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J J Leary

Publications and source records attributed to J J Leary.

At least 19 recordsLinked to original sources
PubMed2001

Absence of rapid selection for acyclovir or penciclovir resistance following suboptimal oral prodrug therapy of HSV-infected mice.

BACKGROUND: Acyclovir (ACV) resistant herpes simplex virus (HSV) isolates can be readily selected in animal infection models receiving suboptimal ACV treatment, however no comparative studies of the emergence of resistance following suboptimal treatment with valacyclovir (VCV) or famciclovir (FCV), the prodrugs of acyclovir and penciclovir, respectively, have been reported. METHODS: Mice (n = 30) were infected with HSV type 1 or 2 in the ear pinnae and administered oral prodrugs at one fifth a dose previously shown to be effective. To select and amplify drug-resistant HSV, a total of seven consecutive in vivo passages with suboptimal treatment were performed for each virus sample and progeny virus from each passage was characterized by the plaque reduction (PRA) and plating efficiency assays (PEA). RESULTS: No drug-resistant HSV-2 and only a single drug-resistant HSV-1 variant were identified. Virus recovered from the first three sequential passages of this HSV-1 sample was susceptible by PRA, although the proportion of resistant virus recovered gradually increased upon passage. The resistant HSV-1 phenotype was confirmed by PRA after four sequential passages in mice. Unexpectedly, this in vivo-selected drug-resistant HSV-1 failed to yield an infection completely refractory to treatment in subsequent passages. CONCLUSIONS: Sub-optimal therapy of immunocompetent mice with either VCV or FCV did not readily select for HSV-mutants resistant to either ACV or PCV, suggesting that selection of resistance with either prodrug remains difficult using this system. Futhermore, this study suggests that the PEA may represent a useful adjunct to the PRA for monitoring alterations in the proportion of drug-resistant virus even when no change in IC50 is apparent.

Acyclovir↗
PubMed2001

Biochemical characterization of a virus isolate, recovered from a patient with herpes keratitis, that was clinically resistant to acyclovir.

In vitro susceptibility assays of herpes simplex virus (HSV) do not necessarily correlate with treatment outcome. An HSV type 1 (HSV-1) isolate, N4, recovered from a patient who presented with herpes keratitis with localized immunosuppression, was characterized for susceptibility. Although the 50% inhibitory concentration (IC(50)) for this isolate was less than the accepted breakpoint for defining resistance to acyclovir (>2.0 microg/mL), the following lines of evidence suggest that the isolate was acyclovir resistant: (1) the clinical history confirmed that the infection was nonresponsive to acyclovir; (2) the in vitro susceptibility was similar to that of a thymidine kinase (TK)-negative, acyclovir-resistant virus SLU360; (3) the IC(50) of acyclovir was more than 10 times the IC(50) for an acyclovir-susceptible control strain; (4) plaque-purified clonal isolates were resistant to acyclovir (IC(50)s, >2.0 microg/mL); and (5) biochemical studies indicated that the HSV-1 N4 TK was partially impaired for acyclovir phosphorylation. Although residue changes were found in both the viral tk and pol coding regions of HSV-1 N4, characterization of a recombinant virus expressing the HSV-1 N4 polymerase suggested that the TK and Pol together conferred the acyclovir-resistance phenotype.

Acyclovir↗
PubMed2001

Frequency of acyclovir-resistant herpes simplex virus in clinical specimens and laboratory isolates.

The proportion of acyclovir (ACV)-resistant herpes simplex virus (HSV) isolates in clinical specimens and laboratory isolates was determined. HSV isolates in clinical specimens and laboratory isolates were cultured in the absence or presence of 5 microg of ACV per ml. The frequency of ACV-resistant HSV was calculated as (average virus titer in the wells with ACV)/(average virus titer in the wells without ACV). The mutation frequency of HSV type 1 isolates in clinical samples (directly from patient lesions) was 7.5 x 10(-4) +/- 2.5 x 10(-4) (mean +/- standard error), and that of HSV type 2 isolates was 15.0 x 10(-4) +/- 4.6 x 10(-4). The mutation frequencies of isolates derived in the laboratory from these clinical samples were very similar. Similarly, the 50% inhibitory concentrations for isolates in clinical samples and laboratory isolates were identical. The frequencies of ACV-resistant HSV types 1 and 2 were in a narrow range of 7.5 x 10(-4) to 15.0 x 10(-4) among isolates in clinical specimens and did not change for laboratory-derived pools of viral isolates.

Acyclovir↗
PubMed2001

Characterization of herpes simplex viruses selected in culture for resistance to penciclovir or acyclovir.

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv(r) clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv(r) mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.

Acyclovir↗
PubMed2000

Effects of antiviral usage on transmission dynamics of herpes simplex virus type 1 and on antiviral resistance: predictions of mathematical models.

Herpes simplex virus type 1 (HSV-1) causes recurrent herpes labialis (RHL), a common disease afflicting up to 40% of adults worldwide. Mathematical models are used to analyze the effect of antiviral treatment on the transmission of, and the prevalence of drug resistance in, HSV-1 in the United States. Three scenarios are analyzed: no antiviral use, the current level of use, and a substantial increase in nucleoside analogue use, such as might occur if topical penciclovir were available over-the-counter for the treatment of RHL. A basic model predicts that present level of nucleoside analogue use has a negligible effect on HSV-1 transmission and that even if use of topical penciclovir for (RHL) increased substantially, the overall prevalence of infectious HSV-1 is unlikely to be reduced by more than 5%. An expanded model, which allows for acquired resistance and includes immunocompromised hosts and other more realistic features, predicts that current antiviral use is unlikely to lead to any noticeable increase in resistance. If antiviral use increases, the resulting rise in resistance in the population will depend primarily on the probability that immunocompetent hosts will acquire permanent resistance upon treatment. This probability is known to be small, but its exact value remains uncertain. If acquired resistance occurs less than once per 2,500 treated episodes, then in the community at large, the frequency of HSV-1 resistance is predicted to increase slowly, if at all (remaining below 0.5% for >50 years), even with extensive nucleoside analogue use. If acquired resistance emerges in 1 of 625 treated episodes (the maximum of an approximate 95% confidence interval derived from the results of several studies of resistance in treated hosts), then the prevalence of infection with resistant HSV-1 could rise from about 0.2% to 1.5 to 3% within 50 years. The limitations of existing data on acquired resistance and the potential impact of acquired resistance if it occurs are discussed, and strategies are suggested for enhancing information on acquired resistance. The predictions of this model contrast with the more rapid increases in antimicrobial resistance anticipated by models and observed for other pathogenic bacteria and viruses. The reasons for these contrasting predictions are discussed.

Aging↗
PubMed2000

Difference in incidence of spontaneous mutations between Herpes simplex virus types 1 and 2.

Spontaneous mutations within the herpes simplex virus (HSV) genome are introduced by errors during DNA replication. Indicative of the inherent mutation rate of HSV DNA replication, heterogeneous HSV populations containing both acyclovir (ACV)-resistant and ACV-sensitive viruses occur naturally in both clinical isolates and laboratory stocks. Wild-type, laboratory-adapted HSV type 1 (HSV-1) strains KOS and Cl101 reportedly accumulate spontaneous ACV-resistant mutations at a frequency of approximately six to eight mutants per 10(4) plaque-forming viruses (U. B. Dasgupta and W. C. Summers, Proc. Natl. Acad. Sci. USA 75:2378-2381, 1978; J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. T. Gelep, and P. A. Schaffer, Virology 132:26-37, 1984). Typically, these resistance mutations map to the thymidine kinase (TK) gene and render the virus TK deficient. To examine this process more closely, a plating efficiency assay was used to determine whether the frequencies of naturally occurring mutations in populations of the laboratory strains HSV-1 SC16, HSV-2 SB5, and HSV-2 333 grown in MRC-5 cells were similar when scored for resistance to penciclovir (PCV) and ACV. Our results indicate that (i) HSV mutants resistant to PCV and those resistant to ACV accumulate at approximately equal frequencies during replication in cell culture, (ii) the spontaneous mutation frequency for the HSV-1 strain SC16 is similar to that previously reported for HSV-1 laboratory strains KOS and Cl101, and (iii) spontaneous mutations in the laboratory HSV-2 strains examined were 9- to 16-fold more frequent than those in the HSV-1 strain SC16. These observations were confirmed and extended for a group of eight clinical isolates in which the HSV-2 mutation frequency was approximately 30 times higher than that for HSV-1 isolates. In conclusion, our results indicate that the frequencies of naturally occurring, or spontaneous, HSV mutants resistant to PCV and those resistant to ACV are similar. However, HSV-2 strains may have a greater propensity to generate drug-resistant mutants than do HSV-1 strains.

Drug Resistance, Microbial↗
PubMed1998

Use of differential display reverse transcription-PCR to reveal cellular changes during stimuli that result in herpes simplex virus type 1 reactivation from latency: upregulation of immediate-early cellular response genes TIS7, interferon, and interferon regulatory factor-1.

The detailed mechanism which governs the choice between herpes simplex virus (HSV) latency and reactivation remains to be elucidated. It is probable that altered expression of cellular factors in sensory neurons leads to induction of HSV gene expression resulting in reactivation. As an approach to identify novel cellular genes which are activated or repressed by stimuli that reactivate HSV from latency and hence may play a role in viral reactivation, RNA from explanted trigeminal ganglia (TG) was analyzed by differential display reverse transcription-PCR (DDRT-PCR). Nearly 50 cDNAs whose mRNA level was modified by the stress of explantation were isolated and sequenced. We present a listing of a spectrum of altered RNAs, including both known and unknown sequences. Five of those differentially displayed transcripts were identified as interferon-related murine TIS7 mRNA. These results were confirmed in both infected and uninfected ganglia by quantitative RNase protection assay and immunostaining. Alpha and beta interferons and interferon regulatory factor-1 (IRF-1) were also induced by explantation. In addition, we have identified sequences that correspond to IRF-1 consensus binding sites in both HSV type 1 origins of replication. Our findings suggest that physiological pathways that include these cellular factors may be involved in modulating HSV reactivation.

Animals↗
PubMed1997

Gene expression during reactivation of herpes simplex virus type 1 from latency in the peripheral nervous system is different from that during lytic infection of tissue cultures.

Herpes simplex virus (HSV) replicates in peripheral tissues and forms latent infections in neurons of the peripheral nervous system. It can be reactivated from latency by various stimuli to cause recurrent disease. During lytic infection in tissue culture cells, there is a well-described temporal pattern of (i) immediate-early, (ii) early, and (iii) late gene expression. However, latency is characterized by little if any expression of genes of the lytic cycle of infection. During reactivation, the pattern of gene expression is presumed to be similar to that during the lytic cycle in tissue culture, though recent work of W. P. Halford et al. (J. Virol. 70:5051-5060, 1996) and P. F. Nichol et al. (J. Virol. 70:5476-5486, 1996) suggests that it is modified in neuronal cell cultures. We have used the mouse trigeminal ganglion explant model and reverse transcription-PCR to determine the pattern of viral and cellular gene expression during reactivation. Surprisingly, the pattern of viral gene expression during lytic infection of cell cultures is not seen during reactivation. During reactivation, early viral transcripts were detected before immediate-early transcripts. The possibility that a cellular factor upregulates early genes during the initial reactivation stimulus is discussed.

Animals↗
PubMed1993

Synergistic drug interactions of an HIV-1 protease inhibitor with AZT in different in vitro models of HIV-1 infection.

Synthetic peptide mimetic inhibitors of HIV-1 protease effectively block spread of infectious virus in acutely infected T-cells. These compounds also inhibit production of infectious virions from chronically infected T-cell lines. In order to determine the potential for drug interaction effects on antiviral activity, an HIV-1 protease inhibitor (SK&F 108922) and AZT were studied in three different in vitro models of HIV-1 infection of T-cell lines, specifically, (1) acutely infected cells infected at low multiplicity, (2) HIV-1 chronically-infected cells and (3) co-cultivations of chronically infected with non-infected cells. Upon co-treatment, these compounds demonstrated synergy in Molt4 or H9 cells acutely infected with HIV-1 strain IIIB. Either compound alone was a potent inhibitor of HIV-1 in co-cultivations of uninfected and chronically infected cells. In combination treatments of co-cultures, SK&F 108922 demonstrated strong synergy with AZT. Treatment of H9/IIIB chronically infected cells demonstrated no inhibitory effect by AZT treatment (EC50 = > 100 microM) whereas SK&F 108922 was inhibitory (EC50 = 3 microM). Upon co-treatment of H9/IIIB chronically infected cultures with both compounds, the antiviral activity was similar to that of the protease inhibitor alone suggesting no drug interaction. In the co-cultivation experiments, AZT's antiviral effect was most likely due to blocking spread of acute infection to uninfected cells in the culture. No antagonistic effects were observed with AZT and SK&F 108922 co-treatments. These results clearly demonstrate that an HIV-1 protease inhibitor can exert a potent antiviral effect on chronically infected T-cells in contrast to AZT and is capable of potent synergy with AZT in acute and co-culture in vitro infection models.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Human immunodeficiency virus type 1 protease inhibitors irreversibly block infectivity of purified virions from chronically infected cells.

Synthetic peptide analog inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were used to study the effects of inhibition of polyprotein processing on the assembly, structure, and infectivity of virions released from a T-cell line chronically infected with HIV-1. Inhibition of proteolytic processing of both Pr55gag and Pr160gag-pol was observed in purified virions from infected T cells after treatment. Protease inhibition was evident by the accumulation of precursors and processing intermediates of Pr55gag and by corresponding decreases in mature protein products. Electron microscopy revealed that the majority of the virion particles released from inhibitor-treated cells after a 24-h treatment had an immature or aberrant capsid morphology. This morphological change correlated with the inhibition of polyprotein processing and a loss of infectivity. The infectivity of virion particles purified from these chronically infected cell cultures was assessed following treatment with the inhibitor for 1 to 3 days. Virions purified from cultures treated with inhibitor for 1 or 2 days demonstrated a 95- to 100-fold reduction in virus titers, and treatment for 3 days resulted in complete loss of detectable infectivity. The fact that virions from treated cultures were unable to establish infection over the 7- to 10-day incubation period in the titration experiments strongly suggests that particles produced by inhibitor-treated cells were unable to reactivate to an infectious form when they were purified away from exogenous protease inhibitor. Thus, a block of HIV-1 protease processing of viral polyproteins by specific inhibitors results in a potent antiviral effect characterized by the production of noninfectious virions with altered protein structures and immature morphologies.

Blotting, Western↗
PubMed1990

Humoral response of cynomolgus macaques to human soluble CD4: antibody reactivity restricted to xeno-human determinants.

The CD4 cell surface glycoprotein which is expressed primarily by a subset of T lymphocytes plays a key role in normal immune responses. In the immunopathogenesis of AIDS, it serves as the high-affinity receptor for HIV, facilitating viral attachment and entry into CD4+ cells. As such, the truncated soluble form of this molecule (sT4) has been proposed as a therapeutic drug for the treatment of AIDS whereby it would act as decoy for viral entry into cells or facilitate elimination of soluble viral envelope glycoprotein. In a study designed to look at the effect of sT4 on immune function, sT4 was administrated to experimentally naive primates. In this report, we show that administration of sT4 to cynomolgus macaque monkeys over a period of up to 3 weeks results in antibody responses with specificities for human CD4 molecules. Antisera thus generated bound sT4 and cell surface CD4 expressed on human T lymphocytes but failed to bind to cynomolgus lymphocytes. These antibodies caused no apparent adverse effects on normal immune functions of the cynomolgus macaques. We conclude from these data that the antibody response to soluble CD4 in cynomolgus monkeys is directed at determinants present on human CD4 but absent on monkey CD4. The restricted xenogeneic specificity of the antibody response indicates that soluble CD4 may not be highly immunogenic in syngeneic hosts. The present study also shows that these antibodies can block HIV-induced syncytium formation indicating that the antibodies bind to regions on the CD4 molecule close to the HIV-env gp120 binding site. The gp120 binding site, which resides within the N-terminal V1 domain of CD4, encompasses a region which corresponds to the complementarity determining regions (CDRs) of immunoglobulins. The CDR-like regions of CD4-V1 manifest the greatest species divergence, are tolerant to experimental in vitro mutagenesis, and generate the predominant antibody response in mice immunized with human CD4 indicating that differences in the V1 sequence between human and other non-human primates may localize to this regions.

Animals↗
PubMed1986

Advanced instrumentation and strategies for metabolic profiling.

This review summarizes the instrumental techniques applicable for metabolic profiling analysis. Since its origin with the work of Roger Williams, use has been made of planar chromatography, column liquid chromatography, electrophoresis, gas-liquid chromatography, and mass spectrometry to obtain analyte patterns. Strategies for the analysis of various metabolites including organic acids, steroids, drugs, bile acids, carbohydrates, and proteins are discussed.

Animals↗
PubMed1985

Use of biotinylated probes for detecting sickle cell anemia.

Earlier, we reported that the 5' end of the normal beta-globin gene (beta) resides on a 1.14-kilobase DNA fragment, whereas the 5' end of the sickle cell gene (beta s) resides on a 1.34-kilobase fragment. In that blot hybridization analysis, we used genomic DNA digested with restricted endonuclease Mst II, and radioactive probes with short half-life. We demonstrate here that, if a biotinylated probe is used instead in a slightly modified procedure, sickle cell anemia can be quickly and directly detected if there is as much as 5 micrograms of total genomic DNA in the sample. This procedure obviates the special precautions necessary when radioactive materials are used.

Anemia, Sickle Cell↗
PubMed1983

Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes.

A method of in situ cytohybridization is described for the detection of specific viral genomes in infected cell cultures or paraffin-embedded tissue sections without the use of radioisotopes. Biotin-labeled analogs of TTP are incorporated into viral DNA in vitro by nick translation and the resultant DNA probes hybridized to cytologic samples. Cells containing viral genetic material are then revealed by standard immunofluorescence, immunoperoxidase, or affinity cytochemical techniques that are based on the specific interaction between biotin and antibiotin IgG or avidin. Hybridization probes containing nucleotides that have an 11- or 16-atom spacer arm between the biotin molecule and the pyrimidine ring interact with these detector proteins more efficiently than probes containing biotin-nucleotides with a 4-atom spacer arm. The total procedure can be performed fairly rapidly (24 hr or less) and numerous samples can be processed simultaneously. Although the detection methods employed to date are not as sensitive as autoradiographic procedures with high specific activity probes, more sensitive protein detector complexes are currently being constructed. The speed, specificity, and resolving power of this technique should be of general utility in screening for the presence of infectious agents in cell or tissue samples. Here we report the visualization of parvovirus, polyomavirus, herpes simplex virus, adenovirus, and retrovirus genetic material in infected cell cultures and herpes simplex and adenovirus DNA in paraffin-embedded autopsy tissues.

Adenoviruses, Human↗
PubMed1983

Improved methods for calculating formation constants for nucleotide--cation complexes.

Two data reduction methods that can be used to calculate formation constants for nucleotide-cation complexes are described. Both methods are used to analyze data obtained by an anion-exchange resin method, and either method can improve the accuracy of the calculated formation constants by more than 50%. The key to this significant improvement in accuracy is the realization that the equation for the mathematical model describing such systems is always nonlinear in terms of the formation constants and, in the general case, is higher order in the cation concentration. There are two major reasons for the improved quality of the results associated with the new model. First, successive linear extrapolations are eliminated, and error propagation is reduced. Second, all of the data are used for the simultaneous calculation of all formation constants, and the uncertainty due to random experimental errors is minimized.

Anion Exchange Resins↗
PubMed1983

Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.

Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1- to 10-pg range after enzyme incubation periods of 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.

Alkaline Phosphatase↗
PubMed1983

Construction of an infectious molecular clone of the autonomous parvovirus minute virus of mice.

The linear single-stranded DNA genome of minute virus of mice, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pBR322. The recombinant clones of minute virus of mice were infectious when transfected into monolayers of human 324K cells and produced virus plaques with an efficiency of about 6% that obtained with duplex replicative-form DNA purified from cells infected with minute virus of mice. Southern blot analysis of transfected cells indicated that the cloned minute virus of mice genome requires both termini to be intact for excision and replication as a linear duplex molecule.

Cells, Cultured↗