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1.
Vet Microbiol ; 242: 108589, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32122593

RESUMO

The CRISPR/CRISPR-associated protein 9 (Cas9) system is a powerful gene-editing tool originally discovered as an integral mediator of bacterial adaptive immunity. Recently, this technology has been explored for its potential utility in providing new and unique treatments for viral infection. Marek's disease virus (MDV) and avian leukosis virus subgroup J (ALV-J), major immunosuppressive viruses, cause significant economic losses to the chicken industry. Here, we evaluated the efficacy of using MDV as a CRISPR/Cas9-delivery system to directly target and disrupt the reverse-transcribed products of the ALV-J RNA genome during its infection cycle in vitro and in vivo. We first screened multiple potential guide RNA (gRNA) target sites in the ALV-J genome and identified several optimized targets capable of effectively disrupting the latently integrated viral genome and providing efficient defense against new infection by ALV-J in cells. The optimal single-gRNAs and Cas9-expression cassettes were inserted into the genome of an MDV vaccine strain. The results indicated that engineered MDV stably expressing ALV-J-targeting CRISPR/Cas9 efficiently resisted ALV-J challenge in host cells. These findings demonstrated the CRISPR/Cas9 system as an effective treatment strategy against ALV-J infection. Furthermore, the results highlighted the potential of MDV as an effective delivery system for CRISPR/Cas9 in chickens.

2.
Hepatology ; 71(2): 463-476, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31278760

RESUMO

Nucleos(t)ide analogues (NAs) have been widely used for the treatment of chronic hepatitis B (CHB). Because viral DNA polymerase lacks proofreading function (3' exonuclease activity), theoretically, the incorporated NAs would irreversibly terminate viral DNA synthesis. This study explored the natures of nascent hepatitis B virus (HBV) DNA and infectivity of progeny virions produced under NA treatment. HBV infectivity was determined by infection of HepG2-NTCP cells and primary human hepatocytes (PHHs). Biochemical properties of HBV DNA in the progeny virions were investigated by qPCR, northern blotting, or Southern blotting hybridization, sucrose gradient centrifugation, and in vitro endogenous DNA polymerase assay. Progeny HBV virions produced under NA treatment were mainly not infectious to HepG2-NTCP cells or PHHs. Biochemical analysis revealed that under NA treatment, HBV DNA in nucleaocapsids or virions were predominantly short minus-strand DNA with irreversible termination. This finding was supported by the observation of first disappearance of relaxed circular DNA and then the proportional decline of HBV-DNA levels corresponding to the regions of PreC/C, S, and X genes in serial sera of patients receiving NA treatment. Conclusion: HBV virions produced under NA treatment are predominantly replication deficient because the viral genomes are truncated and elongation of DNA chains is irreversibly terminated. Clinically, our results suggest that the viral loads of CHB patients under NA therapy vary with the different regions of genome being detected by qPCR assays. Our findings also imply that NA prevention of perinatal and sexual HBV transmission as well as infection of transplanted livers works not only by reducing viral loads, but also by producing noninfectious virions.

3.
Adv Exp Med Biol ; 1179: 17-37, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31741332

RESUMO

Chronic hepatitis B virus (HBV) infection remains to be a serious threat to public health and is associated with many liver diseases including chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma. Although nucleos(t)ide analogues (NA) and pegylated interferon-α (Peg-IFNα) have been confirmed to be efficient in inhibiting HBV replication, it is difficult to eradicate HBV and achieve the clinical cure of CHB. Therefore, long-term therapy has been recommended to CHB treatment under the current antiviral therapy. In this context, the new antiviral therapy targeting one or multiple critical steps of viral life cycle may be an alternative approach in future. In the last decade, the functional receptor [sodium-taurocholate cotransporting polypeptide (NTCP)] of HBV entry into hepatocytes has been discovered, and the immature nucleocapsids containing the non- or partially reverse-transcribed pregenomic RNA, the nucleocapsids containing double-strand linear DNA (dslDNA), and the empty particles devoid of any HBV nucleic acid have been found to be released into circulation, which have supplemented the life cycle of HBV. The understanding of HBV life cycle may offer a new instruction for searching the potential antiviral targets, and the new viral markers used to monitor the efficacy of antiviral therapy for CHB patients in the future.


Assuntos
Antivirais , Vírus da Hepatite B , Hepatite B Crônica , Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatite B Crônica/virologia , Hepatócitos/virologia , Humanos , Interferon-alfa/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
4.
Ann Transl Med ; 7(18): 431, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31700867

RESUMO

Background: The systemic immune-inflammation index (SII) has been used as a prognostic marker for several cancer types, but there is no in-depth study in bladder cancer. This study evaluated the potential utility of the SII as a prognostic factor in patients with bladder cancer after radical cystectomy. Methods: A retrospective analysis of 209 patients with bladder cancer who had undergone radical cystectomy and were randomized into primary (N=139) and validation (N=70) cohorts was conducted. The overall survival (OS) was calculated using the Kaplan-Meier survival curves. The prognostic value of the SII in primary and validation cohorts were analyzed by using the Cox regression model. A SII-based nomogram for bladder cancer was produced in R software. Results: A high SII (>507) was associated with poor prognosis in bladder cancer patients. Univariate and multivariate analyses revealed that the SII was an independent predictor for OS. The SII emerged as an independent prognostic factor that provided more accurate prognostic prediction than neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and C-reactive protein/albumin ratio (CAR), in the primary and validation cohorts. The nomogram had better accuracy and discrimination than tumor, lymph node, metastasis (TNM) classification. The concordance index values of nomogram were 0.82 for the primary cohort and 0.784 for the validation cohort. Conclusions: The SII can serve as an independent predictor of OS in patients who have undergone radical cystectomy for bladder cancer, and was found to be a better predictor of prognosis than NLR, PLR, and CAR. The nomogram is a reliable model for predicting postoperative OS of patients after radical cystectomy.

5.
PLoS Pathog ; 15(9): e1007999, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31539404

RESUMO

The cellular DNA sensor cGMP-AMP synthase (cGAS) detects cytosolic viral DNA via the stimulator of interferon genes (STING) to initiate innate antiviral response. Herpesviruses are known to target key immune signaling pathways to persist in an immune-competent host. Marek's disease virus (MDV), a highly pathogenic and oncogenic herpesvirus of chickens, can antagonize host innate immune responses to achieve persistent infection. With a functional screen, we identified five MDV proteins that blocked beta interferon (IFN-ß) induction downstream of the cGAS-STING pathway. Specifically, the MDV major oncoprotein Meq impeded the recruitment of TANK-binding kinase 1 and IFN regulatory factor 7 (IRF7) to the STING complex, thereby inhibiting IRF7 activation and IFN-ß induction. Meq overexpression markedly reduced antiviral responses stimulated by cytosolic DNA, whereas knockdown of Meq heightened MDV-triggered induction of IFN-ß and downstream antiviral genes. Moreover, Meq-deficient MDV induced more IFN-ß production than wild-type MDV. Meq-deficient MDV also triggered a more robust CD8+ T cell response than wild-type MDV. As such, the Meq-deficient MDV was highly attenuated in replication and lymphoma induction compared to wild-type MDV. Taken together, these results revealed that MDV evades the cGAS-STING DNA sensing pathway, which underpins the efficient replication and oncogenesis. These findings improve our understanding of the virus-host interaction in MDV-induced lymphoma and may contribute to the development of novel vaccines against MDV infection.


Assuntos
Herpesvirus Galináceo 2/imunologia , Herpesvirus Galináceo 2/patogenicidade , Evasão da Resposta Imune , Doença de Marek/imunologia , Doença de Marek/virologia , Animais , Proteínas Aviárias/metabolismo , Carcinogênese , Galinhas , DNA Viral/imunologia , Patos , Herpesvirus Galináceo 2/fisiologia , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade Inata , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/metabolismo , Doença de Marek/metabolismo , Modelos Imunológicos , Nucleotidiltransferases/metabolismo , Proteínas Oncogênicas Virais/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Virais/imunologia , Replicação Viral
6.
Viruses ; 11(8)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394878

RESUMO

Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3'-untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis.

7.
J Virol ; 93(18)2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31243133

RESUMO

Marek's disease virus (MDV), which causes T cell lymphomas in chickens, is economically important and has contributed to knowledge of herpesvirus-associated oncogenicity. The DNA-sensing pathway induces innate immune responses against DNA virus infection, and nuclear factor κB (NF-κB) signaling is critical for the establishment of innate immunity. Here, we report that RLORF4, an MDV-specific protein directly involved in viral attenuation, is an inhibitor of the DNA-sensing pathway. The results showed that ectopically expressed RLORF4 blocked beta interferon (IFN-ß) promoter activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). RLORF4 selectively inhibited the activation of NF-κB but not IFN-regulatory factor 7. RLORF4 was found to bind the endogenous NF-κB subunits p65 and p50, and it also bound to the Rel homology domains of these subunits. Furthermore, RLORF4 suppressed the nuclear translocation of p65 and p50 mediated by tumor necrosis factor alpha and interferon-stimulatory DNA. Finally, deletion of RLORF4 from the MDV genome promoted IFN-ß and interleukin-6 (IL-6) production in vitro and in vivo In the absence of RLORF4, the host cellular immunity was significantly increased, and reduced viral titers were observed during infection of chickens. Our results suggest that the RLORF4-mediated suppression of the host antiviral innate immunity might play an important role in MDV pathogenesis.IMPORTANCE Marek's disease virus (MDV) RLORF4 has been shown to be directly involved in the attenuation of MDV upon serial passages in vitro; however, the exact function of this protein during viral infection was not well characterized. This study demonstrated that RLORF4 significantly inhibits cGAS-STING-mediated NF-κB activation by binding to the Rel homology domains of the NF-κB subunits p65 and p50, interrupting their translocation to the nuclei and thereby inhibiting IFN-ß production. Furthermore, RLORF4 deficiency promoted the induction of IFN-ß and downstream IFN-stimulated genes during MDV infection in chickens. Our results suggest that the contribution of RLORF4 to MDV virulence may stem from its inhibition of viral DNA-triggered IFN-ß responses.

8.
Drugs R D ; 19(3): 255-265, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31197606

RESUMO

BACKGROUND: Two phase I studies assessed the pharmacokinetics of buprenorphine, its metabolite norbuprenorphine, and naloxone following administration of buprenorphine/naloxone sublingual tablets in Chinese participants. METHODS: In the first phase I, open-label, single ascending-dose (SAD) study, 82 opioid-naïve volunteers received a single buprenorphine/naloxone dose ranging from 2 mg/0.5 mg to 24 mg/6 mg while under naltrexone block. In a second phase I, open-label, multiple ascending-dose (MAD) study, 27 patients with opioid dependence in withdrawal received buprenorphine/naloxone doses of either 16 mg/4 mg or 24 mg/6 mg for 9 consecutive days. Serial blood samples were collected after a single dose (SAD study) and at steady-state (MAD study). Pharmacokinetic parameters were calculated using non-compartmental analysis. Safety assessments included adverse events monitoring and laboratory tests. RESULTS: The pharmacokinetic profiles of buprenorphine and naloxone were consistent between single- and multiple-dose studies. Peak plasma concentrations (Cmax) were reached early for buprenorphine (0.75-1.0 h) and naloxone (0.5 h), supporting rapid absorption. In the SAD study, increases in plasma exposures to buprenorphine and naloxone were less than dose proportional, in line with previous observations in Western populations. Buprenorphine-to-naloxone ratios for Cmax and area under the curve (AUC) were constant over the dose range investigated and also consistent with Western populations data. Steady state was reached within 7 days of daily dosing, with slight accumulation over repeated doses. No serious adverse events were observed. CONCLUSIONS: The present data suggest that buprenorphine/naloxone pharmacokinetic profiles in Chinese participants are consistent, overall, with those in Western populations, supporting no differences in dosing. CLINICAL TRIAL REGISTRATION: The protocols were registered on the official website of the China Food and Drug Administration (CFDA): http://www.chinadrugtrials.org.cn/ ; Registration numbers CTR20132963 (RB-CN-10-0012), CTR20140153 (RB-CN-10-0015).


Assuntos
Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/farmacocinética , Buprenorfina/administração & dosagem , Buprenorfina/farmacocinética , Comprimidos/administração & dosagem , Comprimidos/farmacocinética , Administração Sublingual , Adulto , Área Sob a Curva , Grupo com Ancestrais do Continente Asiático , Disponibilidade Biológica , Buprenorfina/análogos & derivados , Feminino , Humanos , Masculino , Naloxona/administração & dosagem , Naloxona/farmacocinética , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Transtornos Relacionados ao Uso de Opioides/metabolismo
10.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30518647

RESUMO

The type I interferon (IFN) response is the first line of host innate immune defense against viral infection; however, viruses have developed multiple strategies to antagonize host IFN responses for efficient infection and replication. Here, we report that Marek's disease virus (MDV), an oncogenic herpesvirus, encodes VP23 protein as a novel immune modulator to block the beta interferon (IFN-ß) activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) in chicken fibroblasts and macrophages. VP23 overexpression markedly reduces viral DNA-triggered IFN-ß production and promotes viral replication, while knockdown of VP23 during MDV infection enhances the IFN-ß response and suppresses viral replication. VP23 selectively inhibits IFN regulatory factor 7 (IRF7) but not nuclear factor κB (NF-κB) activation. Furthermore, we found that VP23 interacts with IRF7 and blocks its binding to TANK-binding kinase 1 (TBK1), thereby inhibiting IRF7 phosphorylation and nuclear translocation, resulting in reduced IFN-ß production. These findings expand our knowledge of DNA sensing in chickens and reveal a mechanism through which MDV antagonizes the host IFN response.IMPORTANCE Despite widespread vaccination, Marek's disease (MD) continues to pose major challenges for the poultry industry worldwide. MDV causes immunosuppression and deadly lymphomas in chickens, suggesting that this virus has developed a successful immune evasion strategy. However, little is known regarding the initiation and modulation of the host innate immune response during MDV infection. This study demonstrates that the cGAS-STING DNA-sensing pathway is critical for the induction of the IFN-ß response against MDV infection in chicken fibroblasts and macrophages. An MDV protein, VP23, was found to efficiently inhibit the cGAS-STING pathway. VP23 selectively inhibits IRF7 but not NF-κB activation. VP23 interacts with IRF7 and blocks its binding to TBK1, thereby suppressing IRF7 activation and resulting in inhibition of the DNA-sensing pathway. These findings expand our knowledge of DNA sensing in chickens and reveal a mechanism through which MDV antagonizes the host IFN response.


Assuntos
Proteínas do Capsídeo/metabolismo , Herpesvirus Galináceo 2/genética , Fator Regulador 7 de Interferon/metabolismo , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Galinhas/genética , DNA Viral/metabolismo , Células HEK293 , Herpesvirus Galináceo 2/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Imunidade Inata , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/metabolismo , Interferon beta/genética , Doença de Marek/genética , Doença de Marek/virologia , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Nucleotidiltransferases , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Proteínas Virais/metabolismo , Replicação Viral/genética
11.
Appl Microbiol Biotechnol ; 103(1): 427-435, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30349931

RESUMO

Avian leukosis virus (ALV) is an avian oncogenic retrovirus that induces leukemia-like proliferative diseases in chickens. ALV infection can result in the development of immunological tolerance and persistent viremia. Since effective vaccines against ALV are not yet available, its current prevention primarily depends on detection and eradication to establish exogenous ALV-free poultry flocks. In this study, a rapid and simple colloidal gold test strip method, specific for the group-specific antigen, p27 protein, was developed and systematically evaluated for the detection of ALV from different samples. The detection limit of this assay was as low as 6.25 ng/ml for p27 protein and 80 TCID50/ml for different subgroups of ALV. Besides, the test strip showed high specificity in the detection of different subgroups of ALV, including ALV-A, ALV-B, ALV-J, and ALV-K, with no cross-reaction with other avian pathogens. Furthermore, we artificially infected specific pathogen-free (SPF) chickens with ALV-J, collected cloacal swabs, and examined viral shedding using both test strips and ELISA. Results from the test strip were highly consistent with that from ELISA. In addition, 1104 virus isolates from anti-coagulant blood samples, 645 albumen samples, and 4312 meconium samples were tested, and the test strip results agreed with those of ELISA kit up to 97.1%. All the results indicated that the colloidal gold test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for eradication of ALV in poultry farms.


Assuntos
Vírus da Leucose Aviária/isolamento & purificação , Leucose Aviária/diagnóstico , Coloide de Ouro , Imunoensaio/métodos , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Leucose Aviária/virologia , Vírus da Leucose Aviária/patogenicidade , Galinhas , Cabras , Imunoensaio/instrumentação , Imunoglobulina G/imunologia , Limite de Detecção , Camundongos , Doenças das Aves Domésticas/virologia , Sensibilidade e Especificidade , Fatores de Tempo
12.
Vet Microbiol ; 228: 32-38, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30593377

RESUMO

Avian metapneumovirus (aMPV), which has been reported in many countries, causes an acute upper respiratory tract disease in chickens and turkeys. Although aMPV was first detected in China in 1999, there has been no further effort to isolate and characterize the aMPV subtype B (aMPV/B) from field outbreaks. In the present study, we used Vero cells to culture a viral strain, LN16, isolated from chickens with swollen head syndrome. The results of RT-PCR, indirect immunofluorescent antibody, and G gene sequence analyses confirmed that strain LN16 corresponds to aMPV/B. We amplified and sequenced the complete genome of strain LN16 and found it to be 13,513 nucleotides in length. Nine viral protein genes of the strain were between 93.2% and 98.4% identical to those of the pathogenic field isolate VCO3/60616. However, insertions and deletions were detected in the intergenic regions. Animal experiments showed that 72.7% of chickens infected with strain LN16 had excess mucus, nasal discharge, and inflammation in the lungs and turbinate. In addition, 27.2% of chickens infected with LN16 shed progeny virions. Viral tissue distribution analysis showed that aMPV could be detected in the turbinate and occasionally in immune organs. This is the first report of the isolation of aMPV/B in China and the first complete genome sequence of aMPV/B from chicken. These findings enrich the epidemiological data on aMPV and may contribute to the development of effective measures to prevent its further spread in China.


Assuntos
Galinhas/virologia , Genoma Viral/genética , Metapneumovirus/genética , Metapneumovirus/patogenicidade , Infecções por Paramyxoviridae/veterinária , Doenças das Aves Domésticas/virologia , Animais , China , Metapneumovirus/isolamento & purificação , Infecções por Paramyxoviridae/patologia , Infecções por Paramyxoviridae/virologia , Filogenia , Doenças das Aves Domésticas/patologia , Células Vero , Proteínas Virais/genética , Virulência
13.
Mol Ther ; 26(6): 1457-1470, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29724685

RESUMO

CH12 is a novel humanized monoclonal antibody against epidermal growth factor receptor variant III (EGFRvIII) for cancer treatment. Unfortunately, in pre-clinical safety evaluation studies, acute thrombocytopenia was observed after administration of CH12 in cynomolgus monkeys, but not rats. More importantly, in vitro experiments found that CH12 can bind and activate platelets in cynomolgus monkey, but not human peripheral blood samples. Cynomolgus monkey-specific thrombocytopenia has been reported previously; however, the underlying mechanism remains unclear. Here, we first showed that CH12 induced thrombocytopenia in cynomolgus monkeys through off-target platelet binding and activation, resulting in platelet destruction. We subsequently found that integrin αIIbß3 (which is expressed on platelets) contributed to this off-target toxicity. Furthermore, three-dimensional structural modeling of the αIIbß3 molecules in cynomolgus monkeys, humans, and rats suggested that an additional unique loop exists in the ligand-binding pocket of the αIIb subunit in cynomolgus monkeys, which may explain why CH12 binds to platelets only in cynomolgus monkeys. Moreover, this study supported the hypothesis that the minor differences between cynomolgus monkeys and humans can confuse human risk assessments and suggests that species differences can help the prediction of human risks and avoid losses in drug development.


Assuntos
Anticorpos Monoclonais/metabolismo , Integrina alfa2/metabolismo , Integrina beta3/metabolismo , Trombocitopenia/imunologia , Trombocitopenia/metabolismo , Animais , Feminino , Humanos , Macaca fascicularis , Masculino , Ratos
14.
Virology ; 519: 121-130, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29698854

RESUMO

Avian leukosis virus subgroup J (ALV-J), a highly oncogenic retrovirus, causes leukemia-like proliferative diseases in chickens. microRNAs post-transcriptionally suppress targets and are involved in the development of various tumors. We previously showed that miR-221 is upregulated in ALV-J-induced tumors. In this study, we analyzed the possible function of miR-221 in ALV-J tumorigenesis. The target validation system showed that CDKN1B is a target of miR-221 and is downregulated in ALV-J infection. As CDKN1B arrests the cell cycle and regulates its progression, we analyzed the proliferation of ALV-J-infected DF-1 cells. ALV-J-infection-induced DF1 cell derepression of G1/S transition and overproliferation required high miR-221 expression followed by CDKN1B downregulation. Cell cycle pathway analysis showed that ALV-J infection induced DF-1 cell overproliferation via the CDKN1B-CDK2/CDK6 pathway. Thus, miR-221 may play an important role in ALV-J-induced aggressive growth of DF-1 cells; these findings have expanded our insights into the mechanism underlying ALV-J infection and tumorigenesis.


Assuntos
Vírus da Leucose Aviária/fisiologia , Ciclo Celular/genética , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/genética , Interações Hospedeiro-Patógeno , MicroRNAs/genética , Animais , Leucose Aviária/virologia , Vírus da Leucose Aviária/classificação , Vírus da Leucose Aviária/genética , Carcinogênese/genética , Ciclo Celular/fisiologia , Linhagem Celular , Galinhas/virologia , Biologia Computacional , Regulação para Baixo , Fibroblastos/fisiologia , Fibroblastos/virologia , Doenças das Aves Domésticas/virologia
15.
J Virol ; 92(1)2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29070685

RESUMO

Chicken Na+/H+ exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry.IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are located in the membrane-proximal region of the N terminus of chECL1, suggesting that the binding site of ALV-J gp85 on chNHE1 is probably located on the apex of the molecule; the receptor-binding mode might be different from that of retroviruses. We also found that soluble chECL1, as well as huECL1 harboring chNHE1 residues 28 to 39, effectively blocked ALV-J infection. These findings contribute to a better understanding of the ALV-J infection mechanism and also provide new insights into the control strategies for ALV-J infection.


Assuntos
Aminoácidos/química , Vírus da Leucose Aviária/metabolismo , Receptores Virais/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo , Ligação Viral , Internalização do Vírus , Aminoácidos/metabolismo , Animais , Leucose Aviária/virologia , Vírus da Leucose Aviária/química , Vírus da Leucose Aviária/genética , Galinhas , Humanos , Mutação Puntual , Receptores Virais/genética , Trocadores de Sódio-Hidrogênio/genética
16.
Oncotarget ; 8(59): 100141-100149, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29245966

RESUMO

Genetic factors play a vital role in the pathogenesis of premature myocardial infarction (PMI). However, current studies explained only small amounts of genetic risk in MI. In this study, we started from a PMI pedigree with three MI patients occurred at the age of 43, 45 and 53 respectively. Sanger sequencing revealed 6 LDLR mutation carriers in the family, but only one was diagnosed with PMI, indicating that the LDLR mutation may not be the reason for PMI. Upon exome-sequencing and bioinformatics analysis, two variants in SCAP and AGXT2 were identified as potential causative mutation for PMI. Further observation revealed that only patients that meet the diagnosis of PMI harbored two variants meantime, while other MI patients or members with no MI carried no more than one of the variants. Screening of the two genes in an independent PMI population identified another variant on SCAP (c.1403 T>C, p.Val468Ala), which was absent in 28, 000 east-Asian population. Further, the two variants on SCAP and AGXT2 were introduced into H293T and EA. hy926 cell lines respectively utilizing CRISPR-Cas9. Functional study revealed that the SCAP mutation impaired SCAP-SREBP feedback mechanism which may lead to a "constitutive activation" effect of cholesterol synthesis related genes, while the AGXT2 mutation reduced its aminotransferase activity leading to a down-regulation of NO production by ADMA accumulation. This study indicates that SCAP and AGXT2 are potential causative genes for PMI. Digenic mutation carriers may manifest a more severe phenotype, namely premature MI.

17.
Sci Rep ; 7(1): 2796, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28584302

RESUMO

The p53 mutation and altered Pten expression are two most common genetic events in Hepatitis B virus (HBV) infection related hepatocellular carcinoma (HCC). To confirm the causative role of p53 and Pten somatic mutation in HCC development, we established CRISPR/Cas9-mediated somatic gene disruption via hydrodynamic tail vein injection, allowing for in vivo targeting p53 and Pten simultaneously in adult HBV transgenic mice. Here we demonstrated that the utility of this approach resulted in macroscopic liver tumors as early as 4 months' post injection and most tumors harbored both p53 and Pten loss-of-function alterations. Immunohistochemical (IHC) and histopathology analysis demonstrated that the tumors were positive for Glutamine synthetase (GS), a marker of HCC and accompanied with prominent lipid accumulation. The study here indicated that CRISPR/Cas9-mediated p53 and Pten somatic mutation accelerated hepatocarcinogenesis in adult HBV transgenic mice. This method also provides a fast and convenient system for generating mouse model of HCC with HBV infection characteristics.


Assuntos
Carcinoma Hepatocelular/etiologia , Vírus da Hepatite B/genética , Hepatite B/complicações , Hepatite B/virologia , Neoplasias Hepáticas/etiologia , PTEN Fosfo-Hidrolase/genética , Proteína Supressora de Tumor p53/genética , Animais , Biópsia , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica , Análise Mutacional de DNA , Expressão Gênica , Ordem dos Genes , Marcação de Genes , Vetores Genéticos/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Transgênicos , Mutação , PTEN Fosfo-Hidrolase/metabolismo , RNA Guia , Transfecção , Carga Tumoral , Proteína Supressora de Tumor p53/metabolismo
18.
Int J Cancer ; 141(3): 540-548, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28470669

RESUMO

Human papillomavirus (HPV) infection is the most important risk factor for cervical cancer development. In HeLa cell line, the HPV viral genome is integrated at 8q24 in one allele of chromosome 8. It has been reported that the HPV fragment integrated in HeLa genome can cis-activate the expression of proto-oncogene MYC, which is located at 500 kb downstream of the integrated site. However, the underlying molecular mechanism of this regulation is unknown. A recent study reported that MYC was highly expressed exclusively from the HPV-integrated haplotype, and a long-range chromatin interaction between the integrated HPV fragment and MYC gene has been hypothesized. In this study, we provided the experimental evidences supporting this long-range chromatin interaction in HeLa cells by using Chromosome Conformation Capture (3C) method. We found that the integrated HPV fragment, MYC and 8q24.22 was close to each other and might form a trimer in spatial location. When knocking out the integrated HPV fragment or 8q24.22 region from chromosome 8 by CRISPR/Cas9 system, the expression of MYC reduced dramatically in HeLa cells. Interestingly, decreased expression was only observed in three from eight cell clones, when only one 8q24.22 allele was knocked out. Functionally, HPV knockout caused senescence-associated acidic ß-gal activity in HeLa cells. These data indicate a long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region, providing an alternative mechanism relevant to the carcinogenicity of HPV integration.


Assuntos
Cromossomos Humanos Par 8/genética , Genoma Viral/genética , Papillomaviridae/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Integração Viral/genética , Sistemas CRISPR-Cas , Senescência Celular , Feminino , Células HeLa , Humanos , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Proteínas Proto-Oncogênicas c-myc/genética
19.
Vet Immunol Immunopathol ; 186: 55-59, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28413051

RESUMO

Infectious bursal disease (IBD) causes significant clinical and economic losses to the poultry industry worldwide. Current vaccine programs using live attenuated and inactivated vaccines have numerous drawbacks. As an alternative solution to control IBD, a Marek's disease virus (MDV) vector vaccine (rMDV-VP2) expressing the VP2 gene of infectious bursal disease virus (IBDV) has been developed. In this study, the protective efficacy of rMDV-VP2 was evaluated in a dose-related experiment which showed that a single dose of 1000 PFU was sufficient to fully protect chickens against IBDV infection. Chickens inoculated with lower doses of rMDV-VP2 (250 or 500 PFU) conferred 80 and 90% protection against IBDV. Next, rMDV-VP2 vaccine provided 90% protection against IBDV in commercial layer chickens with maternal antibodies, which was higher than the protective efficacy using the B87 live vaccine of IBDV. Additionally, rMDV-VP2 conferred effective protection against very virulent MDV challenge in chickens (95% for chickens vaccinated with 250 or 500 PFU and 100% for chickens vaccinated with 1000 or 2000 PFU). These results demonstrated that rMDV-VP2 may be a novel bivalent vaccine against IBD and Marek's disease in chickens.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Birnaviridae/veterinária , Galinhas , Vírus da Doença Infecciosa da Bursa , Vacinas contra Doença de Marek/administração & dosagem , Doenças das Aves Domésticas/prevenção & controle , Proteínas Estruturais Virais/imunologia , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/prevenção & controle , Imunidade Materno-Adquirida , Vírus da Doença Infecciosa da Bursa/genética , Vacinas contra Doença de Marek/genética , Vacinas contra Doença de Marek/imunologia , Doenças das Aves Domésticas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Estruturais Virais/genética
20.
Virus Res ; 235: 82-85, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28263843

RESUMO

Marek's disease virus (MDV) is a preferred vector for recombinant vaccine construction, and insertion site is the main factor influencing foreign gene expression and vaccine efficacy. In this study, recombinant MDVs inserted with the enhanced green fluorescent protein (eGFP) gene at different sites in its genome were generated from overlapping fosmid DNAs and the eGFP expression was compared. The results showed that the eGFP expression levels from the sites in the unique long (UL) region (within UL41, between UL45 and UL46, and between UL55 and LORF10) were comparable, which were significantly higher than those from the sites in the unique short (US) region (US2 and US10), and the eGFP expression level from US2 was significantly higher than that from US10. The identification and comparison of the insertion sites in MDV genome could help elevate the protection efficacy of the recombinant MDVs expressing desired antigens.


Assuntos
Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/análise , Herpesvirus Galináceo 2/genética , Substâncias Luminescentes/análise , Coloração e Rotulagem/métodos , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética
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