Herpes simplex virus (HSV), including HSV-1 and HSV-2, is an important pathogen that can cause many diseases. Usually these diseases are recurrent and incurable. After lytic infection on the surface of peripheral mucosa, HSV can enter sensory neurons and establish latent infection during which viral replication ceases. Moreover, latent virus can re-enter the replication cycle by reactivation and return to peripheral tissues to start recurrent infection. This ability to escape host immune surveillance during latent infection and to spread during reactivation is a viral survival strategy and the fundamental reason why no drug can completely eradicate the virus at present. Although there are many studies on latency and reactivation of HSV, and much progress has been made, many specific mechanisms of the process remain obscure or even controversial due to the complexity of this process and the limitations of research models. This paper reviews the major results of research on HSV latency and reactivation, and discusses future research directions in this field.
Herpes Simplex ; virology ; Herpesvirus 1, Human ; physiology ; Humans ; Virus Activation ; physiology ; Virus Latency ; physiology ; Virus Replication

Human bocavirus 1 (HBoV1) non-structural protein NS1 is a multifunctional protein important for virus replication and induction of apoptosis in host cell. To better understand the function of the NS1 protein, it is urgent to address reducing the toxicity of NS1 to host cells. In the present study, we established a stable cell line that regulates expression of NS1 of HBoV1. The recombinant lentivirus plasmid containing a regulatable promoter fused with ns1 gene was constructed and transfected into HEK 293T cells using transfection reagent. The HEK 293T cell lines stably expressing NS1-100 and NS1-70 proteins were established by screening resistant cells with puromycin and inducing NS1 expression with doxycycline. The expression of NS1 protein was determined by fluorescent labeling protein and Western blotting. HBoV1 promoter was transfected into stably expressing NS1 cell line and its trans-transcriptional activity was analyzed. The results showed that NS1 protein was expressed stably in the established cell lines and had a strong activation activity on the HBoV1 promoter driving luciferase gene. Taken together, this study provides a solid basis for further research on the function of NS1 and the pathogenesis of human bocavirus.
Human bocavirus ; Promoter Regions, Genetic ; Transcriptional Activation ; Viral Nonstructural Proteins ; Virus Replication

OBJECTIVETo explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein.

METHODSFull length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR.

RESULTSThe mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, P<0.01), 66.54% 4.45% (t=42.3, P<0.01), and 73.68% 5.07% (t=51.9, P<0.01) in group 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS, and 2.2.15-CPAS, respectively. The mean inhibitory rates of HBeAg were 4.46% 4.25%, 52.86% 1.32% (t=36.2, P<0.01), 26.36% 1.69% (t=22.3, P<0.01), and 59.28% 2.10% (t=39.0, P<0.01), respectively. The inhibitory rates of HBc related HBV DNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium.

CONCLUSIONSIt has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual expression of them. With the help of wild-type HBV, the modified HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified. The packaging cell line can provide packaging for replication-defective HBV, but with low efficiency.

Cell Line ; Genetic Engineering ; Genetic Vectors ; Hepatitis B virus ; genetics ; Mutation ; Plasmids ; RNA, Antisense ; physiology ; Transfection ; Viral Core Proteins ; physiology ; Virus Assembly ; Virus Replication

Recombinant adeno-associated virus (rAAV)-based vectors that can stably express therapeutic genes in vivo without detectable side-effect have shown great promise for human gene therapy. A major challenge for translation of promising research to clinical development is how to establish clinically compatible purification methods in separating rAAV from potentially pathogenic impurities, especially rAAV vector-related impurities, a class of impurities corresponding to AAV particles that closely resemble bona fide vectors and are difficult to remove. In this review we summarize the assembly process of rAAV vector-related impurities and their characteristics differed with rAAV vectors, and evaluate several current technologies to prevent their formation or separate them from rAAV stocks.
Capsid Proteins ; isolation & purification ; Dependovirus ; genetics ; isolation & purification ; physiology ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; isolation & purification ; Recombination, Genetic ; Virion ; isolation & purification ; Virus Assembly ; genetics ; Virus Replication ; genetics

OBJECTIVESTo understand the hepatic pathology, hepatitis B reactivation rates and serological changes in chronic HBV carriers.

METHODSA 5 year dynamic observation and survey of 220 chronic HBV carriers in Wuxi district was taken, analyzing their clinical symptoms, liver histopathology, virology and HBV immunological markers.

RESULTSThirty-five of the 220 (15.9%) patients, showed hepatitis B reactivation. The hepatitis B reactivation rate of patients with obvious hepatic tissue inflammation (> or = G2) was 27.0% (33/122) and the rate of the patients with mild hepatic tissue inflammation (G0-G1) was 2.0% (2/98), showing significant differences (x2=25.41, P less than 0.01). The reactivation rate of patients with high inflammation was clearly higher than those with mild inflammation. Twenty-seven of the 35 hepatitis B reactivation cases were older than 40 years, showing a significant association between the ages of the patients and hepatitis B reactivation rates (x2=6.72, P less than 0.01), moreover there was no relationship between sex and the hepatitis B reactivation rate. There were differences in the inflammation grades and fibrosis stages between HBeAg positive and anti-HBe positive group cases (Kruskal-Wallis Test, x2=8.68, P less than 0.01, x2=6.84, P less than 0.01), showing inflammation grades and fibrosis stages of the anti-HBe positive group were higher than those of the HBeAg positive group. There were no obvious differences about the inflammation grade between age less than 40 years old and > or = 40 years old group cases (x2=0.62, P more than 0.05), but there were significant statistical differences about the fibrosis stage (x2=7.37, P less than 0.01), showing fibrosis stage of more than 40 years old group cases was clearly higher than the less than 40 years old group cases. Fifty-six cases received a liver biopsy for a second time and 23 for a third time. We found those whose hepatic tissues were normal in their first liver biopsies, then their liver histology continued remaining stable for several years while those with abnormal ones hardly or only recovered slightly. The rate of HBsAg turning to negativity per year was 1.55% and for HBeAg was 5.4%.

CONCLUSIONThe hepatic tissue pathology for most chronic HBV carriers (55%) had significant abnormalities (inflammation grade > or = G2), and the rates of hepatitis B reactivation were highly relevant to the liver inflammation grades and the ages of the patients.

Adolescent ; Adult ; Carrier State ; virology ; Child ; Female ; Follow-Up Studies ; Hepatitis B virus ; physiology ; Hepatitis B, Chronic ; pathology ; virology ; Humans ; Inflammation ; Liver ; pathology ; virology ; Male ; Middle Aged ; Virus Activation ; Virus Replication ; Young Adult

Hepatitis C virus (HCV) is a positive sense, single-stranded RNA virus in the Flaviviridae family. It causes acute hepatitis with a high propensity for chronic infection. Chronic HCV infection can progress to severe liver disease including cirrhosis and hepatocellular carcinoma. In the last decade, our basic understanding of HCV virology and life cycle has advanced greatly with the development of HCV cell culture and replication systems. Our ability to treat HCV infection has also been improved with the combined use of interferon, ribavirin and small molecule inhibitors of the virally encoded NS3/4A protease, although better therapeutic options are needed with greater antiviral efficacy and less toxicity. In this article, we review various aspects of HCV life cycle including viral attachment, entry, fusion, viral RNA translation, posttranslational processing, HCV replication, viral assembly and release. Each of these steps provides potential targets for novel antiviral therapeutics to cure HCV infection and prevent the adverse consequences of progressive liver disease.
Antigens, CD81/metabolism ; Genome, Viral ; Hepacivirus/genetics/*physiology ; Humans ; RNA, Viral/metabolism ; Scavenger Receptors, Class B/metabolism ; Viral Envelope Proteins/chemistry/metabolism ; Viral Nonstructural Proteins/chemistry/metabolism ; Virus Assembly ; Virus Internalization ; Virus Replication

OBJECTIVETo construct a stable cell line with permanent secretion of recombinant hepatitis B virus (HBV) vector, which express blasticidin resistant gene.

METHODSReplication-defective HBV vector, pCH-BsdR, which express blasticidin resistance gene was constructed by deleting the HBV genes and inserting the blasticidin resistance gene into the S region. The G418-resistant, the packaging signal deleted HBV helper plasmid, pcDNA3.1-CH3142, and the HBV vector pCH-BsdR were cotransfected into HepG2 cells. Cell clones were selected by the adding of both blasticidin and G418, then serial detection were done.

RESULTSAfter 36 cell clones were picked and expanded. Three cell clones were defined as the best. Quantity of their HBV DNA were 4.1 x 10(6), 3.6 x 10(6) and 1.2 x 10(6) copies/ml, respectively. Enveloped recombinant, but not wild type HBV were confirmed in the culture medium.

CONCLUSIONSThe stable cell lines can realize large preparation of recombinant HBV virions. This will contribute to the use of HBV vector for gene therapy and HBV susceptible cell lines screening.

Cell Line ; drug effects ; virology ; Clone Cells ; Drug Resistance ; Gene Expression ; Genetic Engineering ; Genetic Vectors ; genetics ; metabolism ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Humans ; Nucleosides ; pharmacology ; Transfection ; Virion ; genetics ; physiology ; Virus Assembly ; Virus Replication

Animals ; Calcium ; metabolism ; DNA-Binding Proteins ; metabolism ; Enzyme Activation ; Focal Adhesion Kinase 2 ; metabolism ; Gene Expression Regulation, Viral ; Hepatitis B virus ; genetics ; physiology ; Humans ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Virus Replication

The late stages of the HIV-1 replication cycle are important to the overall replication cycle. During the late stages, HIV-1 replication undergoes the processes of assembly, release, and maturation, resulting in the production of a mature virus particle capable of infecting a new target cell. The structural protein Gag and its related gene (protein) play a central role in these pathways. The different regions of Gag worked in concert to drive production of a mature infectious particle through protein-protein, protein-RNA and protein-lipid interactions. The designed drug aimed directly at these stages can efficiently block the maturation and infectivity of HIV-1. In this article, the role of structural protein Gag and related gene (protein) in late stages of the HIV-1 replication cycle and related inhibitors is reviewed.
Amphotericin B ; analogs & derivatives ; chemistry ; pharmacology ; Anti-HIV Agents ; chemistry ; pharmacology ; Benzeneacetamides ; chemistry ; pharmacology ; Furans ; chemistry ; pharmacology ; Genes, gag ; HIV-1 ; drug effects ; physiology ; Humans ; Phenylurea Compounds ; chemistry ; pharmacology ; Piperidines ; chemistry ; pharmacology ; Succinates ; chemistry ; pharmacology ; Sulfur Compounds ; chemistry ; pharmacology ; Triterpenes ; chemistry ; pharmacology ; Virus Assembly ; drug effects ; Virus Release ; drug effects ; Virus Replication ; drug effects ; physiology ; gag Gene Products, Human Immunodeficiency Virus ; metabolism ; physiology

BACKGROUND/AIMS: Enhanced replication of hepatitis C virus (HCV) is well described in the setting of moderate to severe immunosuppression. The aims of this retrospective study were to determine the incidence of enhanced HCV replication in hepatocellular carcinoma (HCC) patients undergoing transarterial chemolipiodolization (TACL) and to identify the factors associated with enhanced replication of HCV. The clinical pattern of enhanced HCV replication was compared with hepatitis B virus (HBV) reactivation during TACL. METHODS: This study enrolled 49 anti-HCV-seropositive patients who were diagnosed with HCC between January 2005 and December 2010 and who underwent TACL using epirubicin and/or cisplatin with consecutive HCV RNA copies checked. For comparison, 46 hepatitis B surface antigen1-positive patients with HCC who were treated with TACL were also enrolled. The frequency, associated factors, and clinical outcomes of enhanced HCV replication were analyzed and compared with those of HBV reactivation during TACL. RESULTS: Enhanced replication of HCV occurred in 13 (26.5%) of the 49 anti-HCV-seropositive patients during TACL. Of these 13 patients, 4 developed hepatitis, but none of the subjects developed decompensation due to the hepatitis. No significant clinical factors for enhanced HCV replication during TACL were found. Compared with HBV reactivation, the frequency of hepatitis attributed to enhanced HCV replication was significantly lower than that for HBV reactivation (8.2% vs. 23.9%, P=0.036). CONCLUSIONS: TACL can enhance HCV replication; however, the likelihood of hepatitis and decompensation stemming from enhanced HCV replication was lower than that for HBV reactivation in patients undergoing TACL.
Adult ; Aged ; Antineoplastic Agents/*administration & dosage/adverse effects/pharmacology ; Carcinoma, Hepatocellular/complications/*therapy ; Chemoembolization, Therapeutic/*adverse effects ; Drug Therapy, Combination ; Female ; Hepacivirus/drug effects/*physiology ; Hepatitis B/complications/epidemiology/virology ; Hepatitis B Surface Antigens/blood ; Hepatitis B virus/drug effects/*physiology ; Hepatitis C/complications/epidemiology/virology ; Humans ; Liver Neoplasms/complications/*therapy ; Male ; Middle Aged ; RNA, Viral/analysis ; Retrospective Studies ; Virus Activation ; *Virus Replication