo , Endorribonucleases/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Hospedeiro Imunocomprometido , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Proteínas Serina-Treonina Quinases/genética , Processamento de RNA , Latência Viral , Liberação de Vírus/genética , Proteína 1 de Ligação a X-Box/genética
16.
Res Vet Sci ; 131: 31-37, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32283442

RESUMO

Hepatitis-hydropericardium syndrome (HHS) caused by hypervirulent fowl adenovirus 4 (FAdV-4) have been causing great economic losses to Chinese poultry industry since 2015. Elucidation of the pathogenesis of FAdV-4 will lay solid foundation for developing attenuated FAdV-4 vaccine and vaccine vector. Our previous study has demonstrated that the increased virulence of hypervirulent FAdV-4 was associated with fiber-2 and hexon genes. However, the roles of fiber-1 and penton in virulence of FAdV-4 have never been elucidated. To further investigate the roles of the major structural proteins fiber-1 and penton in the virulence of hypervirulent FAdV-4, the fiber-1- and penton-replaced mutant viruses were constructed based on the FAdV-4 infectious clones of hypervirulent strain HNJZ using Redαß recombineering techniques. The pathogenicity of the rescued viruses was evaluated in 3-week-old SPF chickens. Chickens infected with the rescued recombinant viruses carrying the fiber-1 or penton base gene from a nonpathogenic strain ON1 developed similar clinical signs to the natural hypervirulent FAdV-4 infection, including HHS-indicative gross lesions and histopathological changes in sick/dead chickens. Our results suggested that the increased virulence of hypervirulent FAdV-4 was independent of fiber-1 and penton. The detailed pathogenesis of FAdV-4 and the roles of fiber-1 and penton in the viral replication and infection process need to be further explored.


Assuntos
Infecções por Adenoviridae/veterinária , Adenoviridae/patogenicidade , Doenças das Aves Domésticas/virologia , Proteínas Virais/metabolismo , Adenoviridae/genética , Infecções por Adenoviridae/virologia , Animais , Galinhas/imunologia , Virulência/genética
17.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 763-771, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347070

RESUMO

The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3ß. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.


Assuntos
Adenoviridae , Glucose , MicroRNAs , Adenoviridae/genética , Glucose/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Hep G2 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Transdução de Sinais/genética , Transfecção
18.
J Virol ; 94(10)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32161167

RESUMO

Nuclear import of viral genomes is an important step during the life cycle of adenoviruses (AdV), requiring soluble cellular factors as well as proteins of the nuclear pore complex (NPC). We addressed the role of the cytoplasmic nucleoporin Nup358 during adenoviral genome delivery by performing depletion/reconstitution experiments and time-resolved quantification of adenoviral genome import. Nup358-depleted cells displayed reduced efficiencies of nuclear import of adenoviral genomes, and the nuclear import receptor transportin 1 became rate limiting under these conditions. Furthermore, we identified a minimal N-terminal region of Nup358 that was sufficient to compensate for the import defect. Our data support a model where Nup358 functions as an assembly platform that promotes the formation of transport complexes, allowing AdV to exploit a physiological protein import pathway for accelerated transport of its DNA.IMPORTANCE Nuclear import of viral genomes is an essential step to initiate productive infection for several nuclear replicating DNA viruses. On the other hand, DNA is not a physiological nuclear import substrate; consequently, viruses have to exploit existing physiological transport routes. Here, we show that adenoviruses use the nucleoporin Nup358 to increase the efficiency of adenoviral genome import. In its absence, genome import efficiency is reduced and the transport receptor transportin 1 becomes rate limiting. We show that the N-terminal half of Nup358 is sufficient to drive genome import and identify a transportin 1 binding region. In our model, adenovirus genome import exploits an existing protein import pathway and Nup358 serves as an assembly platform for transport complexes.


Assuntos
Adenoviridae/genética , Adenoviridae/fisiologia , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Genoma Viral , Células HEK293 , Células HeLa , Humanos , Chaperonas Moleculares/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , beta Carioferinas/química
19.
Mol Biotechnol ; 62(4): 260-272, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32144553

RESUMO

Pre-existing immune response against adenovirus could diminish transgene expression efficiency when Ad is employed in humans as gene therapy vector. We previously used Ad-hΔuPA (Recombinant adenovirus expressing human urokinase-type plasminogen activator) as antifibrotic gene therapy in cirrhosis models and demonstrated its effectiveness. As a further clinical approach, transient Cyclosporine A (CsA) immunosuppression was induced in cirrhotic animals to determine whether Ad-hΔuPA administration retained efficacy. Adenovirus sensitization was achieved by systemic administration of non-therapeutic Ad-ßGal (Recombinant adenovirus expressing beta-galactosidase) after 4 weeks of intraperitoneal carbon tetrachloride (CCl4) regimen. Cirrhosis induction continued up to 8 weeks. At the end of CCl4 intoxication, immunosuppression was achieved with three CsA doses (40 mg/kg) as follows: 24 h before administration of Ad-hΔuPA, at the moment of Ad-hΔuPA injection and finally, 24 h after Ad-hΔuPA inoculation. At 2 and 72 h after Ad-hΔuPA injection, animals were sacrificed. Liver, spleen, lung, kidney, heart, brain, and testis were analyzed for Ad-biodistribution and transgene expression. In naïve animals, Ad-hΔuPA genomes prevailed in liver and spleen, while Ad-sensitized rats showed Ad genomes also in their kidney and heart. Cirrhosis and Ad preimmunization status notably diminished transgene liver expression compared to healthy livers. CsA immunosuppression in cirrhotic animals has no effect on Ad-hΔuPA biodistribution, but increments survival.


Assuntos
Adenoviridae/genética , Adenoviridae/metabolismo , Cirrose Hepática/terapia , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Tetracloreto de Carbono/administração & dosagem , Ciclosporina/uso terapêutico , Terapia Genética , Imunização , Imunossupressores/uso terapêutico , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Ratos , Distribuição Tecidual , Transgenes , Ativador de Plasminogênio Tipo Uroquinase/farmacocinética
20.
Emerg Microbes Infect ; 9(1): 586-596, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32174269

RESUMO

Since 2015, the prevalence of severe hepatitis-hydropericardium syndrome, which is caused by the novel genotype fowl adenovirus serotype 4 (FAdV-4), has increased in China and led to considerable economic losses. The replication cycle of FAdV-4, especially the emerging highly pathogenic novel genotype FAdV-4, remains largely unknown. The adenovirus fibre interacts with the cellular receptor as the initial step in adenovirus (AdV) infection. In our previous studies, the complete genome sequence showed that the fibre patterns of FAdV-4 were distinct from all other AdVs. Here, protein-blockage and antibody-neutralization assays were performed to confirm that the novel FAdV-4 short fibre was critical for binding to susceptible leghorn male hepatocellular (LMH) cells. Subsequently, fibre 1 was used as bait to investigate the receptor on LMH cells via mass spectrometry. The chicken coxsackie and adenovirus receptor (CAR) protein was confirmed as the novel FAdV-4 receptor in competition assays. We further identified the D2 domain of CAR (D2-CAR) as the active domain responsible for binding to the short fibre of the novel FAdV-4. Taken together, these findings demonstrate for the first time that the chicken CAR homolog is a cellular receptor for the novel FAdV-4, which facilitates viral entry by interacting with the viral short fibre through the D2 domain. Collectively, these findings provide an in-depth understanding of the mechanisms of the emerging novel genotype FAdV-4 invasion and pathogenesis.


Assuntos
Adenoviridae/imunologia , Receptores Virais/imunologia , Adenoviridae/genética , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Galinhas , Expressão Gênica , Humanos , Receptores Virais/genética , Solubilidade , Proteínas Virais/genética , Proteínas Virais/imunologia
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