Brazilian dengue virus type 1 replication in mosquito cell cultures.

The development of dengue viruses type 1 obtained from acute human sera and inoculated into mosquito cell cultures, was observed by standard transmission electron microscopy and cytochemical staining. It follows the trans-type mechanism already established of other dengue types. Direct passage of single virus particles across the cell membrane seems to be a pathway of entry and exit in dengue-1 infected cells. The nature of numerous electron translucent vesicles and tubules, produced simultaneously during virus replication inside the rough endoplasmic reticulum, was analyzed by cytochemical tests. The largest amount of virus particles was produced inside cell syncytia.     

Ani immunomodulator Hepon inhibits hepatitis C virus replication in human cell cultures in vitro]

Antiviral activity of immunomodulator "Hepon" was evaluated in human cells culture infected with hepatitis C virus. "Hepon" presence protected human cells SW-13 from cytopathogenic effect of hepatitis C virus. Maximum antiviral effect was demonstrated by "Hepon" at concentration 1 mcg/mL. Control antiviral agent reaferon (interferon alfa-2a) was more potent as vitality protecting agent in the case of SW-13 human cells culture. "Hepon" activity is based on changes of cytokins and interferons spectrum so this immunomodulator is expected to be effective against different viruses including herpes virus and encephalocarditis virus.     

Cytokine-sensitive replication of hepatitis B virus in immortalized mouse hepatocyte cultures

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibit hepatitis B virus (HBV) replication by eliminating pregenomic RNA containing viral capsids from the hepatocyte. We have also shown that HBV-specific cytotoxic T lymphocytes that induce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the liver can inhibit HBV gene expression by destabilizing preformed viral mRNA. In order to further study the antiviral activity of IFN-alpha/beta, IFN-gamma, and TNF-alpha at the molecular level, we sought to reproduce these observations in an in vitro system. Accordingly, hepatocytes were derived from the livers of HBV-transgenic mice that also expressed the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met). Here, we show that the resultant well-differentiated, continuous hepatocyte cell lines (HBV-Met) replicate HBV and that viral replication in these cells is efficiently controlled by IFN-alpha/beta or IFN-gamma, which eliminate pregenomic RNA-containing capsids from the cells as they do in the liver. Furthermore, we demonstrate that IFN-gamma, but not IFN-alpha/beta, is capable of inhibiting HBV gene expression in this system, especially when it acts synergistically with TNF-alpha. These cells should facilitate the analysis of the intracellular signaling pathways and effector mechanisms responsible for these antiviral effects.     

Deleterious effects of hepatitis delta virus replication on host cell proliferation

Hepatitis delta virus (HDV) infection and spread in vivo are dependent upon coinfection by hepatitis B virus (HBV), and dual HDV/HBV infection is frequently more severe than HBV infection alone, raising the possibility that HDV infection may be deleterious to cells. Here we have examined the effects of HDV replication on the long-term growth of cultured cells. Our results show that most cells transfected with HDV cDNA do not give rise to stable cell lines expressing viral antigens or replicative intermediates; in addition, cotransfection of HDV replicons with a plasmid vector expressing a hygromycin resistance marker results in a dose-dependent impairment of hygromycin-resistant colony formation. When cells transfected with replication-competent HDV cDNA are followed prospectively, a progressive decline in viral RNA replication and a steady decrease in the proportion of cells expressing delta antigen are observed. However, in transient transfection assays, no evidence was found to link HDV replication to apoptosis or to cell cycle arrest, nor did HDV replication confer on host cells enhanced sensitivity to inducers of apoptosis. Thus, HDV replication does not appear to be acutely cytotoxic. However, in dividing cells HDV replication is associated with a subtler growth disadvantage, leading to selection in culture for cells displaying diminished HDV expression. This effect would not be expected to cause hepatitis in vivo but might contribute to impaired liver regeneration in the setting of ongoing hepatocellular injury.     

戊型肝炎病毒细胞培养模型的研究进展

戊型肝炎病毒（ HEV）体外细胞培养模型的建立有助于阐明HEV的感染、复制及其致病机理,有利于新型抗病毒药物和疫苗的研发与评估。近年来国内外对HEV细胞培养模型的报道很多,但所用细胞系、感染方法及病毒的复制效果等不尽相同。因此,对近年来HEV细胞培养模型的研究进展进行了综述。     

Defective replication units of hepatitis B virus.

Templates for the synthesis of defective hepatitis B virus RNA pregenomes were constructed. Viral sequences in these constructs were replaced by the neomycin resistance gene. Deletions spanned up to 80% of the genome and did not include the cohesive end region. The size of the defective replication units was reduced up to half of the wild-type unit length. After cotransfection with replication competent wild-type DNA, defective pregenomes became included into the pool of replicating viral nucleic acids. A natural template for a defective pregenome was derived from the integrated state in a hepatocellular carcinoma. Owing to a deletion, this unit was devoid of the hepatitis B virus enhancer.     

Antiviral activity of carrageenan on hepatitis A virus replication in cell culture

Sulphated polysaccharides such as iota-, lambda- and kappa-carrageenans showed a potent inhibitory effect on the replication of hepatitis A virus (HAV) in the human hepatoma cell line PLC/PRF/5. No cytotoxic effects were detected with concentrations of carrageenans up to 200 μg/ml. The selectivity indices of these substances, calculated as the ratio of the dose that reduced the number of viable cells to 50 % (CD₅₀) to the effective dose that inhibited 50 % of viral antigen expression (ED₅₀), were > 400 with iota-carrageenan, > 222 with lambda-carrageenan and > 10 with kappa-carrageenan. The selectivity index of ribavirin (reference substance) was only 5. The 3 types of carrageenans resulted in concentration-dependent reduction of HAV-antigen expression and HAV infectivity. Iota-and lambda-carrageenan emerged, from the present study, as promising candidates for chemotherapy of acute hepatitis A.     

Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations

Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.     

Systematic pathogenesis and replication of avian hepatitis E virus in specific-pathogen-free adult chickens

Hepatitis E virus (HEV) is an important human pathogen. Due to the lack of a cell culture system and a practical animal model for HEV, little is known about its pathogenesis and replication. The discovery of a strain of HEV in chickens, designated avian HEV, prompted us to evaluate chickens as a model for the study of HEV. Eighty-five 60-week-old specific-pathogen-free chickens were randomly divided into three groups. Group 1 chickens (n=28) were each inoculated with 5 x 10(4.5) 50% chicken infectious doses of avian HEV by the oronasal route, group 2 chickens (n=29) were each inoculated with the same dose by the intravenous (i.v.) route, and group 3 chickens (n=28) were not inoculated and were used as controls. Two chickens from each group were necropsied at 1, 3, 5, 7, 10, 13, 16, 20, 24, 28, 35, and 42 days postinoculation (dpi), and the remaining chickens were necropsied at 56 dpi. Serum, fecal, and various tissue samples, including liver and spleen samples, were collected at each necropsy for pathological and virological testing. By 21 dpi, all oronasally and i.v. inoculated chickens had seroconverted. Fecal virus shedding was detected variably from 1 to 20 dpi for the i.v. group and from 10 to 56 dpi for the oronasal group. Avian HEV RNA was detected in serum, bile, and liver samples from both i.v. and oronasally inoculated chickens. Gross liver lesions, characterized by subcapsular hemorrhages or enlargement of the right intermediate lobe, were observed in 7 of 28 oronasally and 7 of 29 i.v. inoculated chickens. Microscopic liver lesions were mainly lymphocytic periphlebitis and phlebitis. The lesion scores were higher for oronasal (P=0.0008) and i.v. (P=0.0029) group birds than for control birds. Slight elevations of the plasma liver enzyme lactate dehydrogenase were observed in infected chickens. The results indicated that chickens are a useful model for studying HEV replication and pathogenesis. This is the first report of HEV transmission via its natural route in a homologous animal model.     

Characterization of long-term cultures of hepatitis C virus.

The human T- and B-cell lines HPBMa10-2 and Daudi produced infectious hepatitis C virus (HCV) for more than 1 year after infection. The infectivity titer of the cell culture-grown HCV and its genome titer were comparable. The virion density in sucrose was around 1.12 g/ml. Among the 13 variants detected in the inoculum, 7 were adsorbed by the cells and one particular HCV sequence which was present in minor quantities in the inoculum persisted.     

Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies.

When hepatitis A virus was inoculated into Vero cells, virus-specified protein and RNA synthesis was detected. Production of viral protein was detected by electrophoretic analysis in polyacrylamide gels by using a double-label coelectrophoresis and subtraction method which eliminated the contribution of host protein components from the profiles of virus-infected cytoplasm. Eleven virus-specified proteins were detected in the net electrophoretic profiles of hepatitis A virus-infected cells. The molecular weights of these proteins were very similar to those detected in cells infected with poliovirus type 1. Virus-specified protein synthesis could be detected at 3 to 6 h and continued for at least 48 h postinfection, but no significant effect on host-cell macromolecular synthesis was observed. Limited viral RNA replication occurred between 2 and 6 h postinfection. The genomic RNA of hepatitis A virus was extracted and shown to be capable of infecting cells and inducing the same set of proteins as intact virus, indicating that the RNA genome is positive stranded. Progeny virus was never detected in the supernatant fluids of infected cell cultures, and the cells showed no observable cytopathology, even though hepatitis A virus-specific proteins and antigens were being produced. The nature of the defect in the replicative cycle of hepatitis A virus in this system remains unknown.     

The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication.

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid-cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as beta-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.     

Simian rotavirus SA11 replication in cell cultures.

Understanding the basic virology of rotavirus infections has been hampered by the fastidiousness of most isolates and by the lack of a rapid quantitative assay method. The growth characteristics of the simian rotavirus SA11 were studied because it grows to high titers in tissue culture and infectivity can be quantitated by plaque assay. SA11 replication was analyzed in a variety of primary cell cultures or continuous cell lines derived from both homologous and heterologous hosts. Viral replication was observed in each of the cell cultured examined. The individual cell cultures demonstrated marked variability in their susceptibility to rotavirus infection. The highest titers were obtained with MA104, BSC-1, CV-1, and BGM cells. Observable cytopathic effect was found to correlate with the percentage of infected cells in the culture. This study presents growth curves of the simian rotavirus in a variety of cell cultures.     

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.     

trans-dominant inhibition of human hepatitis delta virus genome replication.

Infection with hepatitis delta virus (HDV) is an important cause of acute and chronic liver disease and can be rapidly fatal. Sequencing of the HDV RNA genome has revealed variability at the C-terminal end of the delta antigen reading frame. One genome type (termed the S genome) synthesizes a 24-kDa protein thought to be required for genome replication. Another genome type (termed the L genome) extends the reading frame by 19 amino acids as a result of a single base change. Replication of the S and L genomes was studied in cultured fibroblasts. While the S genome efficiently initiated genome replication, the L genome did not. Moreover, in a codelivery experiment, L genome RNA inhibited replication of the S genome. Potent trans inhibition was also observed following cotransfection of the S genome and a plasmid encoding the larger delta antigen. Mutational analysis indicated that the inhibitory activity was not a simple function of the large delta antigen reading frame's extra length. Implications for the viral life cycle, clinical infection, and potential treatment are discussed.