Controlled trial of acyclovir for chickenpox evaluating time of initiation and duration of therapy and viral resistance.

BACKGROUND: Chickenpox is prevalent in the US despite the availability of an effective vaccine. Acyclovir treatment is limited by concerns about efficacy if given after the first day of rash and by concerns about induction of viral resistance. OBJECTIVE: Evaluate initiation and duration of acyclovir treatment of chickenpox and its effect on viral resistance. STUDY DESIGN: Randomized, placebo-controlled, double blind trial in immunocompetent patients who were stratified by age at enrollment (children, 2 to 11 years; adolescents, > or = 12 to 18 years; adults, > or = 19 years) and duration of rash (< or = 24 h vs. >24 to 48 h). Lesions were staged, counted and cultured; temperatures and symptoms were recorded daily. INTERVENTION: Subjects presenting within 24 h of rash onset (Group A) were randomly assigned to 5 or 7 days of oral acyclovir treatment, 80 mg/kg/day up to a maximum of 3,200 mg/day in four divided doses. Subjects whose rash was >24 to 48 h old were randomized to receive 5 days of acyclovir treatment beginning on the first (Group B1) or second study day (Group B2). Matching placebos were used to ensure that subjects uniformly received 28 doses of study compound. RESULTS: Of the 177 subjects recruited Group A patients who were treated on the first day of rash had the greatest number of significantly shortened event times with 5 days of therapy being equivalent to 7 days. There also were some shorter times to events for Group B1 patients who began therapy on the second day of rash vs. Group B2 patients who started acyclovir on the third. These included: time to maximum lesion formation (adolescents, P = 0.007; children, P = 0.03); 50% healing in adolescents (P = 0.005); and residual facial lesions in adults (P = 0.047). The probability of viral shedding was significantly reduced for Group A subjects vs. Group B1 subjects (P = 0.006). Viruses shed during therapy remained susceptible to acyclovir and retained normal thymidine kinase function. CONCLUSIONS: Immunocompetent children, adolescents and adults with chickenpox displayed a gradation in their clinical responses to acyclovir that correlated with the time from onset of rash to initiation of therapy. Five days of therapy is sufficient because a 7-day course provided no additional benefit. The susceptibility to acyclovir of viruses shed during treatment did not change; however, the effect of therapy on resistance of latent virus was not assessed.

Characterization of an acyclovir-resistant herpes simplex virus type 2 strain isolated from a premature neonate.

Acyclovir resistance is not a recognized problem among neonates with perinatal herpes simplex virus (HSV) infection. A premature newborn with neurocutaneous HSV infection was treated for 21 days with acyclovir. Disseminated disease recurred 8 days later. A recurrent isolate was resistant to acyclovir and lacked thymidine kinase activity on the basis of a frameshift mutation in the thymidine kinase (tk) gene. Compared with the sensitive isolate obtained during primary infection, replication of the resistant isolate was reduced on primary and permanent cells and even further impaired on cells deleted for cellular tk. The resistant isolate lacked virulence in a murine model of genital infection. Acyclovir-resistant HSV-2 mutants can develop rapidly in neonatal infection and cause clinically significant disease, despite decreased replication in vitro and attenuated virulence in an animal model.

Acyclovir is phosphorylated by the human cytomegalovirus UL97 protein.

Acyclovir (ACV) has shown efficacy in the prophylactic suppression of human cytomegalovirus (HCMV) reactivation in immunocompromised renal transplant patients without the toxicity associated with ganciclovir (GCV). The HCMV UL97 gene product, a protein kinase, is responsible for the phosphorylation of GCV in HCMV-infected cells. This report provides evidence for the phosphorylation of ACV by UL97. Anabolism studies with the HCMV wild-type strain AD169 and with recombinant mutants derived from marker transfer experiments performed by using mutant UL97 DNA from both clinical isolates and a laboratory-derived strain resistant to GCV showed that mutations in the UL97 gene cripple the ability of recombinant virus-infected cells to anabolize both GCV and ACV. These mutant UL97 recombinant viruses were less susceptible to both GCV and ACV than was the wild-type strain. A recombinant herpes simplex virus type 1 strain, in which the thymidine kinase gene is deleted and the UL13 gene is replaced with the HCMV UL97 gene, was able to induce the phosphorylation of ACV in infected cells. Finally, purified UL97 phosphorylated both GCV and ACV to their monophosphates. Our results indicate that UL97 promotes the selective activity of ACV against HCMV.

Induction of lytic Epstein-Barr virus (EBV) infection in EBV-associated malignancies using adenovirus vectors in vitro and in vivo.

The consistent presence of EBV genomes in certain tumor types (in particular, AIDS-related central nervous system lymphomas and nasopharyngeal carcinomas) may allow novel, EBV-based targeting strategies. Tumors contain the latent (transforming) form of EBV infection. However, expression of either of the EBV immediate-early proteins, BZLF1 and BRLF1, is sufficient to induce lytic EBV infection, resulting in death of the host cell. We have constructed replication-deficient adenovirus vectors expressing the BZLF1 or BRLF1 immediate-early genes and examined their utility for killing latently infected lymphoma cells in vitro and in vivo. We show that both the BZLF1 and BRLF1 vectors efficiently induce lytic EBV infection in Jijoye cells (an EBV-positive Burkitt lymphoma cell line). Furthermore, lytic EBV infection converts the antiviral drug, ganciclovir (GCV), into a toxic (phosphorylated) form, which inhibits cellular as well as viral DNA polymerase. When Jijoye cells are infected with the BZLF1 or BRLF1 adenovirus vectors in the presence of GCV, viral reactivation is induced, but virus replication is inhibited (thus preventing the release of infectious EBV particles); yet cells are still efficiently killed. Finally, we demonstrate that the BZLF1 and BRLF1 adenovirus vectors induce lytic EBV infection when they are directly inoculated into Jijoye cell tumors grown in severe combined immunodeficiency mice. These results suggest that induction of lytic EBV infection in tumors, in combination with GCV, may be an effective strategy for treating EBV-associated malignancies.

The Cys607-->Tyr change in the UL97 phosphotransferase confers ganciclovir resistance to two human cytomegalovirus strains recovered from two immunocompromised patients.

Two ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) strains recovered from an AIDS patient (strain VR4990) and a heart transplant recipient (strain VR5474) showed a Cys607-->Tyr change in the UL97-encoded phosphotransferase. No amino acid substitutions were observed in the viral DNA polymerase. Marker transfer experiments showed marked reduction in GCV phosphorylation and drug susceptibility of the recombinant HCMV strain VR4990rec2-1-1. These results further extend the region of the carboxy-terminal domain of the UL97 phosphotransferase involved in GCV substrate recognition.

Novel mutation in the UL97 gene of a clinical cytomegalovirus strain conferring resistance to ganciclovir.

Characterization of a ganciclovir-resistant cytomegalovirus strain from a patient with AIDS showed a histidine-to-glutamine change at residue 520 of UL97 (Q520 mutation). In anabolism studies, Q520 was associated with impaired phosphorylation of ganciclovir. Transfer of Q520 to a recombinant virus resulted in a ganciclovir-resistant phenotype.

Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance.

The UL97 phosphotransferase coding sequences of clinical cytomegalovirus (CMV) isolates, 10 resistant and 11 sensitive to ganciclovir, were compared to define mutations associated with drug resistance. In each ganciclovir-resistant isolate, a mutation was found that resulted in an amino acid substitution at codon 460 (4 isolates), codon 594 (2 isolates), or codon 595 (4 isolates). No sensitive isolate carried any of these mutations. Marker transfer studies showed that each mutation was capable of conferring ganciclovir resistance to the laboratory CMV strain AD169. Rapid diagnostic tests based on DNA amplification and restriction enzyme analysis were developed for these mutations. Specific mutant DNAs were detected when they constituted at least 10% of the population in the specimen. Several mutations in UL97 appear to be common markers for ganciclovir resistance, and their detection may be a rapid alternative to conventional cell culture susceptibility testing.

A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate.

Multiple human cytomegalovirus (HCMV) strains frequently coexist in patients with AIDS, and chronic ganciclovir treatment may favor the emergence of ganciclovir-resistant viral mutants. We report the molecular and biochemical characterization of a HCMV ganciclovir-resistant strain (VR3480) previously recovered from a patient with AIDS who was undergoing multiple courses of ganciclovir treatment (G. Gerna, F. Baldanti, M. Zavattoni, A. Sarasini, E. Percivalle, and M. G. Revello, Antiviral Res. 19:333-345, 1992). Ganciclovir resistance of strain VR3480 was related to impaired ability to monophosphorylate the drug, as indicated by the finding that ganciclovir phosphorylation values for VR3480 were 30% of those shown by the HCMV reference strain AD169 in an in vitro activity assay. Sequencing of the UL97 gene of VR3480, which encodes the viral kinase responsible for ganciclovir phosphorylation, showed an in-frame deletion of three nucleotides resulting in the loss of a leucine at position 595 in the polypeptide. Mutant VR3480 UL97 DNA was able to transfer resistance to the AD169 strain in marker rescue experiments. Analysis of virus isolates and blood polymorphonuclear leukocyte samples spanning the 2-year follow-up period of the patient showed that ganciclovir-resistant strain VR3480 arose ex novo during prolonged antiviral treatment and accounted for the majority of virus load circulating in blood during the period of clinical resistance to ganciclovir treatment.

Phenotypic and genotypic characterization of acyclovir-resistant varicella-zoster viruses isolated from persons with AIDS.

Phenotypic and genotypic analyses were done on 17 varicella-zoster virus (VZV) isolates recovered from 10 persons with AIDS (mean CD4 cell count, 16.4/mm3) who had chronic VZV lesions. Eleven acyclovir-resistant isolates were recovered from 10 patients after a mean of 20.1 weeks of therapy. Six susceptible isolates were recovered before acyclovir treatment (n = 1), early during therapy (n = 4; mean time, 4.2 weeks), or after discontinuation of acyclovir (n = 1). Acyclovir-resistant VZV isolates were deficient in thymidine kinase (TK) or induced a TK with altered substrate specificity; all isolates were susceptible to foscarnet. Ten of 11 acyclovir-resistant mutants contained tk gene mutations, including single nucleotide substitutions in highly conserved binding sites (n = 2) as well as nucleotide deletions (n = 4) and insertions (n = 4). These findings suggest that multiple, nonuniform mutations within the tk gene are associated with acyclovir-resistant VZV phenotypes.

Analysis of the thymidine kinase genes from acyclovir-resistant mutants of varicella-zoster virus isolated from patients with AIDS.

Patients with AIDS often experience recurrent infections with varicella-zoster virus (VZV) requiring repeated or prolonged treatment with acyclovir (ACV), which may lead to the development of ACV resistance. The ACV resistance of isolates recovered from such patients is associated with diminished VZV thymidine kinase (TK) function. We determined the nucleotide sequences of the TK genes of 12 ACV-resistant VZV strains purified from nine patients with AIDS. Five VZV strains contained nucleotide deletions in their TK genes, introducing a premature termination codon which is expected to result in the production of a truncated protein. No detectable full-length TK protein could be immunoprecipitated from extracts of cells infected with these virus strains. These TK-deficient strains were cross resistant to the TK-dependent antiviral agents ACV, 9-(4-hydroxy-3-hydroxymethylbutyl-yl)guanine (penciclovir), and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil (BVaraU). The remaining seven strains each contained a nucleotide change that resulted in an amino acid substitution in the TK protein. These substitutions occurred throughout the TK protein, namely, in the ATP-binding site, the nucleoside-binding site, between the two binding sites, and at the carboxy terminus of the protein. We determined the effects of these mutations on the stability of TK protein expression in virus-infected cells and on the sensitivity of mutants to the TK-dependent antiviral agents ACV, BVaraU, and penciclovir.

A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) is a major pathogen in immunosuppressed individuals, including patients with acquired immune deficiency syndrome. The nucleoside analogue ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)-guanine) is one of the few drugs available to treat HCMV infections, but resistant virus is a growing problem in the clinic and there is a critical need for new drugs. The study of ganciclovir-resistant mutants has indicated that the selective action of ganciclovir depends largely on virus-controlled phosphorylation in HCMV-infected cells. The enzyme(s) responsible have not been identified. Here we report that the HCMV gene UL97, whose predicted product shares regions of homology with protein kinases, guanylyl cyclase and bacterial phosphotransferases, controls phosphorylation of ganciclovir in HCMV-infected cells. A four-amino-acid deletion of UL97 in a conserved region, which in cyclic AMP-dependent protein kinase participates in substrate recognition, causes impaired ganciclovir phosphorylation. The implications of these results for antiviral drug development and drug resistance are discussed.

Gorlin's syndrome with associated odontogenic cysts.

Multiple maxillary and mandibular cysts are principle features of basal cell nevus syndrome (Gorlin-Goltz). We present cases from an affected family in which magnetic resonance imaging (MRI) was helpful in evaluation of the cystic lesions. A middle ear anomaly was identified which may represent an additional abnormality associated with the syndrome.