Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication.

Nitric oxide (NO) has antimicrobial activity against a wide spectrum of infectious pathogens, but an antiviral effect has not been reported. The impact of NO, from endogenous and exogenous sources, on herpes simplex virus type 1 (HSV 1) replication was studied in vitro. HSV 1 replication in RAW 264.7 macrophages was reduced 1,806-fold in monolayers induced to make NO by activation with gamma IFN and LPS. A competitive and a noncompetitive inhibitor of nitric oxide synthetase substantially reduced the antiviral effect of activated RAW macrophages. S-nitroso-L-acetyl penicillamine (SNAP) is a donor of NO and was added to the media of infected monolayers to assess the antiviral properties of NO in the absence of gamma IFN and LPS. A single dose of S-nitroso-L-acetyl penicillamine 3 h after infection inhibited HSV 1 replication in Vero, HEp2, and RAW 264.7 cells in a dose-dependent manner. Neither virucidal nor cytocidal effects of NO were observed under conditions that inhibited HSV 1 replication. Nitric oxide had inhibitory effects, comparable to that of gamma IFN/LPS, on protein and DNA synthesis as well as on cell replication. This report demonstrates that, among its diverse properties, NO has an antiviral effect.

Structural and kinetic analyses of herpes simplex virus type 1 latency-associated transcripts in human trigeminal ganglia and in cell culture.

Only one herpes simplex virus type 1 (HSV-1) gene is expressed in sensory neurons of latently infected animals and humans, yielding two RNAs, called latency-associated transcripts (LATs). The LATs appear to modulate virus reactivation. In mice and rabbits the 5' origins, kinetics of synthesis, and splicing pattern of the LATs are well established. Because these details of LAT structure and expression have not been defined in humans, we sought to do so. Using primer extension and Northern hybridization analyses, we demonstrate that in human trigeminal ganglia, the smaller (1.35 kb) HSV-1 transcript differs from the larger (1.85 kb) LAT by excision of an intron near its 5' end; they are otherwise colinear, and 5' coterminal. In infected cells only the 1.85 kb LAT is detected. Its expression is inhibited by cycloheximide or acyclovir, indicating this LAT is synthesized late in the viral replicative cycle. All of these features of the LATs in humans are consistent with those reported in rabbits and mice and further validate the animal models of human HSV-1 infection.

Herpes simplex virus type 1 transcription is not detectable in quiescent human stromal keratitis by in situ hybridization.

PURPOSE: The purpose of this study was to determine whether herpes simplex virus (HSV) transcripts are present in the corneas of patients with chronic herpetic stromal keratitis. METHODS: Corneal buttons from patients with a history of stromal keratitis, but no ongoing active disease, together with positive and negative control tissues, were analyzed by in situ hybridization using single-stranded RNA probes for all three classes of viral lytic cycle transcripts as well as for the latency-associated transcripts (LATs). Tissues also were screened for presence of HSV genomic DNA using the polymerase chain reaction (PCR). RESULTS: HSV DNA was detected in 7 of 13 quiescent corneas by PCR, but no viral transcripts were detected in any of these corneas by in situ hybridization. CONCLUSIONS: At the level of detection afforded by in situ hybridization, HSV persistent in scarred human corneas after stromal keratitis appears to be transcriptionally dormant. This contrasts with the situation in neurons of latently infected sensory ganglia, in which LATs are present at high levels.

Detection of respiratory syncytial virus (RSV) infected cells by in situ hybridization in the lungs of cotton rats immunized with formalin-inactivated virus or purified RSV F and G glycoprotein subunit vaccine and challenged with RSV.

The replication of RSV in unimmunized cotton rats was evaluated by quantitating the amount of infectious virus in the lung and the number of RSV infected cells in a histopathological section of lung by in situ hybridization. RSV infected cells were detected only in alveoli and bronchioles and constituted only a small minority of the cell population. The temporal patterns of rise to the peak number of infected cells (day 4) and the peak titer of infectious virus (day 3) were similar. The clearance of both infected cells and infectious virus was nearly complete by day 7. In animals previously immunized with purified RSV glycoproteins or formalin-inactivated RSV there also was a good correlation between the number of infected cells detected by in situ hybridization and the amount of infectious virus recovered. It was previously demonstrated that cotton rats immunized with formalin-inactivated vaccine developed enhanced pulmonary histopathology following challenge with RSV. In such animals, there was approximately a 90% reduction in the number of infected cells compared to control unimmunized, RSV-challenged animals. Formalin-inactivated RSV vaccine-enhanced lung histopathology developed despite the effective elimination of virus and virus-infected cells suggesting that the enhanced pathology is the result of an exaggeration of normal immune mechanisms involved in clearance of virus infection, an aberrant immune response during infection, or both.

Latency of the human herpesviruses.

The herpesviruses are ubiquitous pathogens that establish latent infections and can reactivate to produce recurrent disease. The fact that clinical recurrences are infrequent in most healthy individuals indicates that potent regulatory mechanisms must exist to ensure viral latency. Rapid progress is being made toward understanding the pathogenesis of latency and reactivation and is the subject of this review.

Patterns of gene expression and sites of latency in human nerve ganglia are different for varicella-zoster and herpes simplex viruses.

The cellular localization and viral transcription patterns of acute and latent varicella-zoster virus (VZV) infections of human sensory nerve ganglia were studied by in situ hybridization and compared with those of latent herpes simplex virus (HSV) infection. Trigeminal and dorsal root ganglia obtained at autopsy were hybridized with 35S-labeled single-stranded RNA probes homologous to VZV or HSV fragments. We have reported that HSV persists in human sensory neurons and expresses only one family of transcripts that overlap extensively with, but are opposite in polarity to, the mRNA encoding the immediate early protein termed infected cell protein 0 (ICP0). In the present study we find that latent VZV infection involves nonneuronal cells, and multiple, but not all, VZV genes are transcribed. In contrast, during varicella both neuronal and nonneuronal cells are infected, with all regions of the VZV genome analyzed being expressed. Thus, the patterns of gene expression and cellular locations of VZV and HSV infections of human ganglia differ. The differences may underlie clinical features that distinguish these infections.

Detection and preliminary characterization of herpes simplex virus type 1 transcripts in latently infected human trigeminal ganglia.

RNA extracted from human trigeminal ganglia was examined for the presence of herpes simplex virus type 1 (HSV-1) transcripts by Northern hybridization (RNA blot) analysis. By using cloned DNA and single-stranded RNA probes, two abundant colinear HSV-1 transcripts (1.85 and 1.35 kilobases) were detected in ganglia from 9 of 17 individuals. These RNAs overlap the 3' end of the transcript for immediate-early gene ICP0 but are transcribed from the opposite strand; thus, they are antisense relative to the ICP0 mRNA. We also report evidence by in situ hybridization that these latently infected ganglia contain HSV-1 RNA homologous to the BamHI SP region of the genome which is transcribed in the same direction as the other latency transcripts. In Vero cells productively infected with laboratory strain HSV-1 KOS and in cultures infected with different low-passage clinical isolates, only the 1.85-kilobase transcript was detected, and there was variation in the size of this larger latency transcript. These novel transcripts may play a role in maintaining HSV-1 latency in human ganglia.

Characterization of herpes simplex virus type 2 latency-associated transcription in human sacral ganglia and in cell culture.

The ability of herpes simplex virus type 2 (HSV-2) to establish latency in and reactivate from sacral dorsal root sensory ganglia is the basis for recurrent genital herpes. The expression of HSV-2 genes in latently infected human sacral ganglia was investigated by in situ hybridization. Hybridizations with a probe from the long repeat region of HSV-2 revealed strong nuclear signals overlying neurons in sacral ganglia from five of nine individuals. The RNA detected overlaps with the transcript for infected cell protein O but in the opposite, or "anti-sense," orientation. These observations mimic those made previously with HSV-1 in human trigeminal ganglia and confirm the recent findings during latency in HSV-2-infected mice and guinea pigs. Northern hybridization of RNA from infected Vero cells showed that an HSV-2 latency-associated transcript was similar in size to the larger (1.85 kb) latency transcript of HSV-1. Thus, HSV-1 and HSV-2 latency in human sensory ganglia are similar, if not identical, in terms of their cellular localization and pattern of transcription.

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.

D-arabitol metabolism in Candida albicans: studies of the biosynthetic pathway and the gene that encodes NAD-dependent D-arabitol dehydrogenase.

Candida albicans produces large amounts of the pentitol D-arabitol in culture and in infected mammalian hosts, but the functional and pathogenic significance of D-arabitol in C. albicans is not known. In this study, we sought to elucidate the pathway by which C. albicans synthesizes D-arabitol and to identify and characterize key enzymes in this pathway. C. albicans B311 produced D-[14C-1]arabitol from [14C-2]glucose; this finding implies on structural grounds that D-ribulose-5-PO4 from the pentose pathway is the major metabolic precursor of D-arabitol. NAD- or NADP-dependent pentitol dehydrogenases catalyze the final steps in D-arabitol biosynthesis in other fungi; therefore, lysates of C. albicans B311 were tested for enzymes of this class and were found to contain a previously unknown NAD-dependent D-arabitol dehydrogenase (ArDH). The ArDH structural gene was cloned by constructing a new D-arabitol utilization pathway in Escherichia coli. The C. albicans ArDH gene expressed in E. coli and Saccharomyces cerevisiae an enzyme that catalyzes the reaction D-arabitol + NAD <-->D-ribulose + NADH; this gene was present as a single copy per haploid genome, and its deduced peptide sequence was homologous with sequences of several members of the short-chain dehydrogenase family of enzymes. These results suggest that (i) C. albicans synthesizes D-arabitol by dephosphorylating and reducing the pentose pathway intermediate D-ribulose-5-PO4 and (ii) ArDH catalyzes the final step in this pathway.

Varicella-zoster virus transcription in human trigeminal ganglia.

Our laboratory previously reported in situ hybridization analyses showing varicella-zoster virus (VZV)-specific RNA transcripts in latently infected human trigeminal ganglia. These transcripts were found exclusively in non-neuronal cells and emanated from at least three separate regions of the VZV genome, those encompassing restriction fragments BamHI-EY, EcoRI-B, and BamHI-J. Extending these in situ studies, we now report that VZV RNAs colinear with open reading frames (ORFs) 29 and 62 are demonstrable in non-neuronal cells; however, neither VZV RNA colinear with ORF 28 nor antisense RNAs of ORFs 29 and 62 are detected. Northern hybridization analyses of poly(A)+ RNA from human trigeminal ganglion pools also reveal VZV-specific transcripts corresponding to ORFs 29 and 62, but not those of ORFs 28 or 61. The ORF 29- and 62-specific transcripts are similar to those expressed in productive infection, with regard to size and polyadenylation. The cumulative findings suggest that VZV latency is characterized by selected expression of multiple genes, which appear to include some but not all putative immediate-early and early genes.

Latent herpes simplex virus in human trigeminal ganglia. Detection of an immediate early gene "anti-sense" transcript by in situ hybridization.

We used in situ hybridization to study the expression of herpes simplex virus type 1 genes during latent infections of human sensory ganglia. Trigeminal ganglia were recovered at autopsy from 24 subjects with no evidence of an active herpetic infection. These ganglia were hybridized to 35S-labeled single-stranded RNA probes spanning 72 percent of the herpes simplex genome. In the ganglia of 16 subjects, 0.2 to 4.3 percent of the neuronal cells contained abundant nuclear signals for viral RNA. Ganglia from three patients with low or undetectable levels of antibodies to herpes simplex type 1 lacked viral RNA signals, whereas the ganglia from all of six patients with elevated antibody titers showed viral RNA signals. Transcription was detected only from the region of the viral genome containing a gene that encodes an immediate early protein known as the infected-cell protein number zero (ICP0). However, normal ("sense") transcripts of this gene, which are prominent in an acute infection, were not detected. In contrast, a novel transcript was found overlapping with, but opposite in direction to, the ICP0 transcript (and was therefore "anti-sense"). Although this transcript has been only partially characterized, we believe that it may have a role in maintaining the latency of herpes simplex virus.

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.

Acyclovir suppression of frequently recurring genital herpes. Efficacy and diminishing need during successive years of treatment.

Forty-seven patients with frequently recurring genital herpes participated in one or more of five sequential trials of oral suppressive therapy with 200 mg of acyclovir three times daily from four to 12 months' duration. The prolonged use of acyclovir was extremely well tolerated, and treatment efficacy was sustained through successive studies. Recurrences in eight patients with repeated treatment "failures" were more effectively suppressed with higher doses of acyclovir. All patients experienced recurrent infections after the treatments were completed; however, the mean time to recurrence following each treatment period became progressively longer, and resumption of suppressive therapy was no longer warranted for ten patients. These data indicate the efficacy and safety of chronic suppressive therapy with acyclovir and the value of interrupting prolonged treatment to assess its further need.

A novel latency-active promoter is contained within the herpes simplex virus type 1 UL flanking repeats.

Herpes simplex virus type 1 (HSV-1) expresses a unique series of RNA molecules, the latency-associated transcripts or LATs, during latent infection of neuronal tissues. Previous studies by others have described a TATA box-containing latency-active promoter, referred to here as LAP1, located approximately 700 bp upstream of the 5' end of the major 2.0-kb LAT. In this report, transient gene expression assays were employed to identify a second, novel latency-active promoter (LAP2) present within a region downstream of LAP1 and 5' proximal to the major 2.0-kb LAT. In contrast to LAP1, this promoter lacks a TATA box but possesses cis-acting regulatory elements and other features frequently observed within eukaryotic housekeeping gene promoters. Unlike most other HSV promoters, LAP2 was down-regulated by the viral transcriptional activators ICP4 and ICP0. The majority of LAP2-positive regulatory elements were located within sequences from -257 to -58 relative to the 5' end of the 2.0-kb LAT, and the basal promoter mapped within sequences from -14 to +28. RNase protection experiments demonstrated that chimeric LAT-chloramphenicol acetyltransferase transcripts produced in the transient assays initiated at or near the 5' end of the major 2-kb LAT. Tn5 insertional mutagenesis of the ICP4 regulatory gene determined that down-regulation of LAP2 required the ICP4 transactivating domain and targeted the minimal promoter region as the site of action by ICP4. Replicating recombinant viruses containing a LAP2-lacZ reporter gene cassette in an ectopic site (glycoprotein C locus) were shown to be active in mouse trigeminal ganglia. Taken together, these experiments suggest that the LAT region of the HSV-1 genome contains at least two latency-active promoters which may play different roles in expressing the various LATs. Alternatively, these promoters may comprise a larger promoter-regulatory complex which may influence transcription during latency.