Jeffrey M. Ostrove

NIH conference. Varicella-zoster virus infections. Biology, natural history, treatment, and prevention.

During the last 10 years, there have been major advances in the understanding of varicella-zoster virus and the diseases it causes. The molecular biology of the virus is being unraveled with the aid of new molecular technologies. Varicella, usually a benign manifestation of primary infection, and zoster, a result of reactivation of latent virus, can cause considerable morbidity in patients with immune impairment. Antiviral drugs, especially acyclovir, ameliorate severe infections but still have little role in the treatment of most normal patients with varicella or zoster. Varicella can be prevented when necessary by patient isolation and passive prophylaxis with varicella-zoster immune globulin. An experimental live vaccine also prevents varicella, but problems regarding its virulence for immunosuppressed patients and the durability of the protective response are still being addressed.Abstract During the last 10 years, there have been major advances in the understanding of varicella-zoster virus and the diseases it causes. The molecular biology of the virus is being unraveled wi...

Acquisition of Genital Herpes from an Asymptomatic Sexual Partner

THE incidence of genital herpes simplex infection is increasing in the United States.1 Although there have been substantial advances in the diagnosis and treatment of this infection, several import...

Molecular Analysis of the Pyrimidine Deoxyribonucleoside Kinase Gene of Wild-type and Acyclovir-resistant Strains of Varicella-Zoster Virus

The pyrimidine deoxyribonucleoside kinase (dPK) genes from five wild-type and four acyclovir-resistant varicella-zoster virus (VZV) strains were studied. One of the acyclovir-resistant strains was isolated from a patient receiving chronic acyclovir therapy. Acyclovir-resistant strains expressed the 1.8 kb VZV dPK transcript but lacked dPK activity. To determine the basis for the lack of enzyme activity the dPK gene from each strain was cloned and its DNA sequence determined. The VZV dPK gene was found to be highly conserved among strains, with greater than 99% nucleotide and amino acid homology. Each acyclovir-resistant VZV strain differed from its wild-type parent in only a single amino acid. The dPK genes from the acyclovir-resistant strains contained either point mutations near the putative thymidine-binding site of the enzyme or ones that resulted in the premature termination of protein synthesis. Single point mutations were sufficient to render these strains dPK-negative and highly resistant to acyclovir. The molecular basis for acyclovir resistance at the dPK locus of VZV is similar to that previously noted to render herpes simplex viruses resistant to acyclovir.

Effect of oral acyclovir treatment on symptomatic and asymptomatic virus shedding in recurrent genital herpes

Twenty-six men and women with recurrent genital herpes maintained diaries of their symptoms and signs of infection and submitted 6,515 self-collected cultures during a one-year study of acyclovir therapy. As compared with periods before or after treatment, the mean rates of experiencing symptoms or lesions, and of shedding virus were significantly lower during treatment. Acyclovir treatment reduced the rate of symptomatic shedding from 95 positive cultures to six per 1,000 cultures, but the rate of asymptomatic shedding remained relatively constant, averaging eight per 1,000 cultures. Among the isolates of herpes simplex virus studied, there was no differences in sensitivity to acyclovir between strains recovered on or off therapy or during symptomatic or asymptomatic recurrences. The endonuclease cleavage profiles of asymptomatically shed viruses were essentially the same as those of the symptomatically shed viruses from the same individual. Chronic acyclovir therapy significantly reduced the symptoms and signs of recurrent genital herpes but did not eliminate virus shedding, nor, therefore, the possibility of disease transmission.

Molecular biology of varicella zoster virus.

Publisher Summary This chapter focuses on varicella zoster virus (VZV), a member of the subfamily Alphaherpesvirinae. The virus causes varicella, or chickenpox, and, on reactivation from a latent state, zoster, or shingles. The construction of restriction endonuclease maps for VZV DNA has been useful for the elucidation of the genome structure, for molecular epidemiological studies, and for providing the foundation for the understanding of the molecular biology of VZV through the cloning and sequencing of the entire VZV genome. Studies aimed at characterizing VZV-encoded proteins began shortly after VZV was determined to be morphologically similar to HSV. The study of VZV biology and molecular biology still lags well behind that of other members of the herpesvirus family. A major advance has been in the publishing of the complete DNA sequence for both VZV and HSV. This should allow us to better understand the expression of individual VZV genes and to place them in the context of viral replication.

Mapping of the varicella zoster virus deoxypyrimidine kinase gene and preliminary identification of its transcript

The varicella-zoster virus (VZV) deoxypyrimidine kinase (dPK) gene was mapped by transfection of cloned viral DNA fragments into thymidine kinase-deficient mouse L (LTK-) cells and subsequent biochemical transformation of these cells to the LTK+ phenotype. Such transforming activity was limited to the BamHI-H and EcoRI-D fragments of the VZV genome, which overlap by 2.2 kb between map units 0.50 and 0.52. Biochemically transformed cells were shown to contain a high copy number of viral DNA sequences that had integrated into the cellular DNA. Extracts of these cells showed a higher level of dPK activity than did extracts of parental LTK- cells. With the use of Northern hybridization analysis of transformed and VZV-infected cell RNAs, it was possible to tentatively assign a 1.8-kb transcript to the VZV dPK.

DETECTION AND CHARACTERIZATION OF LATENT HSV RNA BY IN SITU AND NORTHERN BLOT HYBRIDIZATION IN GUINEA PIGS

Following intravaginal infection of guinea pigs, herpes simplex virus establishes a latent infection in the sensory lumbosacral ganglia. Using the techniques of in situ and Northern blot hybridization, we have characterized this latent HSV-2 virus and compared it to latent HSV-1 at the same anatomical site. For HSV-2, a single 1.8-kb latency-associated transcript (LAT) was detected. In contrast, as described for latent HSV-1 in the trigeminal ganglia of rabbits and mice, two HSV-1 LAT species were detected in the lumbosacral ganglia, an abundant transcript of 1.8 kb and a less abundant transcript of 1.55 kb. Despite these differences in LAT expression, the clinical course of the acute and recurrent genital disease was similar for both viruses. LAT was detected in 0.3-6.0% of the sensory neurons of sacral but not in lumbar ganglia. The abundance of LAT correlated with the severity of the initial infection, but not with the frequency of recurrent disease. Thus, vaccination strategies that substantially reduced or eliminated symptomatic disease following challenge infection appeared to block the establishment of a latent infection.

Directionality and further mapping of varicella zoster virus transcripts

Our laboratory previously identified and preliminarily mapped 58 viral RNA transcripts in varicella zoster virus (VZV) infected cells (Ostrove et al., 1985). This study was initiated to more precisely map these transcripts, to identify additional transcripts, and to determine transcript directionality. To accomplish this, 32 overlapping BamHI, EcoRI, and SmaI fragments representing 99.7% of the genome were cloned into pGEM-2, a plasmid which contains a multiple cloning site flanked by SP6 and T7 RNA polymerase promoters. Each of these clones was used to produce 32P-labeled double-stranded DNA probes to detect transcripts homologous to either strand of the VZV insert, and single-stranded [32P]RNA probes in order to detect RNAs of either polarity. These probes were hybridized to Northern blots of VZV-infected cell RNA. In all, 77 RNAs were detected with both DNA and RNA probes. The direction of transcription and localization of 57 of the 58 previously identified RNAs and of 20 newly recognised abundant transcripts were determined. Thirty-three additional low-abundance transcripts were detected only by the relatively more sensitive RNA probes. A map indicating the directionality and approximate locations of the abundant VZV transcripts was constructed.

VARICELLA-ZOSTER VIRUS INFECTION OF HUMAN ASTROCYTES, SCHWANN CELLS, AND NEURONS

Human fetal cell cultures enriched for astrocytes, Schwann cells, or dorsal root ganglia neurons were infected with cell-free varicella-zoster virus (VZV), and the course of these infections was compared with that in fetal fibroblasts. Virus replication was detected in each neural cell type as early as 10-16 hr postinfection. Permissiveness of each cell type was confirmed by electron microscopy. However, the kinetics of virus spread varied between the neural cell types. Moreover, the accumulation, progression, and localization of VZV putative immediate early (IE), early (E), and late proteins was neural cell-type specific. VZV replication was slower in Schwann cells and neurons than in astrocytes. In Schwann cells and neurons VZV E proteins could be detected before IE proteins, a reversal of the order of accumulation noted with astrocytes and fibroblasts. There was also relatively more VZV IE protein in the perinuclear cytoplasm of Schwann cells and neurons, suggesting a delay in the transport of this antigen to its nuclear site. The permissiveness of non-neuronal cell types suggests that they could play a role in the pathogenesis of herpes zoster.

MOLECULAR ANALYSIS OF ACYCLOVIR-RESISTANT VARICELLAZOSTER VIRUS ISOLATES

Many serious varicella-zoster virus (VZV) infections are treated with acyclovir (ACV). While there has not been clinical evidence of VZV resistance to ACV, its possibility warrants our understanding of its molecular basis. The majority of laboratory derived resistant VZV strains are deficient in thymidine kinase (TK) which.phosphorylates ACV. We studied the TK locus of 3 wild-type (TK+) and 3 ACV-resistant (TK-) strains. The 2.6 Kb Pst I-P VZV DMA fragment encoding TK was cloned from each strain and sequenced by the dideoxy chain termination method. The TK open reading frame is highly conserved with > 99% nucleotide homology among all strains. All TK+ strains are identical in predicted amino acid sequence in and around the putative enzyme binding sites for ATP and thymidine. However, one TK- mutant has a single base change near the thymidine binding site. The two other mutants contain premature stop codons which presumably result in a truncated, nonfunctional polypeptide. We propose that one mechanism of ACV resistance in VZV involves single base substitutions that lead to alterations in the substrate binding sites or overall secondary structure of the viral TK.