Treatment of opportunistic viral infections.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
C S Crumpacker
Publications and source records attributed to C S Crumpacker.
Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates.
The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.
Cloning, Molecular↗
Expression of herpes simplex virus type 1 DNA polymerase gene by in vitro translation and effects of gene deletions on activity.
A cloned herpes simplex virus type 1 DNA polymerase gene which is biologically functional was inserted into SP6 and T7 promoter-containing vectors for in vitro transcription-translation. pol-specific RNA synthesized in vitro will direct the synthesis of a 140-kilodalton polypeptide in rabbit reticulocyte lysates. RNAs prepared from pol templates linearized at internal restriction sites specified deleted polypeptides with sizes consistent with colinearity of the pol gene and the 140-kilodalton primary translation product. The in vitro translated pol gene product was enzymatically active, with salt resistance and sensitivity to acyclovir triphosphate, similar to the enzyme activity in crude extracts of herpes simplex virus type 1-infected Vero cells. An in-frame deletion of 78 residues (amino acids residues 881 to 959) was introduced into the expression vectors to investigate the function of a region of the polypeptide (amino acids residues 881 to 895) which is conserved in nine other DNA polymerases. In a complementation assay, this mutation abolished biological activity as well as the enzymatic activity of the in vitro translated product. A BAL31 mutation deleting the upstream open reading frame of pol had no effect on biological activity in a complementation assay but was found to increase the efficiency of in vitro translation of pol RNA. Two amino-terminal deletions of 27 and 67 residues were found to greatly enhance the enzymatic activity of the in vitro translated product, while all carboxy-terminal deletions examined (the smallest being 164 residues) abolished in vitro enzymatic activity. Expression of the 67-residue amino-terminal deleted pol gene in Escherichia coli, using a bacteriophage T7-based system, resulted in accumulation of large amounts of an insoluble fusion protein. An antiserum prepared against this fusion protein precipitated the 140-kilodalton DNA polymerase from herpes simplex virus type 1-infected cell lysates.
Animals↗
Serial immunologic identification of lymphocyte subsets in murine coxsackievirus B3 myocarditis: different kinetics and significance of lymphocyte subsets in the heart and in peripheral blood.
To elucidate possible immune mechanisms in the pathogenesis of myocarditis, we examined, by immunofluorescence techniques, the serial changes in lymphocyte subsets in the heart, spleen, and peripheral blood of C3H/He mice inoculated coxsackievirus B3 (experiment I). B cells were identified by staining with fluorescein isothiocyanate-labeled rabbit antimouse immunoglobulin. T cell subsets were demonstrated with rat anti-Thy 1.2, anti-Lyt 1, anti-Lyt 2, and anti-L3T4 monoclonal antibodies, respectively, plus fluorescein isothiocyanate-labeled antimouse immunoglobin. The percent of Thy 1.2+, Lyt 1+, and L3T4+ cells was decreased in the peripheral blood on days 3, 7, and 14. B cells were increased on day 3. In contrast, Thy 1.2+ (pan T) or Lyt 1+ cells appeared to occupy the major portion of the myocardium on days 7 and 14, when myocarditis was most severe, while B cells were sparse. For confirmation, serial immunohistologic studies (immunoperoxidase staining) of the hearts of C3H/He mice with coxsackievirus B3 myocarditis were performed (experiment II). Most of the stained cells in the heart were Thy 1.2, Lyt 1, and Lyt 2 positive on days 7 and 14. Thus, independent methods demonstrate that specific antigenic markers on lymphocytes at the site of inflammation in the acute stage of viral myocarditis differ from those on corresponding peripheral lymphocytes, and suggest the possible involvement of Thy 1.2+ (pan T) cells, mainly the Lyt 1+ and Lyt 2+ subsets (immature T cells), in the development of myocarditis in this preparation.
Animals↗
The effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine on DNA repair and mutagenesis of herpes simplex virus type 1.
Growth of HSV-1 in (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) results in incorporation of the analog into HSV-1 DNA. Employing the technique of Weigle-type reactivation (WR), HSV-1 inactivated by uv irradiation but not by growth in BVDU is a substrate for induced cellular repair pathways. Growth of mammalian cells in BVDU does not induce cellular repair pathways detected by WR. HSV-1 temperature sensitive (ts) mutants demonstrate an increased reversion frequency to the nonpermissive phenotype (ts+) following growth in a high concentration of BVDU. A ts mutant did not display an increased reversion frequency following growth in equally inhibitory concentrations of an HSV-1 polymerase inhibitor, aphidicolin. These findings suggest that BVDU incorporated into HSV-1 DNA may not be readily excised and may be a mutagenic lesion in viral DNA, although other explanations are possible.
Animals↗
Prevention of viral myocarditis with recombinant human leukocyte interferon alpha A/D in a murine model.
Effects of recombinant human leukocyte interferon alpha A/D on experimental myocarditis due to encephalomyocarditis virus were investigated. Plaque reduction assays revealed that 50% of plaque formation in vitro in human amnion (FL) cells was inhibited by interferon alpha A/D (9.7 U/ml) when it was administered 24 hours before infection with the encephalomyocarditis virus. Four week old male DBA/2 mice were inoculated intraperitoneally with 10 plaque-forming units (pfu) of encephalomyocarditis virus. Interferon alpha A/D was administered subcutaneously (10(2) U/g body weight per day in Group 1, 10(3) U/g per day in Group 2 and 10(4) U/g per day in Group 3) starting 1 day before infection. It was also administered starting the same day in Group 4 and 1 day after virus inoculation in Group 5 (10(4) U/g per day in both groups). Control mice were injected with saline solution. Each group consisted of 10 mice; they were killed on day 4 for evaluation. Myocardial virus titers were significantly lower in Group 3 (8.2 +/- 25.2 X 10(2) pfu/mg, p less than 0.05) and Group 4 (3.0 +/- 5.5 X 10(3) pfu/mg, p less than 0.05) than in control mice (5.6 +/- 4.1 X 10(4) pfu/mg). Histologic examination showed extensive myocardial necrosis and cellular infiltration in all control mice, but no myocardial necrosis or cellular infiltration in Group 3 and less severe necrosis and infiltration in Group 4. There were no significant differences in myocardial virus titers or histologic changes between control mice and Group 1, 2 or 5.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Virus vaccine and passive immunization for the prevention of viral myocarditis in mice.
The effect of an inactivated virus vaccine and passive immunization was studied in an animal model of viral myocarditis. Encephalomyocarditis (EMC) virus was inactivated by 1:4000 formalin. Four-week-old CD-1 mice were injected subcutaneously and after 7 days intravenously with 0.1 ml vaccine. Seven days after the second vaccine, live EMC virus was inoculated intraperitoneally. None of ten mice with vaccine treatment died or showed myocardial lesions after challenge with EMC virus, whereas all ten mice without vaccine died 5-7 days after EMC virus inoculation and showed severe myocardial lesions. Hyperimmune rabbit serum, made by inactivated EMC virus, had a neutralizing antibody titer of 1:3000. Four-week-old BALB/c mice were injected intramuscularly with 1:10 dilution of hyperimmune rabbit serum in 6 groups (each, n = 10) on day -2, -1, 0, 1, 2, or 3, respectively, after inoculation with 10 plaque-forming units of EMC virus, and observed for 14 days. Eight mice infected with the virus without immune serum died and all ten mice showed severe myocardial lesions. No death or myocardial lesion was seen in mice with hyperimmune rabbit serum given on days -2 to 2. In this model, the virus vaccine prevented development of myocarditis, and passive immunization had a protective effect if given early after infection.
Animals↗
Pathogenesis and preventive and therapeutic trials in an animal model of dilated cardiomyopathy induced by a virus.
An animal model of dilated cardiomyopathy following encephalomyocarditis (EMC) virus has been developed. Virus was isolated from mouse hearts and viral antigens were detected in the myocardium until the second week of infection, but neither was found thereafter. Differences were found among different strains of mice in the frequency of occurrence and severity of myocarditis, and even in the character of the pathologic lesions. Thus, genetic factors may play an important role in the pathogenesis. Autoantibodies against heart developed and the distribution of cardiac myosin isoenzymes was altered during the course of myocarditis. Virus, vaccine, maternal vaccination, recombinant interferon alpha A/D and ribavirin were effective in protecting the mice from developing myocarditis. This animal model is suitable for studying the pathogenesis of viral myocarditis and evaluating preventive and therapeutic interventions of the condition.
Animals↗
Drugs five years later: acyclovir.
In the 5 years since its release for clinical use, acyclovir (9-[2-hydroxyethoxymethyl]guanine) has proved to be a safe and effective agent for therapy of herpes simplex and varicella-zoster infections. The drug's availability in topical, oral, and intravenous preparations has allowed its use in a range of clinical situations. Acyclovir must be phosphorylated by viral thymidine kinase in infected cells, where it then acts to inhibit viral DNA replication specifically. Epstein-Barr virus and human cytomegalovirus infections do not seem to respond to acyclovir therapy, although in-vitro effects on these viruses may be seen. Acyclovir is well absorbed and distributed, with cerebrospinal fluid levels 50% that of plasma. Clearance is almost entirely by the renal route, with a half-life of 20 hours in the anuric patient. Acyclovir has an excellent safety profile, its major adverse effect being transient serum creatinine elevations during high-dose intravenous use. Major uses include treatment of primary and recurrent genital herpes and herpes encephalitis and prophyllaxis and therapy of mucocutaneous herpes and varicella-zoster infections in immunocompromised patients. Resistance to acyclovir in herpes simplex virus is rarely encountered and does not seem to be due to long-term chronic suppressive therapy.
Acyclovir↗
Incorporation of 1-beta-D-arabinofuranosylcytosine into DNA and mutagenesis of herpes simplex virus type 1.
To investigate the association between incorporation of 1-beta-D-arabinofuranosylcytosine (ara-C) into herpes simplex virus type 1 (HSV-1) DNA and mutagenesis at selected genetic loci, we grew virus in the presence or absence of ara-C and compared the mutation frequencies. Both the forward mutation frequency of HSV-1 (KOS) to the tk- phenotype and the reversion frequency of a temperature-sensitive mutant to the non-temperature-sensitive (ts+) phenotype were significantly increased following growth in ara-C. When the same viruses were grown in an inhibitor of the HSV-1 DNA polymerase that is not incorporated into DNA, no significant increase in the frequency of tk- or ts+ particles was observed. Furthermore, HSV-1 araAr, a mutant resistant to ara-C on the basis of reduced incorporation of the analog into viral DNA, did not demonstrate an increase in the number of tk- particles following growth in ara-C. These results suggest that mutagenesis of HSV-1 following growth in ara-C correlates with incorporation of the nucleoside analog into viral DNA.
Cytarabine↗
Therapy with cyclosporine in experimental murine myocarditis with encephalomyocarditis virus.
To explain the progression from infectious viral myocarditis to congestive cardiomyopathy an infection/immune hypothesis has been proposed stating that the primary viral process incites an excessive or disordered immunologic response against the myocardium. To test whether one form of immunosuppressive therapy might ameliorate this process, we used cyclosporine in a murine preparation of infectious myocarditis (encephalomyocarditis [EMC] virus), which has been shown to result in a congestive cardiomyopathy pathologically similar to that seen in man. Eight-week-old male DBA-2 mice were infected with EMC virus and randomized to a treatment or control group. Cyclosporine (25 mg/kg/day) was administered subcutaneously for 3 weeks, starting (1) at 1 week after infection during viral replication, and (2) at 3 weeks after infection, after the period of active viral replication. In mice treated during viral replication there was a significantly higher mortality rate compared with that of control mice (15/21 vs 9/29, p = .01). There was no evident reduction in myocardial pathology (inflammation, necrosis, or calcification) in the treated compared with the control groups. In mice treated after the period of viral replication, there was no improvement in mortality (8/22 vs 2/19, NS) compared with control. Treated mice showed no reduction in myocardial histopathologic lesions. Furthermore, treated mice had significantly greater heart weight/body weight ratios (1.3 +/- 0.4% vs 1.0 +/- 0.3%, p less than .005), lung weight/body weight ratios (1.1 +/- 0.5% vs 0.8 +/- 0.3%, p less than .05), and liver weight/body weight ratios (6.0 +/- 0.8% vs 5.4 +/- 0.6%, p less than .005) than control mice, suggesting more severe myocardial failure.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Progressive polyradiculopathy in acquired immune deficiency syndrome.
We studied three patients with acquired immune deficiency syndrome (AIDS) and progressive polyradiculopathy. Postmortem examination of one patient disclosed extensive necrosis, inflammatory infiltrates, and focal vasculitis of spinal roots. Typical cytomegaloviral (CMV), intranuclear, and intracytoplasmic inclusions were noted within enlarged endoneurial and endothelial cells. Progressive polyradiculopathy is an unusual complication of AIDS; CMV may be the causative agent in certain cases.
Acquired Immunodeficiency Syndrome↗
Intravenous trimethoprim-sulfamethoxazole and ataxia.
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Adult↗
Nonresponsiveness to hepatitis B vaccine in health care workers. Results of revaccination and genetic typings.
Twenty-eight health care workers who had a poor antibody response when initially vaccinated with hepatitis B vaccine were revaccinated with three additional 20-microgram doses. Eight of the twenty nonresponders, who had levels of antibody to hepatitis B surface antigen (anti-HBs) of less than 8 estimated radioimmunoassay (RIA) units, and all 8 of the hyporesponders, who had anti-HBs levels of 8 or 16 RIA units, attained anti-HBs levels of 36 RIA units or more after revaccination. Tests for HLA-A, B, C, and DR; for complement proteins C2, C4A, C4B, and BF; and for the erythrocyte enzyme glyoxalase I were done in 17 nonresponders and 3 hyporesponders. Nine (45%) had HLA-DR7 and 8 (40%) had HLA-DR3, compared with an expected rate of 23% in the general population. At least one of two extended haplotypes (B44, DR7, FC31 or B8, DR3, SCO1) were detected in 6 of the 9 who did not respond to revaccination, compared with 2 of 11 who responded to a second course of vaccine. Poor responders to vaccine may benefit from revaccination, and genetic factors may modulate the immune response to vaccination.
Adult↗
Treatment of viral myocarditis with ribavirin in an animal preparation.
The therapeutic effects of ribavirin, a broad-spectrum, antiviral agent, on experimental myocarditis caused by encephalomyocarditis (EMC) virus were investigated. Four-week-old DBA/2 mice were inoculated with 10 plaque-forming units (pfu) of EMC virus. Ribavirin in a dose of 100 (group 1, n = 20), 200 (group 2, n = 10), or 400 mg/kg/day (group 3, n = 10) was administered subcutaneously on days 0 to 12 after virus inoculation, and animals were observed for 12 days. Control animals were injected with saline (n = 20). Mice treated with ribavirin survived longer than controls (mean survival 6.7 days for group 1, 7.4 days for group 2, 7.7 days for group 3, and 5.2 days for control; p less than .005). Myocardial virus titers on days 6 to 8 were significantly lower in group 2 (3.24 +/- 0.49 log10pfu/mg; p less than .005) and in group 3 (1.70 +/- 0.65 log10 pfu/mg; p less than .001) compared with controls (4.09 +/- 0.57 log10 pfu/mg). The incidence of gross myocardial lesions was significantly lower in group 1 (13/20, 65%), group 2 (2/10, 20%), and group 3 (0/20, 0%) compared with controls (20/20, 100%) (p less than .05). Histologic examination showed extensive myocardial necrosis and cellular infiltration in untreated groups; there was less infiltration in groups 2 and 3 (p less than .01) and less severe necrosis in group 3 (p less than .01). Thus ribavirin effectively inhibited myocardial virus replication and reduced the inflammatory response and myocardial damage in an experimental preparation of viral myocarditis.
Animals↗
Isolated antibody to hepatitis B surface antigen and response to hepatitis B vaccination.
To determine the need for immunization of health workers with antibody to hepatitis B surface antigen (anti-HBs) as their only serologic marker of previous hepatitis B exposure, we studied the level, persistence, and immunologic specificity of isolated anti-HBs in 46 persons identified during screening for hepatitis B vaccine. We rescreened these persons 1 year later, administered a single dose of hepatitis B vaccine, and determined the anti-HBs level at 1, 2, and 8 weeks after vaccination. Isolated anti-HBs levels were low and antibody did not persist; 22 subjects tested had lost detectable anti-HBs within 19 months even though immunologic specificity was shown in vitro in 34. Anamnestic responses suggesting previous exposure and immunity were seen in only 10 subjects; 5 of these subjects had moderate-level, persistent anti-HBs. Although some persons with naturally acquired, isolated anti-HBs may be protected from hepatitis B, the immunologic specificity and protective value of anti-HBs, especially when levels are low, remain questionable.
Antibody Specificity↗
Resistance of herpes simplex virus to 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine: physical mapping of drug synergism within the viral DNA polymerase locus.
A herpes simplex virus type 2 (HSV-2) mutant TS6 (strain HG52) induces a heat-labile viral DNA polymerase at the nonpermissive temperature and is markedly resistant to 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-guanine [2'-nor-2'-deoxyguanosine; 2'NDG]. This antiviral drug requires HSV thymidine kinase for phosphorylation to an active inhibitor (2'NDG-triphosphate), and thymidine kinase-deficient mutants of HSV exhibit varying degrees of resistance to 2'NDG, with the HSV type 1 (HSV-1) B2006 mutant (Kit) being markedly resistant. The ts6 mutation and the 2'ndgR-1 mutation within the viral DNA polymerase locus have been physically mapped by marker rescue and generation of HSV-1/HSV-2 intertypic recombinants. The physical map limits for the ts6 mutation and 2'ndgR-1 mutation are closely linked within a 2.2-kilobase-pair region of DNA sequences and are physically separate from the paaR-1 and acvR-1 mutations. Resistance to 2'NDG by HSV-2 ts6 can be overcome in the presence of combinations of 2'NDG and phosphonoacetic acid, indicating drug synergism within the viral DNA polymerase locus. These physical mapping studies expand the limits of DNA sequences defining an active center in the viral polymerase to 3.5 kilobase pairs, indicating that regions spanning the entire polymerase polypeptide may contribute to a specialized surface able to interact with nucleotides of different structure.
Acyclovir↗
Virus replication and induction of interferon in human epidermal keratinocytes following infection with herpes simplex virus.
Keratinocyte cultures derived from surgical skin specimens of healthy newborns and adults were infected with herpes simplex virus (HSV) type 1 or 2. Typical HSV cytopathic effects involved all cell layers in stratified colonies, and paralleled the production of infectious virus. Virus growth curves and production of virus were comparable in newborn and adult keratinocytes. Interferon (IF) production by keratinocytes paralleled the yield of virus over at least 72 h, and was greater in cultures of adult cells than cultures from newborns. UV irradiation of HSV resulted in progressive virus inactivation and a parallel reduction in induced IF. This suggests that IF production was related to virus replication, and that irradiated (noninfectious) HSV DNA did not contribute significantly to the generation of IF in this system. These results establish that human epidermal keratinocytes can serve as a model system for quantitative assessment of herpes simplex virus infection.
Adult↗