BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.
Adult ; Asian Continental Ancestry Group ; Capsid Proteins/genetics ; Carcinoma, Hepatocellular/etiology/*pathology/virology ; DNA, Viral/chemistry/genetics/metabolism ; DNA-Binding Proteins/genetics ; Dependovirus/*genetics/isolation & purification/pathogenicity ; Female ; Humans ; Incidence ; Inverted Repeat Sequences/genetics ; Liver Neoplasms/etiology/*pathology/virology ; Male ; Middle Aged ; Parvoviridae Infections/complications/epidemiology ; Polymerase Chain Reaction ; Republic of Korea ; Sequence Analysis, DNA ; Viral Proteins/genetics

Avian leukosis virus subgroup J (ALV-J) is an avian retrovirus that can induce myelocytomas. A high-frequency mutation in gene envelope endows ALV-J with the potential for cross-species transmission. We wished to ascertain if the ALV-J can spread across species under selection pressure in susceptible and resistant hosts. First, we inoculated (in turn) two susceptible host birds (specific pathogen-free (SPF) chickens and turkeys). Then, we inoculated three resistant hosts (pheasants, quails and ducks) to detect the viral shedding, pathologic changes, and genetic evolution of different isolates. We found that pheasants and quails were infected under the selective pressure that accumulates stepwise in different hosts, and that ducks were not infected. Infection rates for SPF chickens and turkeys were 100% (16/16), whereas those for pheasants and quails were 37.5% (6/16) and 11.1% (3/27). Infected hosts showed immune tolerance, and inflammation and tissue damage could be seen in the liver, spleen, kidneys and cardiovascular system. Non-synonymous mutation and synonymous ratio (NS/S) analyses revealed the NS/S in hypervariable region (hr) 2 of pheasants and quails was 2.5. That finding suggested that mutation of isolates in pheasants and quails was induced by selective pressure from the resistant host, and that the hr2 region is a critical domain in cross-species transmission of ALV-J. Sequencing showed that ALV-J isolates from turkeys, pheasants and quails had moved away from the original virus, and were closer to the ALV-J prototype strain HPRS-103. However, the HPRS-103 strain cannot infect pheasants and quails, so further studies are needed.
Amino Acid Sequence ; Animals ; Avian Leukosis ; transmission ; virology ; Avian Leukosis Virus ; classification ; genetics ; physiology ; Chickens ; Ducks ; virology ; Galliformes ; virology ; Host Specificity ; Molecular Sequence Data ; Poultry Diseases ; transmission ; virology ; Quail ; virology ; Sequence Alignment ; Turkeys ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

Amiloride ; analogs & derivatives ; chemistry ; metabolism ; Binding Sites ; Genotype ; Hepacivirus ; genetics ; metabolism ; Humans ; Magnetic Resonance Spectroscopy ; Molecular Dynamics Simulation ; Protein Structure, Tertiary ; Viral Proteins ; antagonists & inhibitors ; metabolism

Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451-739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451-739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.
Ebolavirus ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Nucleocapsid ; chemistry ; genetics ; metabolism ; RNA, Viral ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Virus Assembly

The rabies virus (RABV) is an enveloped RNA virus. It mainly damages the central nervous system and causes anencephaly in mammals and humans. There is now compelling evidence that enveloped virions released from infected cells can carry many host proteins, some of which may play an important part in viral replication. Several host proteins have been reported to be incorporated into RABV particles. However, a systematic study to reveal the proteomics of RABV particles has not been conducted. In the present study, after virus culture and purification by sucrose density gradient ultracentrifugation, a proteomics approach was used to analyze the protein composition of purified RABV particles to understand the molecular mechanisms of virus-cell interactions. Fifty host proteins, along with five virus-encoded structural proteins, were identified in purified RABV particles. These proteins could be classified into ten categories according to function: intracellular trafficking (14%), molecular chaperone (12%), cytoskeletal (24%), signal transduction (8%), transcription regulation (12%), calcium ion-binding (6%), enzyme binding (6%), metabolic process (2%), ubiquitin (2%) and other (14%). Of these, four proteins (beta-actin, p-tubulin, Cofilin, Hsc70) were validated by western blotting to be present in purified RABV particles. This novel study of the composition of host proteins in RABV particles may aid investigation of the mechanism of RABV replication.
Animals ; Humans ; Molecular Sequence Data ; Proteomics ; Rabies ; genetics ; metabolism ; virology ; Rabies virus ; chemistry ; genetics ; metabolism ; Viral Proteins ; analysis ; chemistry ; genetics ; metabolism ; Virion ; chemistry ; genetics ; metabolism

To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.
Amino Acid Sequence ; Enterovirus D, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics ; metabolism

To prepare virus-like particles containing FluA/B mRNA as RNA standard and control in Influenza RNA detection, the genes coding the coat protein and maturase of E. coli bacteriophage MS2 were amplified and cloned into D-pET32a vector. Then we inserted 6 histidines to MS2 coat protein by QuikChange Site-Directed Mutagenesis Kit to construct the universal expressing vector D-pET32a-CP-His. In addition, the partial gene fragments of FluA and FluB were cloned to the down-stream of expressing vector. The recombinant plasmid D-pET32a-CP-His-FluA/B was transformed to BL21 with induction by IPTG. The virus-like particles were purified by Ni+ chromatography. The virus-like particles can be detected by RT-PCR, but not PCR. They can be conserved stably for at least 3 months at both 4 degrees C and -20 degrees C. His-tagged virus-like particles are more stable and easier to purification. It can be used as RNA standard and control in Influenza virus RNA detection.
Escherichia coli ; genetics ; metabolism ; Influenza A virus ; genetics ; metabolism ; Influenza B virus ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Ribonucleases ; chemistry ; Virion ; genetics ; metabolism

The replicase genes of five isolates of Cucumber green mottle mosaic virus from Jiangsu, Zhejiang, Hunan and Beijing were amplificated, sequenced and analyzed. The similarities of nucleotide acid sequences indicated that 129 kD and 57 kD replicase genes of CGMMV-No. 1, CGMMV-No. 2, CGMMV-No. 3, CGMMV-No. 4 and CGMMV-No. 5 were 99.64% and 99.74%, respectively. The similarities of 129 kD and 57 kD replicase genes of CGMMV-No. 1, CGMMV-No. 3 and CGMMV-No. 4 were 99.95% and 99.94%, while they were lower between CGMMV-No. 2 and the rest of four reference sequences, just from 99.16% to 99.27% and from 99.04% to 99.18%. All reference sequences could be divided into six groups in neighbor-joining (NJ) phylogenetic trees based on the replicase gene sequences of 129 kD, 57 kD protein respectively. CGMMV-No. 1, CGMMV-No. 3 and CGMMV-No. 4 were clustered together with Shandong isolate (Accession No. KJ754195) in two NJ trees; CGMMV-No. 5 was clustered together with Liaoning isolate (Accession No. EF611826) in two NJ trees; CGMMV-No. 2 was clustered together with Korea watermelon isolate (Accession No. AF417242) in phylogenetic tree of 129 kD replicase gene of CGMMV; Interestingly, CGMMV-No. 2 was classified as a independent group in phylogenetic tree of 57 kD replicase gene of CGMMV. There were no significant hydrophobic and highly coiled coil regions on 129 kD and 57 kD proteins of tested CGMMV isolates. Except 129 kD protein of CGMMV-No. 4, the rest were unstable protein. The number of transmembrane helical segments (TMHs) of 129 kD protein of CGMMV-No. 1, CGMMV-No. 2, CGMMV-No. 3 and CGMMV-No. 5 were 6, 6, 2 and 4, respectively, which were 13, 13 and 5 on the 57 kD protein of CGMMV-No. 2, CGMMV-No. 4 and CGMMV-No. 5. The glycosylation site of 129 kD protein of tested CGMMV isolates were 2, 4, 4, 4 and 4, and that of 57 kD protein were 2, 5, 2, 5 and 2. There were difference between the disorders, globulins, phosphorylation sites and B cell antigen epitopes of 129 kD and 57 kD proteins of tested CGMMV isolates. The current results that there was no significant difference between the replicase gene sequences, it was stable and conservative for intra-species and clearly difference for inter-species. CGMMV-No. 1, CGMMV-No. 3, CGMMV-No. 4 and CGMMV-No. 5 had. a close genetic relationship with Shandong and Liangning isolates (Accession No. KJ754195 and EF611826), they are potentially originate from the same source. CGMMV-No. 2 was closer with Korea isolate. High sequence similarity of tested samples were gathered for a class in phylogenetic tree. It didn't show regularity of the bioinformatics analysis results of 129 kD and 57 kD proteins of tested CGMMV isolates. There was no corresponding relationship among the molecular phylogeny and the bioinformatics analysis of the tested CGMMV isolates.
Computational Biology ; Cucumis sativus ; chemistry ; classification ; enzymology ; genetics ; Molecular Sequence Data ; Phylogeny ; Plant Diseases ; virology ; RNA Replicase ; chemistry ; genetics ; metabolism ; Sequence Homology, Nucleic Acid ; Viral Proteins ; chemistry ; genetics ; metabolism

The core protein (CP) of the classical swine fever virus (CSFV) is one of its structural proteins. Apart from forming the nucleocapsid to protect internal viral genomic RNA, this protein is involved in transcriptional regulation. Also, during viral infection, the CP is involved in interactions with many host proteins. In this review, we combine study of this protein with its disorders, structural/functional characteristics, as well as its interactions with the non-structural proteins NS3, NS5B and host proteins such as SUMO-1, UBC9, OS9 and IQGAP1. We also summarize the important part played by the CP in CSFV pathogenicity, virulence and replication of genomic RNA. We also provide guidelines for further studies in the CP of the CSFV.
Animals ; Classical Swine Fever ; virology ; Classical swine fever virus ; genetics ; metabolism ; pathogenicity ; Genome, Viral ; Swine ; Viral Core Proteins ; chemistry ; genetics ; metabolism ; Virulence

Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease (HFMD) and various neurological complications, including aseptic meningitis and neurogenic pulmonary edema in young children. HFMD caused by EV-A71 have broken out several times in the Asia-Pacific region since 2007. And it has been a serious threat to public health. There is no effective vaccine or antiviral drug. The pathogenesis of EV-A71 infection is unknown, and EV-A71 3C protein plays an irreplaceable role in replication and anti - innate immunity. Further research on EV-A71 3C protein is conducive to understand the pathogenesis of EV-A71 infection and antiviral drug.
Animals ; Antiviral Agents ; pharmacology ; Enterovirus ; drug effects ; immunology ; metabolism ; physiology ; Humans ; Immunity, Innate ; Viral Proteins ; chemistry ; genetics ; metabolism ; Virus Replication ; drug effects