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Modulation of herpes simplex virus replication in adenovirus transformed cells.

The ability of herpes simplex virus 1 to replicate in cells transformed by adenovirus type 5 is strongly dependent on the origin of the cells. Studies show that adenovirus transformed rat cells lose their permissiveness while cells of hamster or human origin retain their ability to replicate HSV although at a reduced level when compared to the untransformed parent cells. One line of adenovirus transformed rat cells, 107, demonstrates thermosensitive events, allowing HSV to replicate at 34 degrees C but not at 37 degrees C. Analysis of the biochemical events taking place at 37 degrees C showed that virus-specific DNA synthesis was greatly reduced but that all of the late virus structural proteins could be observed after SDS-polyacrylamide gel electrophoresis. It was also demonstrated that shut-off of host macromolecular synthesis appeared to be less efficient after HSV infection of 107 cells than after infection of more permissive cells such as the non-transformed REF line. Collectively the data show that interactions between HSV and the host cell are perturbed when the cell is transformed by type 5 adenovirus. The degree of perturbation ranges from a slight reduction in number of progeny to a completely abortive infection.

Adenoviridae↗

Inhibition of hepatitis B virus replication in 2.2.15 cells by expressed shRNA.

Hepatitis B virus (HBV) infection is a worldwide health problem. To determine whether RNA interference (RNAi) could inhibit ongoing HBV replication in 2.2.15 cells, we constructed shRNA-producing vector pU6P based on the mouse U6 RNA promoter and cloned 12 targeted sequences against HBV into the vector, resulting in a series of pU6-siHBV vectors. The recombinant vectors were transfected into 2.2.15 cells, HBsAg and HBeAg in cultured media were assayed using enzyme-linked immunosorbent assay at various days after transfection. The amount of HBV DNA in the culture medium was quantitated by real-time polymerase chain reaction. HBsAg and HBeAg expression were inhibited by 72.8 +/- 5.4% (P = 0.00003) and 55.8 +/- 6.2% (P = 0.000026), respectively, 4 days after transfection with pU6-siHBV5. The greatest inhibition of HBV DNA was decreased by approximately 1.9-fold (P = 0.013) on day 6 post transfection with pU6-siHBV11 compared with that of empty vector. No change was found for HBV protein expression and DNA replication on pU6-siGFP (negative control) transfected cells. Our data demonstrate that the transfection of HBV-targeted shRNA-producing vector in 2.2.15 cells could inhibit the HBV protein expression and HBV DNA replication specifically. RNAi may be considered as a potential antiviral approach for human HBV infection.

Animals↗

"Inapparent" hepatitis B virus infection and hepatitis C virus replication in alcoholic subjects with and without liver disease.

We evaluated hepatitis B virus DNA and hepatitis C virus RNA in sera from 110 HBsAg and IgM HBc antibody-negative heavy drinkers (50 cirrhosis, 13 chronic active hepatitis, 25 fatty liver with or without mild to moderate fibrosis, alcoholic hepatitis or both and 22 healthy alcoholic subjects) with polymerase chain reaction. Results of hepatitis C virus polymerase chain reaction were compared with those obtained with two tests (second generation recombinant immunoblot assay and enzyme-linked immunosorbent assay) used to detect hepatitis C virus antibodies. Hepatitis B virus DNA was found in three (2.7%) patients. Hepatitis C virus RNA was detected in 29 (29.8%) of the 97 subjects whose sera were well preserved for RNA extraction (42.5% cirrhosis, 83.3% chronic active hepatitis, 8% fatty liver and 0% healthy alcoholic subjects). Results obtained with second-generation recombinant immunoblot assay and enzyme-linked immunosorbent assay had a high degree of agreement with polymerase chain reaction as expected, the kappa indexes being 0.76 and 0.61, respectively. Nevertheless, five hepatitis C virus RNA-positive patients had negative recombinant immunoblot assay results, whereas all hepatitis C virus RNA-positive patients had positive or borderline enzyme-linked immunosorbent assay results. We conclude that, in Italian HBsAg-negative alcoholic patients, "inapparent" hepatitis B virus infection is rare. On the contrary, hepatitis C virus infection, as detected on hepatitis C virus polymerase chain reaction, is quite frequent, especially in patients who have cirrhosis and chronic active hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Inhibition of influenza A virus replication by rifampicin and selenocystamine.

The effects of selenocystamine, an inhibitor of influenza virus RNA-dependent RNA polymerase in vitro activity, in the antibiotic rifampicin were studied on influenza A/PR/8/34 (HON1) infection in embryonated eggs. Both drugs completely inhibited hemagglutinating and infective virus yields when added at relatively early times postinfection. Maximal inhibition was produced by apparently noncytotoxic concentrations of 50 microgram of selenocystamine, or of 400 microgram of rifampicin, per egg.

Animals↗

Evidence that nucleocapsid disassembly and a later step in virus replication are inhibited in transgenic tobacco protoplasts expressing TMV coat protein.

Tobacco mosaic virus (TMV)-like pseudovirus particles containing mRNA for Escherichia coli beta-glucuronidase (GUS) were electroporated into mesophyll protoplasts from control or TMV coat protein (CP)-transgenic tobacco (Nicotiana tabacum cv. Xanthi). GUS-particles were expressed 100-fold less efficiently in CP-transformed than in control protoplasts whereas unencapsidated GUS mRNA was expressed only 2.8-fold less efficiently. Lower transient expression of packaged GUS mRNA is probably due to inhibited disassembly of nucleocapsids in CP-transgenic protoplasts. Control and U1 CP-transformed protoplasts are equally susceptible to infection by cowpea strain TMV (Cc), as well as unencapsidated Cc or U1 RNA. In contrast, native or in vitro reconstituted U1 TMV particles result in 5- to 6-fold fewer infected CP-transgenic than control protoplasts. When Cc RNA was transcapsidated in U1 CP in vitro, the hybrid virions were equally infectious in both classes of protoplasts. We conclude that although compatible U1 protein-protein interactions significantly inhibit (GUS) nucleocapsid disassembly in CP-transgenic protoplasts, the endogenous CP must also interfere with a later stage of infection involving the homologous viral RNA.

Capsid↗

Antiviral immunotoxins: antibody-mediated delivery of gelonin inhibits Pichinde virus replication in vitro.

Immunotoxins were produced and evaluated for antiviral activity against Pichinde virus, a member of the family Arenaviridae. Immunoglobulins were conjugated to the ribosome-inactivating protein, gelonin, through a disulfide linkage to form the immunotoxins. Immunotoxins were produced utilizing monoclonal antibodies, immunoglobulin-binding proteins and hyperimmune sera. An immunotoxin consisting of hyperimmune rabbit sera conjugated with gelonin displayed strong antiviral activity against Pichinde virus, as did a protein G-gelonin indirect immunotoxin in combination with nonconjugated hyperimmune sera. Hyperimmune rabbit sera conjugated with gelonin caused no detectable cytotoxicity in non-infected Vero cells as measured by [3H]leucine incorporation. The 50% effective dose for the immunotoxin was 0.018 microM compared with 86 microM for ribavirin.

Animals↗

Hepatitis C virus replication and pathogenesis.

The mechanism involved in the development of persistent hepatitis C virus (HCV) infection and the pathogenesis remain unclear. The present review is an accumulation of evidence gathered to date. In addition, it discusses the system developed to characterize HCV.

Animals↗

A 37 base sequence in the leader region of human T-cell leukaemia virus type I is a high affinity dimerization site but is not essential for virus replication.

Mutagenesis has demonstrated a region in the human T-cell leukaemia virus type I (HTLV-I) 5' leader RNA which, when deleted, abolishes stable RNA dimer formation in vitro. We have further mapped, using both in vitro transcribed and synthesized RNA, this site to a 37 base region, which dimerizes with high affinity. When deleted from an HTLV-I Gag-Pol-expressing plasmid which was co-transfected with an envelope protein expressor to produce virions capable of single round infection, the dimer linkage deletion did not affect viral protein production. In addition, virus infectivity was only slightly reduced, to approximately 75-80% of the wild-type.

5' Untranslated Regions↗

Inhibitory effect of anti-pyretic and anti-inflammatory herbs on herpes simplex virus replication.

The increasing clinical use of acyclovir, ganciclovir, and foscarnet against herpes simplex virus (HSV), varicella-zoster virus, and cytomegalovirus has been associated with the emergence of drug-resistant herpesvirus strains. To develop anti-HSV compounds from plants, 31 herbs used as antipyretic and anti-inflammatory agents in Chinese medicine were screened. Five different preparations (cold aqueous, hot aqueous, ethanolic, acid ethanolic, and methanolic) from 31 herbs were analyzed by plaque reduction assay, and 7 extracts. which showed significant antiviral activities, were further elucidated for their antiviral mechanisms. Our results showed that ethanolic extract of Rheum officinale and methanolic extract of Paeonia suffruticosa prevented the process of virus attachment and penetration. Aqueous extract of P. suffruticosa and ethanolic extract of Melia toosendan inhibited virus attachment to cell surface. Aqueous extract of Sophora flavescens and methanolic extract of M. toosendan showed no effect on virus attachment and penetration. These data indicated that these 4 herbs have a potential value as a source of new powerful anti-HSV compounds.

Alcohols↗

Delta infection and hepatitis B virus replication in Danish patients with fulminant hepatitis B.

The presence of hepatitis B virus and delta agent markers was investigated in 41 patients referred during the years 1970-1985 with fulminant hepatitis classified as type B or non-A non-B and compared to findings in patients with uncomplicated hepatitis B and chronic hepatitis B infection. 13 patients had no markers of hepatitis B and delta infection and were classified as non-A non-B hepatitis. The remaining 28 patients were all HBsAg and IgM anti-HBc positive and 14 (50%) had evidence of delta infection. In contrast, only 13/71 patients (18%) with acute benign hepatitis B had evidence of delta coinfection (p less than 0.005). This corresponds to an odds ratio of 4.5 for development of fulminant hepatitis among patients with hepatitis B and delta coinfection. In 100 chronic HBsAg carriers 29% were positive for delta markers. 12 of the delta infected patients with fulminant hepatitis were positive for total antibody to the delta antigen, and 2 were delta antigen positive. Three were HBeAg positive/anti-HBe negative. None had hepatitis B virus DNA. Among the 14 patients without delta infection, hepatitis B virus DNA was found in 2/4 HBeAg positive/anti-HBe negative patients and in 1/8 patients negative for both markers. The present data indicate that a high proportion of Danish patients with fulminant hepatitis B have hepatitis B and delta agent coinfection. Further, the findings suggest that hepatitis B and delta coinfection may be associated with an increased risk of development of fulminant hepatitis as compared to that of hepatitis B alone.

Adolescent↗

Inefficient human immunodeficiency virus replication in mobile lymphocytes.

Cell-to-cell viral transfer facilitates the spread of lymphotropic retroviruses such as human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV), likely through the formation of "virological synapses" between donor and target cells. Regarding HIV replication, the importance of cell contacts has been demonstrated, but this phenomenon remains only partly characterized. In order to alter cell-to-cell HIV transmission, we have maintained cultures under continuous gentle shaking and followed viral replication in this experimental system. In lymphoid cell lines, as well as in primary lymphocytes, viral replication was dramatically reduced in shaken cultures. To document this phenomenon, we have developed an assay to assess the relative contributions of free and cell-associated virions in HIV propagation. Acutely infected donor cells were mixed with carboxyfluorescein diacetate succinimidyl ester-labeled lymphocytes as targets, and viral production was followed by measuring HIV Gag expression at different time points by flow cytometry. We report that cellular contacts drastically enhance productive viral transfer compared to what is seen with infection with free virus. Productive cell-to-cell viral transmission required fusogenic viral envelope glycoproteins on donor cells and adequate receptors on targets. Only a few syncytia were observed in this coculture system. Virus release from donor cells was unaffected when cultures were gently shaken, whereas virus transfer to recipient cells was severely impaired. Altogether, these results indicate that cell-to-cell transfer is the predominant mode of HIV spread and help to explain why this virus replicates so efficiently in lymphoid organs.

CD4-Positive T-Lymphocytes↗

Suppression of bovine viral diarrhea virus replication by small interfering RNA and short hairpin RNA-mediated RNA interference.

Bovine viral diarrhea virus (BVDV) is a ubiquitous viral pathogen that affects cattle herds' worldwide causing significant economic loss. The current strategies to control BVDV infection include vaccination (modified-live or killed) and control of virus spread by enhanced biosecurity management, however, the disease remains prevalent. With the discovery of the sequence-specific method of gene silencing known as RNA interference (RNAi), a new era in antiviral therapies has begun. Here we report the efficient inhibition of BVDV replication by small interfering (siRNA) and short hairpin RNA (shRNA)-mediated gene silencing. siRNAs were generated to target the 5' non-translated (NTR) region and the regions encoding the C, NS4B and NS5A proteins of the BVDV genome. The siRNAs were first validated using an EGFP/BVDV reporter system and were then shown to suppress BVDV-induced cytopathic effects and viral titers in cell culture with surprisingly different activities compared to the reporter system. Efficient viral suppression was then achieved by bovine 7SK-expressed BVDV-specific shRNAs. Overall, our results demonstrated the use of siRNA and shRNA-mediated gene silencing to achieve efficient inhibition of the replication of this virus in cell culture.

Animals↗

[Hepatitis B virus replication status and its response to surgery for tumor in hepatocellular carcinoma patients with positive markers of HBV].

OBJECTIVE: To investigate the status of hepatitis B virus (HBV) activity in patients with hepatocellular carcinoma (HCC) and its response to surgical interventions for tumor in HBV positive HCC patients. METHOD: We analyzed HBV marker results and detected HBV DNA in sera of 97 HCC patients (pre-operation 66 cases, post-operation 31 cases) with polymerase chain reaction (PCR) assay. We also investigated the HBV DNA titer in sera before and one week after operation in 20 HCC patients with positive HBV markers by quantitative polymerase chain reaction (Q-PCR). RESULT: HBV DNA positive rate in sera was 40.9% and 64.52% in the two subgroup respectively (P < 0.05). HBV DNA titer in sera increased after operation (P < 0.01). CONCLUSION: The infection rate of HBV in Chinese HCC patients is very high. The replication of hepatitis B virus is strong in partial HBV marker positive HCC patients and surgical interventions may promote the virus replication.

Carcinoma, Hepatocellular↗

Enterovirus receptors and virus replication in human leukocytes.

Although enteroviruses cause a great variety of diseases including meningitis, paralysis and myocarditis, the life-cycle of these viruses in man is still quite poorly understood. The role of human leukocytes as a target for enterovirus infections was studied in this report. Despite great similarity in the structure and replication of coxsackievirus B3 (CBV3), echovirus 1 (EV1), and poliovirus 1 (PV1), the ability of these viruses to infect human peripheral blood mononuclear cells (PBMC), and B (Raji), T (Molt-4) and monocytic (U-937) cell lines differed markedly. CBV3 attached both to PBMC and the three haematopoietic cell lines studied, whereas EV1 bound only to PBMC. Generally, the attachment of PV1 was very poor. The binding assays mostly correlated with the expression of CD55 (decay accelerating factor, DAF) and alpha2beta1-integrin (VLA-2), which are known to act as receptors for CBV3 and EV1, respectively, as well as with the expression of the PV receptor on the cell surface. To analyse virus replication in the cells, viral protein and nucleic acid syntheses were studied by immunoprecipitation and RT-PCR. CBV3 was able to replicate in Raji and Molt-4 cells, even though no expression of DAF was detected, whereas in the monocytic cell line, viral protein synthesis was detected only after transfection of virus RNA. Neither CBV3 nor EV1 replicated in PBMC and all haematopoietic cells studied seemed to be poorly permissive for PV1 replication.

Cell Line↗

Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice.

We previously showed that the intrahepatic induction of cytokines such as alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibits hepatitis B virus (HBV) replication noncytopathically in the livers of transgenic mice. The intracellular pathway(s) responsible for this effect is still poorly understood. To identify interferon (IFN)-inducible intracellular genes that could play a role in our system, we crossed HBV transgenic mice with mice deficient in IFN regulatory factor 1 (IRF-1), the double-stranded RNA-activated protein kinase (PKR), or RNase L (RNase L) (IRF-1(-/-), PKR(-/-), or RNase L(-/-) mice, respectively), three well-characterized IFN-inducible genes that mediate antiviral activity. We showed that unmanipulated IRF-1(-/-) or PKR(-/-) transgenic mice replicate HBV in the liver at slightly higher levels than the respective controls, suggesting that both IRF-1 and PKR individually appear to mediate signals that modulate HBV replication under basal conditions. These same animals were responsive to the antiviral effects of the IFN-alpha/beta inducer poly(I-C) or recombinant murine IFN-gamma, suggesting that under these conditions, either the IRF-1 or the PKR genes can mediate the antiviral activity of the IFNs or other IFN-inducible genes mediate the antiviral effects. Finally, RNase L(-/-) transgenic mice were undistinguishable from controls under basal conditions and after poly(I-C) or IFN-gamma administration, suggesting that RNase L does not modulate HBV replication in this model.

Animals↗

Transinhibition of herpes simplex virus replication by an inducible cell-resident gene encoding a dysfunctional VP19c capsid protein.

This study demonstrates that cells expressing a dysfunctional analog of a herpes simplex virus (HSV) capsid protein inhibits HSV replication. Vero cell lines expressing HSV-1 capsid protein VP19c/beta-galactosidase fusion proteins were constructed and tested for their kinetics of expression, intracellular location, and ability to interfere with HSV replication. Two chimeric genes were constructed for these studies. The larger chimeric gene encodes the amino terminal 327 amino acids (aa) of VP19c fused to the carboxy terminal 1026 aa of beta-galactosidase, and the shorter chimeric gene encodes VP19c aa 1-30 and 302-327 fused to the carboxy-terminal 1026 aa of beta-galactosidase. Cell lines V32G-1 and V32G-2 containing the larger and the shorter chimeric genes, respectively, were isolated after cotransfection with plasmid pSV2-neo DNA, cell selection, and limiting-dilution cloning. The chimeric VP19c/beta-galactosidase genes resident in V32G-1 and V32G-2 cell lines were induced by early gene products of superinfecting wild-type HSV-1 and HSV-2, but were not constitutively expressed. The hybrid proteins expressed in infected V32G-1 and V32G-2 cells both colocalized with infected cell protein 8 (ICP8) into virus-replicative compartments in the cell nuclei. HSV-1 and HSV-2 growth in V32G-1 cells (which express the larger chimeric gene) was significantly reduced compared to growth in V32G-2 and control Vero cells. The data suggest that the larger VP19c/beta-galactosidase hybrid protein interferes with virus capsid assembly or morphogenesis in a competitive manner. Results also demonstrate that a small portion of VP19c containing the predicted endoplasmic reticulum signal sequence for this capsid protein (aa 1-30) promotes incorporation of the VP19c/beta-galactosidase fusion proteins into nuclear viral replication compartments.

Amino Acid Sequence↗

Correlation of virus replication, cytokine (TNF-alpha and IL-1) producing cells, neuronal necrosis and inflammation after intranasal infection of mice with herpes simplex virus strains of different virulence.

The number of TNF-alpha and IL-1 beta producing cells was investigated during the acute replication phase of herpes simplex virus (HSV) in trigeminal ganglia after intranasal infection with strains of different virulence. The highly virulent strain WAL replicated strongly and induced many cytokine producing cells early in the ganglia. The low virulent strain HFEM replicated less, only few cytokine producing cells were detected late. The thymidine-kinase negative (TK-) virus 1301 did not replicate but produced some lymphocytic inflammation. The higher the virulence of strains of HSV-1 or -2 was, the stronger was the extent of histopathological lesions; moreover, a dissociation in time between replication and cellular reaction (granulocytic and lymphocytic) could be observed after infection with strains HFEM and TK- virus 1301. CD4 and CD8 positive cells could be detected mainly at the rim of necrotic areas, TNF-alpha and IL-1 beta producing cells, however, were scattered throughout the ganglia.

Administration, Intranasal↗

Synergy of small molecular inhibitors of hepatitis C virus replication directed at multiple viral targets.

Chronic hepatitis C virus (HCV) infection is a significant worldwide health problem with limited therapeutic options. A number of novel, small molecular inhibitors of HCV replication are now entering early clinical trials in humans. Resistance to small molecular inhibitors is likely to be a significant hurdle to their use in patients. A systematic assessment of combinations of interferon and/or novel anti-hepatitis C virus agents from several different mechanistic classes was performed in vitro. Combinations of inhibitors with different mechanisms of action consistently demonstrated more synergy than did compounds with similar mechanisms of action. These results suggest that combinations of inhibitors with different mechanisms of action should be prioritized for assessment in clinical trials for chronic hepatitis C virus infection.

Antiviral Agents↗