Virus Replication In Cell Culture Research Articles

Efficient replication and expression of murine leukemia virus with major deletions in the enhancer region of U 3

The effect of deletions within the enhancer region in the U 3 part of the LTR derived from the murine retrovirus Akv was studied. The deletions were stably transmitted through normal virus replication as shown by sequence analysis of cloned polymerase chain reaction product of the cDNA copy of the viral RNA. Genetic tagging of the retrovirus with lacO facilitated the analysis. Among the individual mutated LTRs an over 100-fold difference in a transient expression assay was previously detected. This difference was not revealed in studies of viral replication in cell culture, where the expression level of virus with the deleted LTRs all reached the level of virus with the intact LTR. We propose that stimulatory cis-acting sequences either adjacent to the site of proviral integration or in the coding regions of the provirus may compensate for deletions in the LTR.

Activity of Penciclovir in Combination with Azido-Thymidine, Ganciclovir, Acyclovir, Foscarnet and Human Interferons against Herpes Simplex Virus Replication in Cell Culture

Combinations of penciclovir (PCV) with other antiviral agents (acyclovir, ACV; ganciclovir, GCV; foscarnet, PFA; azido-thymidine, AZT) or with human interferons (HulFN-α,β,γ) were tested for inhibitory activity against herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) in cell culture. The antiviral interactions observed between combinations of PCV with ACV or GCV were purely additive. Combinations of PCV with HulFNs demonstrated highly synergistic anti-herpesvirus activity; some synergy was also detected between PCV and PFA against HSV-1. High concentrations of AZT inhibited the antiviral activity of PCV; this antagonism was competitive. In more detailed studies it was demonstrated that high concentrations of AZT also inhibited the antiviral activity of ACV, and that ACV was more sensitive to this antagonism than PCV. It was concluded that the antagonism was unlikely to have clinical significance.

Human immunodeficiency virus type 1 mutants resistant to nonnucleoside inhibitors of reverse transcriptase arise in tissue culture.

We have recently described a nonnucleoside compound that specifically inhibits the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS. This compound, nevirapine (BI-RG-587), interacts with highly conserved tyrosine residues at positions 181 and 188 in the reverse transcriptase to inhibit the recombinant enzyme and virus replication in cell culture with 50% inhibitory concentrations in the 40 nM range. HIV-1 variants resistant to nevirapine emerged with passage in cell culture in the presence of drug. This resistant phenotype was stable with continued passage in the absence of drug. These mutants had a substitution of cysteine for the tyrosine at position 181. Introduction of this mutation into the recombinant enzyme increased the inhibitory concentration of nevirapine 100-fold. Substitution of cysteine for tyrosine at residue 181 into the wild-type viral genome conferred a similar reduction in susceptibility to nevirapine. Mutants were also resistant to a tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione derivative and two 6-phenylthiouracil derivatives but retained their sensitivity to the other reverse transcriptase inhibitors, 3'-azido-3'-deoxythymidine and foscarnet.

Kinetics of Rabies Virus Replication in Cell Cultures

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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 % (CD 50) to the effective dose that inhibited 50 % of viral antigen expression (ED 50), 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.

Isolation of a viable herpesvirus (pseudorabies virus) mutant specifically lacking all four known nonessential glycoproteins

Recently we described the isolation and characterization of a pseudorabies virus (PrV) mutant lacking the nonessential glycoproteins gI, gp63, and gIII. Using insertional mutagenesis with a functional gX-β-galactosidase fusion gene we describe here the isolation of a PrV mutant specifically lacking all four known nonessential glycoproteins, gl, gp63, gIII, and gX. The quadruple mutant did not show any significant alterations in the in vitro growth characteristics compared to its triple mutant parent. These results prove that PrV nonessential glycoproteins are dispensable for viral replication in cell culture altogether.

Inhibition of human immunodeficiency virus replication in cell culture by endogenously synthesized antisense RNA

Antisense RNA, which has a sequence complementary to mRNA, may provide the basis for antiviral therapies of high selectivity. We have explored the inhibitory effect of six antisense RNAs upon the replication of human immunodeficiency virus (HIV) in cell culture. We chose regions of the HIV genome to test whether sequences required for splicing or for translation initiation were more susceptible to antisense RNA interference. Our results suggest that inhibitory antisense RNAs contain sequences complementary to the AUG initiation codon of the tat gene and have a comparatively low tendency to form intramolecular base pairs which would interfere with intermolecular duplex formation. Inhibition can be substantial (over 70%) but is transient. Transience does not result from mutation of the input virus. Inhibition was not a consequence of the induction of interferon by antisense RNA-mRNA duplex formation. Our results suggest that at least part of the inhibitory effect is at the posttranscriptional level.

In situ molecular hybridization for detection of Aleutian mink disease parvovirus DNA by using strand-specific probes: identification of target cells for viral replication in cell cultures and in mink kits with virus-induced interstitial pneumonia.

Strand-specific hybridization probes were utilized in in situ molecular hybridization specifically to localize replicative form DNA of Aleutian mink disease parvovirus (ADV). Throughout in vitro infection, duplex replicative form DNA of ADV was located in the cell nuclei. Single-stranded virion DNA and capsid proteins were present in the nuclei early in infection, but were later translocated to the cytoplasm. In neonatal mink, ADV causes acute interstitial pneumonia, and replicative forms of viral DNA were found predominantly in alveolar type II cells of the lung. Viral DNA was also found in other organs, but strand-specific probes made it possible to show that most of this DNA represented virus sequestration. In addition, glomerular immune complexes containing intact virions were detected, suggesting that ADV virions may have a role in the genesis of ADV-induced glomerulonephritis.

Rapid identification of nonessential genes of herpes simplex virus type 1 by Tn5 mutagenesis.

The large genome of herpes simplex virus type of (HSV-1) encodes at least 80 polypeptides, the majority of which have no recognized function. A subgroup of these gene products appears to be nonessential for virus replication in cell culture, but contributes to the complex life cycle of the virus in the host. To identify such functions, a simple insertional mutagenesis method has been used for selective inactivation of individual HSV-1 genes. The bacterial transposon Tn5 was allowed to insert randomly into cloned restriction fragments representing the entire short unique (US) region of the HSV-1 genome. Of the 12 open reading frames that were mutagenized with Tn5, mutant derivatives of US2, US4, and US5 were recombined into the virus. These three genes proved to be nonessential for HSV-1 replication in Vero (African Green monkey kidney) cells and the US4 gene appeared to be involved in viral pathogenesis in the central nervous system of mice. This rapid mutagenesis procedure should prove useful in exploring the entire HSV-1 genome as well as the genomes of other complex animal viruses.

Inhibiting effect of (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine on varicella-zoster virus replication in cell culture.

The activity and mode of action of the new nucleoside analog (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (2HM-HBG) against varicella-zoster virus (VZV) were determined. In cell culture, replication of different strains of VZV was inhibited to 50% by 0.4 to 0.7 microM 2HM-HBG, while 685 microM was required to inhibit 50% of the DNA synthesis in uninfected human lung fibroblasts. A thymidine kinase-negative VZV strain was not inhibited by 100 microM 2HM-HBG. Inhibition of VZV replication was not reversible after 7 to 14 days of incubation, depending on the multiplicity of VZV. 2HM-HBG was shown to be selectively phosphorylated by purified VZV thymidine kinase, with an inhibition constant of 32.5 microM. The antiviral activity of 2HM-HBG in cell culture was decreased by the addition of deoxythymidine and deoxycytidine but not by other ribo- or deoxyribonucleosides.

Efficacy of 5-methoxymethyl-2′-deoxyuridine in combination with arabinosyladenine for the treatment of primary herpes simplex genital infection of mice and guinea pigs

The relative efficacy of 5-methoxymethyl-2′-deoxyuridine (MMdUrd), arabinosyl-adenine (ara-A) and the combination of MMdUrd and ara-A in the treatment of experimental genital herpes (GH) was investigated using mouse and guinea pig models. The infection was initiated by intravaginal inoculation using either HSV-2, strain X-265 or HSV-2, strain MS. Treatment was initiated 3 h post virus inoculation. The parameters used to evaluate efficacy were: (i) percent mortality; (ii) mean day of death; (iii) virus yield from the vaginal secretions; and (iv) mean lesion score. The simultaneous application of 5% MMdUrd and 5% ara-A was an effective treatment for controlling primary GH in both animal models. Combination chemotherapy was also effective in preventing recurrence of infection as well as the emergence of drug resistant virus. At 20% concentration, ara-A was effective in providing protection against GH. However, lesions due to recurrent GH appeared after cessation of treatment and the virus isolated from vaginal secretions of ara-A treated animals required higher concentration of drug for inhibition of virus replication in cell culture. 20% MMdUrd was only partially effective in controlling GH. The production of infectious virus particles (virus yield) in cell culture after exposure to either ara-A of MMdUrd alone or in combination was determined. When MMdUrd and ara-A were used together, a substantially lower amount of each drug was needed to inhibit virus production completely and removal of drugs did not result in an increase in virus yield.

The laboratory selection of antiviral agents.

The major disease targets for antiviral chemotherapy are influenza, rhinovirus colds and herpetic infections Some success has been achieved against influenza with 1-amino adamantane (amantadine), and several new alicyclic amines are at the development stage. There are many organic compounds, covering a wide range of chemical types, which will inhibit very effectively most or all strains of rhinovirus in vitro, but all have failed to inhibit significantly disease development in infected human volunteers. Most success has been achieved with anti-herpetic drugs, and a variety of nucleoside analogues ar now available for clinical use. Primary testing methods for potential antiviral agents rely on high-through-put methods for detecting inhibition of virus replication in cell cultures. Secondary screening of active compounds is often done in organ culture e.g. tracheal tissue explants. In-vivo evaluation is done in animal models of the human disease, where available. Prolonged safety testing is required before human trials can be started.

Rapid detection of Semliki Forest virus replication in cell culture with peroxidase-labeled monoclonal antibodies: Determination of inhibitory concentrations of monoclonal antibodies and ribavirin

Rapid detection of Semliki Forest virus replication in cell culture with peroxidase-labeled monoclonal antibodies: Determination of inhibitory concentrations of monoclonal antibodies and ribavirin

Influence of various experimental conditions on the inhibitory effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine on varicella-zoster virus replication in cell culture.

Inhibition of varicella-zoster virus (VZV) replication by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) has been examined in vitro under various experimental conditions. The 50% inhibitory dose (ID50) of BVDU for VZV replication in human embryonal fibroblast (HEF) cultures was 0.016 micrograms/ml, if the assay was based on the reduction of visible foci. If the assay was based on the reduction of immunofluorescent foci, the ID50 was 3 times higher. There was no significant difference in ID50, whether the HEF cultures were infected with cell-free or cell-associated VZV. When the HEF cells were infected with VZV at different multiplicities of infection (MOI), the ID50 of BVDU increased in parallel with the increase of MOI. BVDU was normally added immediately after virus infection. However, the addition of BVDU could be delayed until 8 hr after infection without substantial decrease of activity. On the other hand, BVDU did not cause any inhibition of focus formation when it was removed within 8 hr after VZV infection. If removed at 24 or 48 hr after infection, BVDU caused a significant reduction in focus formation. BVDU had no effect on focus formation if added to the HEF cells before virus infection. BVDU was also found to inhibit VZV focus formation in Vero cells, but only at an ID50 that was 5-10 times higher than the ID50 noted in HEF cells.

Inhibition of viral replication in cell cultures treated with prostaglandin E1.

AbstractA study has been initiated to determine the influence of prostaglandins on virus replication and its relationship to the interferon system. In the present study, data are presented which show that pretreatment of cells in culture with prostaglandin E1 results in (i) a reduction in the yields of Mengo, MM and polio viruses, (ii) enhanced yields of interferon, and (iii) inhibition of cell division.

Synthesis of 5-[(methylthio)methyl]-2'-deoxyuridine, the corresponding sulfoxide and sulfone, and their 5'-phosphates: antiviral effects and thymidylate synthetase inhibition.

Substitution on the alpha position of thymidine with methylthio (3) and methylsulfonyl (5) groups gave antiviral agents that were specific and relatively nontoxic inhibitors of herpes simplex virus replication in cell culture. The thioether (3) was effective against both types 1 and 2 of herpes simplex virus, whereas the activity of the sulfone derivative (5) was restricted to herpes simplex virus type 1. The sulfoxide derivative 1-(2-deoxy-beta-D-ribofuranosyl)-alpha-(methylsulfinyl)thymine (4) was inactive as an antiviral agent. The 5'-phosphates of these three thymidine derivatives were relatively potent inhibitors of thymidylate synthetase (Ki values range from 7.8 to 1.9 microM). It is improbable that the inhibition of this enzyme accounts for the anti-herpes activity of compounds 3 and 5.

Susceptibility of different strains of influenza a virus to the inhibitory effects of 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA)

Susceptibility to the neuraminidase-inhibiting effects of a synthetic analog of neuraminic acid, 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA) was found to vary among different strains of influenza A virus according to the neuraminidase they contain. In particular, NWS (H0N1) virus and recombinant strains which derive their neuraminidase from NWS are especially susceptible to FANA as measured by the concentrations of inhibitor which reduce rates of elution from red cells, in vitro neuraminidase activity using fetuin as a substrate, and virus replication in cell culture under agar. Growth in tissue culture of X-7 (H0N2) virus and all recombinant viruses containing N2 neuraminidase is approximately 50–200 times less susceptible to inhibition by FANA.These results provide further evidence that the inhibitory effects of FANA on the replication of influenza viruses are mediated by its specific neuraminidase-inhibitory activity and confirm that neuraminidase activity is necessary for the replication of influenza viruses.