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

To study the complete genomic sequence, genomic characteristics, and genetic variation of the bovine papillomavirus 2 genotype (BPV-2) Aks-01 strain at the molecular level, genotyping of this strain from the skin samples of cows in southern Xinjiang (China) was first detected by the polymerase chain reaction with FAP59/FAP64 primers. Based on the complete genome of the BPV-2 reference strain, specific primers and sequencing primers were designed, and the complete genome of the Aks-01 strain amplified and sequenced. Sequence analyses showed that genotyping of the Aks-01 strain belonged to BPV-2. The Aks-01 strain had the structural characteristics of BPV-2. The 7944-bp full-length genomic sequence of the Aks-01 strain was compiled using DNAStar™. The sequence of the Aks-01 strain had 98% similarity to the reference strain from GenBank. The Aks-01 strain was most closely related to BPV-1 and BPV-13. BPV-2, BPV-1 and BPV-13 were grouped within the genus Deltapapillomavirus. The Aks-01 strain is the first BPV-2 strain reported in southern Xinjiang.
Amino Acid Sequence ; Animals ; Base Sequence ; Bovine papillomavirus 1 ; genetics ; Cattle ; China ; Evolution, Molecular ; Female ; Genome, Viral ; genetics ; Genomics ; Genotype ; Molecular Sequence Data ; Oncogene Proteins, Viral ; chemistry ; genetics ; metabolism ; Phylogeny ; Sequence Analysis, DNA ; Skin ; virology

To meet the needs of detection of infectious bursal disease virus (IBDV) under high efficient culture, a SYBR Green I real-time RT-PCR (qRT-PCR) was developed using a pair of primers specific to the conserved region of VP4 gene of IBDV and compared with TCID50 method by monitoring the proliferation dynamics of IBDV in DF-1 cell line adherent to micro carrier in tubular reactor. The results showed that the RT-PCRassay was linear in the range of 4. 03 X 10(1)-10(9) copies/microL. The IBDV RNA detection limit was 40 copies/microL, which was 1 000 times more sensitive than conventional PCR. No cross-reactions with other viruses was observed. The intra-assay coefficient of variation was less than 0.05%. There was a parallel correlation of IBDV proliferation dynamics in DF-1 cell under Micro carrier suspension and static adherent culture by the qRT-PCR assay and TCID50 method. The detection results of the IBDV samples from tubular and flask culture showed the differences of the micro carrier and adherent culture by both methods. In conclusion, the qRT-PCR assay is more rapid and sensitive than the TCID50 method, which is more appropriate for the real time detection of IBDV.
Animals ; Calibration ; Cell Line ; Conserved Sequence ; DNA Primers ; genetics ; Infectious bursal disease virus ; genetics ; isolation & purification ; Organic Chemicals ; chemistry ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Spectrometry, Fluorescence ; Viral Proteins ; genetics ; Virus Replication

To understand the HA1 genetic variation characterization of influenza H3N2 virus isolates in Zhu-hai during 2008-2009, we selected 20 of H3N2 Influenza strains cultured in MDCK cell. Viral RNAs were extracted and amplified by using RT-PCR. The amplified products were purified after identified by gel electrophoresis and then the nucleotide sequences of the amplicons were determined. The results were analyzed by the software ClustalX and MEGA4. 1. When compared with the amino acid sequences of the epitopes of HA1 district of H3N2 influenza vaccine recommended by WHO in 2008, changes were found in those of H3N2 influenza strains in Zhuhai in 2008: K140I in all of H3N2 influenza strains, L157S in 08-0343 and 08-0677, K158R in 08-0466, 08-0620 and 08-0667, K173E in 08-0466 and 08-0620, K173N in 08-0667, and I192T in 08-0667. The epitopes of HA1 district of H3N2 influenza strains in Zhuhai in 2009 are different from that of H3N2 influenza vaccine during the same time: K173Q and P194L occur in all of H3N2 influenza strains, N144K, K158N, and N189K occur in the strains except the strain 09-0056. HA1 domain of H3N2 influenza strains in 2009 has lost a glycosylation site at amino acid position 144 while the glycosylation sites of HA1 domain of H3N2 influenza stains isolated in 2008 remained. This study suggested that H3N2 influenza virus in Zhuhai in 2008 was not evolved a novel variant and H3N2 influenza variant in 2009 was attributed to antigenic drift in HA1 district.
Animals ; Antigens, Viral ; immunology ; Cell Line ; China ; Dogs ; Epitopes ; immunology ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; metabolism ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; immunology ; isolation & purification ; Mutation ; Phylogeny ; Sequence Analysis, DNA

Animals ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA-Directed RNA Polymerases ; antagonists & inhibitors ; Drug Discovery ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; metabolism ; Humans ; Influenza A virus ; drug effects ; genetics ; metabolism ; Influenza, Human ; drug therapy ; Molecular Targeted Therapy ; Neuraminidase ; antagonists & inhibitors ; RNA-Binding Proteins ; antagonists & inhibitors ; Signal Transduction ; drug effects ; Viral Core Proteins ; antagonists & inhibitors ; Viral Matrix Proteins ; antagonists & inhibitors

Murine gammaherpesvirus 68 (MHV-68), a member of the gammaherpesvirus family, replicates robustly in permissive cell lines and is able to infect laboratory mice. MHV-68 has emerged as a model for studying the basic aspects of viral replication and host-virus interactions of its human counterparts. Herpesvirus genome replication is mediated through a cis-element in the viral genome called the origin of lytic replication (oriLyt). A family of transcription factors, CCAAT/enhancer binding proteins (C/EBPs), assists in oriLyt-mediated DNA replication during gammaherpesvirus reactivation. In this study, we examined the role of C/EBPs in gammaherpesvirus DNA replication during de novo infection, using MHV-68 as a model. We found that C/EBP α and β bind to the CCAAT boxes in the MHV-68 oriLyt core region both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. A dominant negative form of C/EBPs significantly impaired the lytic replication efficiency of MHV-68 on both the plasmid and genome levels in a replication assay, indicating that functional C/EBPs are required for maximal MHV-68 genome DNA replication. Collectively, our data demonstrate that C/EBPs interact with the oriLyt core region and play an important role in MHV-68 lytic DNA replication during de novo infection.
Animals ; Base Sequence ; CCAAT-Enhancer-Binding Proteins ; genetics ; metabolism ; Cell Line ; Chromatin Immunoprecipitation ; Cricetinae ; DNA Replication ; DNA, Viral ; chemistry ; genetics ; metabolism ; Electrophoretic Mobility Shift Assay ; Genome, Viral ; Herpesviridae Infections ; genetics ; metabolism ; virology ; Humans ; Mice ; Molecular Sequence Data ; Plasmids ; Promoter Regions, Genetic ; Protein Isoforms ; genetics ; metabolism ; Replication Origin ; Rhadinovirus ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Latency ; genetics

The purpose of this study is to elucidate the molecular mechanism of one-way serological reaction between XJ-160 virus and SINV by recombinant viruses which exchanged the glycoprotein genes individually or simultaneously. Three recombinant viruses were obtained based on the whole-length infectious cDNA clone of XJ-160 virus. The infectivity and pathogenesis to BHK-21 cells and animals were studied and the gene which controlled this one-way serological reaction phenomenon was searched by MCPENT. The results showed that the E2 glycoprotein was the main factor which influenced the growth rate, plaque morphology and pathogenicity of BHK-21 cells and suckling mice. The results of MCPENT showed that the E2 glycoprotein of SINV played a major role in this one-way serological reaction phenomenon. Our study identified the SINE2 gene was the determined gene for one way serological reaction between XJ-160 virus and SINV, and this research laid the foundation for further analysis of the genomic structure and function of SINV.
Alphavirus ; genetics ; immunology ; physiology ; Amino Acid Sequence ; Animals ; Cell Line ; DNA, Recombinant ; genetics ; Female ; Genetic Engineering ; Glycoproteins ; chemistry ; metabolism ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Neutralization Tests ; Sindbis Virus ; immunology ; Viral Load ; Viral Proteins ; chemistry ; metabolism

Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45 Å and 2.2 Å, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 Å, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1.
Animals ; Catalytic Domain ; genetics ; Cattle ; DNA ; genetics ; DNA, Viral ; HIV-1 ; genetics ; metabolism ; Humans ; Immunodeficiency Virus, Bovine ; enzymology ; genetics ; Integrases ; chemistry ; genetics ; metabolism

Previously, we have established an epsilon library and selected out a series of RNA aptamers with higher affinity to P protein based on the in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) in duck hepatitis B virus (DHBV) system. In order to study the structural elements within the epsilon that is essential for initiating priming of HBV reverse transcriptase (P protein), all selected aptamers were subjected to in vitro priming assay and RNA secondary structure probing. We found that all those aptamers supporting priming had an undamaged bulge, while those lacking of the bulge no more support priming. Our results suggest an undamaged bulge within Depsilon is indispensable for initiating priming of P protein.
Base Sequence ; Hepatitis B Virus, Duck ; chemistry ; enzymology ; genetics ; Molecular Sequence Data ; Nucleic Acid Conformation ; RNA, Viral ; chemistry ; genetics ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Reverse Transcription ; Sequence Alignment ; Viral Proteins ; genetics ; metabolism

External Guide Sequences (EGSs) represents a novel nucleic acid based gene interference approach to modulate gene expression. They are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. DNA-based EGS1386 with a size of 12 nt was chemically synthesized to target the mRNA coding for the UL49 gene of human cytomegalovirus (HCMV). The DNA-based EGS1386 molecule efficiently directed human RNase P to cleave the target mRNA sequence in vitro. A reduction of more than 50% in the levels of UL49 expression was observed in human cells treated with the DNA-based EGS1386 targeted UL49 assayed by fluorescent quantization PCR and Western blotting. This results showed that the DNA-EGS1386 can effectively guide the RNase P cut the target mRNA. Therefore, DNA-EGS can develop into a new gene silencing technology and potential of the anti-viral reagents.
Base Sequence ; Cytomegalovirus ; drug effects ; genetics ; metabolism ; Cytomegalovirus Infections ; enzymology ; virology ; DNA, Viral ; genetics ; Directed Molecular Evolution ; methods ; Gene Expression Regulation, Viral ; Humans ; Nucleic Acid Conformation ; Oligodeoxyribonucleotides ; genetics ; pharmacology ; RNA, Guide ; chemistry ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Ribonuclease P ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism