Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.
Ebolavirus/physiology* ; HEK293 Cells ; HeLa Cells ; Humans ; Nucleocapsid Proteins/metabolism* ; RNA, Viral/metabolism* ; Viral Matrix Proteins/metabolism* ; Virion/metabolism* ; Virus Assembly

No abstract available.
Adolescent ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; Burkitt Lymphoma/*pathology ; Child, Preschool ; Cyclophosphamide/therapeutic use ; Doxorubicin/therapeutic use ; Female ; Gene Rearrangement ; Herpesvirus 4, Human/metabolism ; Humans ; Immunohistochemistry ; Lymphoma, B-Cell/*diagnosis/drug therapy ; Lymphoma, Large B-Cell, Diffuse/*pathology ; Male ; Prednisone/therapeutic use ; Proto-Oncogene Proteins c-myc/genetics ; Tomography, X-Ray Computed ; Vincristine/therapeutic use ; Viral Matrix Proteins/immunology/metabolism

Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.
Epstein-Barr Virus Infections/complications/virology ; Epstein-Barr Virus Nuclear Antigens/genetics/metabolism ; *Genes, Viral ; Herpesvirus 4, Human/*physiology ; Humans ; MicroRNAs/genetics ; Neoplasms/etiology ; Protein Binding ; RNA, Viral/genetics ; Viral Matrix Proteins/genetics/metabolism ; *Virus Latency

BACKGROUND: Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. METHOD: 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. RESULTS: The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. CONCLUSIONS: Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients.
Antiviral Agents/therapeutic use ; Cytomegalovirus/*genetics ; Cytomegalovirus Infections/drug therapy/pathology/virology ; DNA, Viral/*blood/metabolism ; Ganciclovir/therapeutic use ; Humans ; *Immunoassay ; Organ Transplantation ; Phosphoproteins/genetics/immunology/*metabolism ; *Real-Time Polymerase Chain Reaction ; Viral Matrix Proteins/genetics/immunology/*metabolism ; Virology/*methods

The purpose of this study was to investigate the genetic features of canine coronavirus (CCV) strains detected in Korea. M gene sequences obtained for isolates from 22 dogs with enteritis over a 5-year period were evaluated. Sequence comparison revealed that the 22 Korean CCV strains had an 87.2 to 100% nucleotide homology. Comparing to the typical reference CCV strains (type II), the nucleotide sequence of Korean strains had homology ranged from 86.3% to 98.3% (89.1% to 99.2% for the amino acid sequence) and 87.7% to 97.8% (92.4% to 100% for the amino acid sequence) when compared to FCoV-like CCV strains (type I). Three amino acid variations in the M gene were characteristic for the Korean CCV strains. Phylogenetic analysis demonstrated that the 22 Korean CCV strains belonged to four typical CCV clusters (i.e., a unique Korean CCV cluster, a type II and transmissible gastroenteritis virus cluster, an intermediate cluster between type I and II, and a type I cluster). This study was the first to identify genetic differences of the M gene from Korean CCV strains and provided a platform for molecular identification of different Korean CCV strains.
Amino Acid Sequence ; Animals ; Coronavirus Infections/epidemiology/*veterinary/virology ; Coronavirus, Canine/*isolation & purification ; Dog Diseases/*epidemiology/virology ; Dogs ; Female ; Male ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction/veterinary ; Republic of Korea/epidemiology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Viral Matrix Proteins/*genetics/metabolism

OBJECTIVETo profile the clinicopathologic features of a series of grey zone lymphoma (GZL) cases with hybrid features of diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (CHL), with a purpose to gain an in-depth understanding of the borderline B-cell neoplasm.

METHODSThe clinical, morphologic and immunophenotyical characteristics of 16 cases were retrospectively analyzed.

RESULTSThe patients were mostly male adults, with a male to female ratio of 1.7: 1.0 and a mean age of 40.2 years. Eight patients presented with peripheral nodal lesions and five cases with mediastinal involvement. Histologically and immunophenotypically, the 16 cases were classified into three sub-categories. In 4 cases, the morphologic features resembled CHL more closely, but the neoplastic cells showed uniform and intense positive staining of CD20 (pattern 1). Although the initial impression of the other 8 cases was that of DLBCL, the expression levels of CD20 and PAX5 were variable, and CD30 or CD15 was positive (pattern 2). A characteristic feature of pattern 3, observed in the remaining 4 cases, demonstrated a broad spectrum of morphology with hybrid features of both CHL and DLBCL. The neoplastic cells in pattern 3 were positive for CD20, CD30 and CD15. EBV-LMP1 was detected in 6 of the 11 tested cases. Clinically, most patients with GZL seemed insensitive to immuno-chemotherapy of the R-CHOP regimen.

CONCLUSIONSThe diagnostic criteria for GZL with features intermediate between DLBCL and CHL is proposed by the three histologic patterns commonly seen in these lesions. Cases presented with peripheral lesions might differ from those with mediastinal presentation pathologically. At current time, there is no effective treatment for these borderline B-cell lymphomas and the prognosis is poor.

Adolescent ; Adult ; Aged ; Antibodies, Monoclonal, Murine-Derived ; therapeutic use ; Antigens, CD20 ; metabolism ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Cyclophosphamide ; therapeutic use ; Diagnosis, Differential ; Doxorubicin ; therapeutic use ; Female ; Hodgkin Disease ; drug therapy ; metabolism ; pathology ; Humans ; Ki-1 Antigen ; metabolism ; Lewis X Antigen ; metabolism ; Lymphoma, Large B-Cell, Diffuse ; drug therapy ; metabolism ; pathology ; Male ; Middle Aged ; PAX5 Transcription Factor ; metabolism ; Prednisone ; therapeutic use ; Prognosis ; Retrospective Studies ; Rituximab ; Vincristine ; therapeutic use ; Viral Matrix Proteins ; metabolism ; Young Adult

This study was purposed to investigate the expression of latent membrane protein 1 (LMP-1) and CD68 in Hodgkin's lymphoma (HL) patients with EB virus infection and to analyze the relation of LMP-1 expression and CD68(+) tumor-associated macrophage count with clinical features and prognosis of HL patients. The expression of LMP1 and count of CD68(+) TAM were detected by immunohistochemical staining in tissue specimens of 72 HL patients; their correlation with clinical features and prognosis of HL patients was analyzed by using statistical method. The results showed that among tissue specimens of 72 HL patients, the positive rate of LMP-1 expression was 18.1% (13/72), the CD68(+) TAM count was more higher in LMP-1 positive expression [250 of CD68(+) TAM/high power field (hpf) is used as demarcation point] (P = 0.003). The statistical analysis showed that the LMP-1 positive expression was more observed in mixed type HL patients (P = 0.000); the positive rate of LMP-1 expression was much high in HL patients with albumin <40 g/L and age ≥ 45 years (P < 0.05). There was no relation of LMP-1 expression and CD68(+) TAM count with the short term therapeutic efficacy of HL patients, but the overall survival time of LMP-1 positive patients among patients followed-up for ≥ 5 years was short (P < 0.05). Moveover, no correlation of CD68(+) TAM count with the overall survival time of HL patients was found. It is concluded that the high count of CD68(+) TAM is more observed in LMP-1 positive expression of HL tissue, the LMP-1 expression states relates both with the pathological types, age and albumin level of patient with HL. The HL patients with LMP-1 positive expression have poor prognosis, suggesting that LMP-1 may be a new prognostic marker for HL patients.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Biomarkers, Tumor ; metabolism ; Child ; Epstein-Barr Virus Infections ; Female ; Hodgkin Disease ; diagnosis ; virology ; Humans ; Male ; Middle Aged ; Prognosis ; Viral Matrix Proteins ; metabolism ; Young Adult

OBJECTIVETo investigate Epstein-Barr virus(EBV) infection in Hodgkin's lymphoma (HL) of Uygur patients and related clinicopathological characteristics.

METHODSEBV-encoded small RNA (EBER) was detected in 40 cases of HL and 20 cases of lymphoid reactive hyperplasia by in-situ hybridization. Expression of LMP2A in HL was investigated by immunohistochemistry.

RESULTSEBV was detected in 26/40 (65.0%) of HL and 5/20 of lymphoid reactive hyperplasia (P < 0.05). The expression level of EBER showed significant difference among various histological subtypes of HL (P < 0.05) and between patients with and without B symptom (P = 0.02). However, no difference was found in relation to gender, clinical stage and tumor burden. The expression of LMP2A in the mixed cellularity and nodular sclerosis classical HL associated with EBV infection was 57.7% (15/26). Expression of LMP2A was not detected in lymphoid reactive hyperplasia cases.

CONCLUSIONUyghur patients with Hodgkin's lymphoma have a high infection rate of EBV and distinct clinicopathologic characteristics.

Adolescent ; Adult ; Child ; Child, Preschool ; China ; ethnology ; Epstein-Barr Virus Infections ; Female ; Herpesvirus 4, Human ; isolation & purification ; Hodgkin Disease ; metabolism ; pathology ; virology ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Lymphatic Metastasis ; Male ; Middle Aged ; Pseudolymphoma ; metabolism ; pathology ; virology ; RNA, Viral ; metabolism ; Viral Matrix Proteins ; metabolism ; Young Adult

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.
Animals ; Antibodies, Viral ; blood ; Bacteriophage T7 ; genetics ; immunology ; metabolism ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Viral ; Immunization ; Influenza A virus ; genetics ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; immunology ; metabolism ; prevention & control ; Peptides ; genetics ; immunology ; metabolism ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Specific Pathogen-Free Organisms ; Viral Matrix Proteins ; genetics ; immunology ; metabolism

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus with pathogenic mechanisms that may be driven by innate immune pathways. The goal of this study is to characterize the expression of the structural (S, E, M, N) and accessory (ORF 3, ORF 4a, ORF 4b, ORF 5) proteins of MERS-CoV and to determine whether any of these proteins acts as an interferon antagonist. Individual structural and accessory protein-coding plasmids with an N-terminal HA tag were constructed and transiently transfected into cells, and their native expression and subcellular localization were assessed using Wes tern blotting and indirect immunofluorescence. While ORF 4b demonstrated majorly nuclear localization, all of the other proteins demonstrated cytoplasmic localization. In addition, for the first time, our experiments revealed that the M, ORF 4a, ORF 4b, and ORF 5 proteins are potent interferon antagonists. Further examination revealed that the ORF 4a protein of MERS-CoV has the most potential to counteract the antiviral effects of IFN via the inhibition of both the interferon production (IFN-β promoter activity, IRF-3/7 and NF-κB activation) and ISRE promoter element signaling pathways. Together, our results provide new insights into the function and pathogenic role of the structural and accessory proteins of MERS-CoV.
Cell Line ; Coronavirus ; genetics ; pathogenicity ; Genes, Viral ; Humans ; Interferons ; antagonists & inhibitors ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Regulatory and Accessory Proteins ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism