Autoimmunity Links Vinculin to the Pathophysiology of Chronic Functional Bowel Changes Following Campylobacter jejuni Infection in a Rat Model.

BACKGROUND: Acute gastroenteritis can precipitate irritable bowel syndrome (IBS) in humans. Cytolethal distending toxin is common to all pathogens causing gastroenteritis. Its active subunit, CdtB, is associated with post-infectious bowel changes in a rat model of Campylobacter jejuni infection, including small intestinal bacterial overgrowth (SIBO). AIM: To evaluate the role of host antibodies to CdtB in contributing to post-infectious functional sequelae in this rat model. METHODS: Ileal tissues from non-IBS human subjects, C. jejuni-infected and control rats were immunostained with antibodies to CdtB, c-Kit, S-100, PGP 9.5 and vinculin. Cytosolic and membrane proteins from mouse enteric neuronal cell lysates were immunoprecipitated with anti-CdtB and analyzed by mass spectrometry. ELISAs were performed on rat cardiac serum using CdtB or vinculin as antigens. RESULTS: Anti-CdtB antibodies bound to a cytosolic protein in interstitial cells of Cajal (ICC) and myenteric ganglia in C. jejuni-infected and naïve rats and human subjects. Mass spectrometry identified vinculin, confirmed by co-localization and ELISAs. Anti-CdtB antibodies were higher in C. jejuni-infected rats (1.27 ± 0.15) than controls (1.76 ± 0.12) (P < 0.05), and rats that developed SIBO (2.01 ± 0.18) vs. rats that did not (1.44 ± 0.11) (P = 0.019). Vinculin expression levels were reduced in C. jejuni-infected rats (0.058 ± 0.053) versus controls (0.087 ± 0.023) (P = 0.0001), with greater reductions in rats with two C. jejuni infections (P = 0.0001) and rats that developed SIBO (P = 0.001). CONCLUSIONS: Host anti-CdtB antibodies cross-react with vinculin in ICC and myenteric ganglia, required for normal gut motility. Circulating antibody levels and loss of vinculin expression correlate with number of C. jejuni exposures and SIBO, suggesting that effects on vinculin are important in the effects of C. jejuni infection on the host gut.

Staphylococcal leukotoxins trigger free intracellular Ca(2+) rise in neurones, signalling through acidic stores and activation of store-operated channels.

Headache, muscle aches and chest pain of mild to medium intensity are among the most common clinical symptoms in moderate Staphylococcus aureus infections, with severe infections usually associated with worsening pain symptoms. These nociceptive responses of the body raise the question of how bacterial infection impinges on the nervous system. Does S. aureus, or its released virulence factors, act directly on neurones? To address this issue, we evaluated the potential effects on neurones of certain bi-component leukotoxins, which are virulent factors released by the bacterium. The activity of four different leukotoxins was verified by measuring the release of glutamate from rat cerebellar granular neurones. The bi-component γ-haemolysin HlgC/HlgB was the most potent leukotoxin, initiating transient rises in intracellular Ca(2+) concentration in cerebellar neurones and in primary sensory neurones from dorsal root ganglia, as probed with the Fura-2 Ca(2+) indicator dye. Using pharmacological antagonists of receptors and Ca(2+) channels, the variations in intracellular Ca(2+) concentration were found independent of the activation of voltage-operated Ca(2+) channels or glutamate receptors. Drugs targeting Sarco-Endoplasmic Reticulum Ca(2+)-ATPase (SERCA) or H(+)-ATPase and antagonists of the store-operated Ca(2+) entry complex blunted, or significantly reduced, the leukotoxin-induced elevation in intracellular Ca(2+). Moreover, activation of the ADP-ribosyl cyclase CD38 was also required to initiate the release of Ca(2+) from acidic stores. These findings suggest that, prior to forming a pore at the plasma membrane, leukotoxin HlgC/HlgB triggers a multistep process which initiates the release of Ca(2+) from lysosomes, modifies the steady-state level of reticular Ca(2+) stores and finally activates the Store-Operated Calcium Entry complex.

Redefining the genus Spraguea based on ultrastructural and phylogenetic data from Spraguea gastrophysus n. sp. (phylum Microsporidia), a parasite found in Lophius gastrophysus (Teleostei) from Brazil.

The ultrastructure of the fish-infecting microsporidium Spraguea gastrophysus found in the dorsal ganglia and kidney of the anglerfish, Lophius gastrophysus (family Lophiidae) collected on the Brazilian Atlantic coast is described. Each whitish xenoma (up to 3.1 × 1.8 mm) contains several groups of parasites. The host cells are hypertrophied and contain various parasite life stages including mature spores and several developmental stages with unpaired nuclei. Monomorphic spores are ellipsoidal, lightly curved and measure about 3.35 × 1.71 µm. The spore contains a gradually tapering isofilar polar filament with five to six coils arranged in a single row. The nucleus occupies a central zone of the sporoplasm where also several polyribosomes are presented. The posterior vacuole contains a voluminous spherical and granular posterosome measuring up to ~0.65 µm in diameter. The partial small subunit, intergenic spacer and partial large subunit rRNA gene were sequenced and the phylogenetic analysis places the microsporidian described here in the clade that includes all sequences of the Spraguea genus. The ultrastructural morphology of the xenoma and the spores of this microsporidian parasite, as well as the molecular and phylogenetic analysis, suggest the description of a new species. A redefining of the genus Spraguea is also done.

Ultrastructure and molecular characterization of a microsporidium, Tubulinosema hippodamiae, from the convergent lady beetle, Hippodamia convergens Guérin-Méneville.

Hippodamia convergens, the convergent lady beetle, is available for aphid control in home gardens and in commercial food production systems throughout the United States and Canada. Beetles received from commercial insectaries for biological control are occasionally infected with a microsporidium. The objective of this study was to describe the pathogen by means of ultrastructure, molecular characterization and tissue pathology. All stages of the microsporidium were in direct contact with the host cell cytoplasm. Early developmental stages were proximal to mature spores and both were observed throughout the tissue sections that were examined. Merogony resulted from binary fission. Early-stage sporoblasts were surrounded by a highly convoluted plasma membrane and contained an electron-dense cytoplasm and diplokaryon. Ovoid to elongated late-stage sporoblasts were surrounded by a relatively complete spore wall. The polar filament, polaroplast, and anchoring disk were readily observed within the cell cytoplasm. Mature spores were typical of terrestrial microsporidia, with a thickened endospore surrounded by a thin exospore. Spores contained well-defined internal structures, including a diplokaryon, lamellar polaroplast and a slightly anisofilar polar filament with 10-14 coils arranged in a single or double row. A prominent indentation was evident at the apical end of the spore wall proximal to the anchoring disk. Aberrant spores were also observed. These had a fully developed endospore and exospore but lacked any discernable internal spore structures, and were, instead, filled with lamellar or vesicular structures. Typical and aberrant spores measured 3.58 ± 0.2 × 2.06 ± 0.2 µm (n=10) and 3.38 ± 0.8 × 2.13 ± 0.2 µm (n=10), respectively. Spores were observed in longitudinal muscle surrounding the midgut and within the fat body, Malpighian tubules, pyloric valve epithelium, ventral nerve cord ganglia, muscles and ovaries. The hindgut epithelium was often infected but the connective tissues were rarely invaded. The life cycle and pathology of the microsporidium bears some resemblance to Nosema hippodamiae, the only microsporidium reported from H. convergens by Lipa and Steinhaus in 1959. Molecular characterization of the pathogen genomic DNA revealed that it is 99% similar to Tubulinosema acridophagus and T. ratisbonensis, two pathogens that infect Drosophila melanogaster and 98% similar to T. kingi from D. willistoni. Based on similarities in pathogen ultrastructure and the molecular information gained during this study, we propose that the microsporidium in H. convergens be given the name Tubulinosema hippodamiae.

[African histoplasmosis with ganglionic localisation. Apropos of 1 case in an HIV negative patient]. / L'histoplasmose africaine à localisation ganglionnaire. A propos d'une observation chez un sujet VIH négatif.

This study concerns one case of ganglionary African histoplasmosis observed in a pregnant woman. The diagnosis of this histoplasmosis case has been based on histological presentation. This patient has a HIV negative serologic reaction. The histoplasmosis clinical presentation is like tuberculosis, so its diagnosis is difficult. The prevalence of this pathology is unknown in our region but it is increasing since the discovery of AIDS.

Quantity of latency-associated transcript produced by herpes simplex virus is not predictive of the frequency of experimental recurrent genital herpes.

The role of the latency-associated transcript (LAT) in control of recurrent herpes simplex virus type 2 (HSV-2) infection was investigated by examining whether LAT concentration in vitro during productive infection or in ganglia during latency correlated with frequency of recurrent genital herpes. Clinical HSV-2 isolates from frequent or infrequent recurrent genital disease produced comparable amounts of glycoprotein D and infected cell polypeptide 0 RNA, but the isolate from frequent disease produced about seven times more LAT. The guinea pig model of genital herpes was used to determine whether the quantity of LAT produced during acute infection in vitro correlated with recurrence phenotype; the frequency of recurrent disease was similar for the 2 clinical isolates. Likewise, there was no correlation between the recurrence phenotype of individual animals and LAT concentration in their ganglia. Thus, while absence of LAT may impair HSV reactivation and recurrence, once a threshold concentration is exceeded, LAT has no further effect on recurrence frequency.

[Anatomo-clinical aspects of ganglionic tuberculosis in Senegal: comparative study between the 60's and 80-90's apropos of 139 cases]. / Aspects anatomo-cliniques des tuberculoses ganglionnaires en milieu sénégalais: une étude comparative entre les années 60 et 80-90 à propos de 139 cas.

The authors report the results of a retrospective and comparative study of anatomo-clinical aspects of ganglionic tuberculosis carried out over two periods of five years each: first period: 1962-1966; second period: 1988-1992. This study shows a higher prevalence of this disease among children during the first period with an odd ratio of 4.36. In the second period, young adults are the most affected particularly the age group between 30 and 39. The possible role of the HIV virus in this phenomenon has been pointed out. The most common histological forms were the subacute ones with a distinct prevalence during the second period, on the opposite of the acute and chronic forms.

Decreased reporter gene expression during latent infection with HSV LAT promoter constructs.

The latency-associated transcripts (LAT), which code from an 8.5 kb segment of the internal repeat region of the HSV genome, are the only viral transcripts that are present during HSV latent infection. However, little is known about the relative contribution of promoter activity, degradative processes, and elements or regions affecting long term expression of these transcripts in latently infected neurons. To begin to address this question we investigated LAT promoter activity during acute and latent infection. Mouse footpads were infected with KOS/62-3, an engineered herpes simplex virus in which both copies of the LAT promoter are used to drive expression of the Escherichia coli lac Z gene. Four days post-inoculation (p.i.) abundant beta-galactosidase (beta-gal) protein and transcripts were present within ganglionic neurons as assayed by enzyme histochemistry and in situ hybridization. In contrast, by Day 21 (at which time a latent infection had been established) no beta-gal transcripts were present in infected ganglia, even when assayed by the polymerase chain reaction (PCR). These findings indicate a significant drop in LAT promoter activity between Day 4 and Day 21 p.i. To provide confirmatory evidence for this conclusion we infected mice with a second viral construct, KOS/67-7, in which the LAT promoter was used to drive expression of the nerve growth factor (NGF) gene. Four days p.i., abundant NGF antigen and transcripts were present in infected ganglionic neurons, but no evidence of transcription of the cloned NGF gene could be found in latently infected ganglia. Our findings suggest that LAT promoter activity is severely restricted during the latent phase of ganglionic infection.

Immunization with the immediate-early tegument protein (open reading frame 62) of varicella-zoster virus protects guinea pigs against virus challenge.

The IE62 protein, the primary regulatory protein of varicella-zoster virus (VZV) and the major component of the virion tegument, was an effective immunogen in the guinea pig model of VZV infection, whereas the ORF 29 gene product, a nonstructural DNA replication protein, did not elicit protection. All animals immunized with the ORF 29 protein had cell-associated viremia compared with 2 of 11 guinea pigs given the IE62 protein (P = 0.005). VZV was detected in ganglia from 38% of the animals given the ORF 29 protein and 44% of the control animals compared with 9% of the animals immunized with the IE62 protein (P = 0.04). In contrast to the IE62 protein, immunization with the ORF 29 protein did not prime the animals for an enhanced T-cell response upon challenge with infectious virus. The VZV IE62 protein has potential value as a vaccine component.

The herpes simplex virus type 1 regulatory protein ICP0 enhances virus replication during acute infection and reactivation from latency.

ICP0 is a potent activator of herpes simplex virus type 1 gene expression in transient assays and in productive infection. A role for ICP0 in reactivation from latency in vivo has also been suggested on the basis of the observation that viruses with mutations in both copies of the diploid gene for ICP0 reactivate less efficiently than wild-type virus. Because the ICP0 gene is contained entirely within the coding sequences for the latency-associated transcripts (LATs), ICP0 mutants also contain mutations in LAT coding sequences. This overlap raises the question of whether mutations in ICP0 or the LATs, which have also been implicated in reactivation, are responsible for the reduced reactivation frequencies characteristic of ICP0 mutants. Two approaches were taken to examine more definitively the role of ICP0 in the establishment and reactivation of latency. First, a series of ICP0 nonsense, insertion, and deletion mutant viruses that exhibit graded levels of ICP0-specific transactivating activity were tested for parameters of the establishment and reactivation of latency in a mouse ocular model. Although these mutants are ICP0 LAT double mutants, all nonsense mutants induced the synthesis of near-wild-type levels of the 2-kb LAT, demonstrating that the nonsense linker did not disrupt the synthesis of this LAT species. All mutants replicated less efficiently than the wild-type virus in mouse eyes and ganglia during the acute phase of infection. The replication efficiencies of the mutants at these sites corresponded well with the ICP0 transactivating activities of individual mutant peptides in transient expression assays. All mutants exhibited reduced reactivation frequencies relative to those of wild-type virus, and reactivation frequencies, like replication efficiencies in eyes and ganglia, correlated well with the level of ICP0 transactivating activity exhibited by individual mutant peptides. The amount of DNA of the different mutants varied in latently infected ganglia, as demonstrated by polymerase chain reaction analysis. No correlation was evident between reactivation frequencies and the levels of viral DNA in latently infected ganglia. Thus, replication and reactivation efficiencies of ICP0 mutant viruses correlated well with the transactivating efficiency of the corresponding mutant peptides. In a second approach to examining the role of ICP0 in latency, a single copy of the wild-type gene for ICP0 was inserted into the genome of an ICP0- LAT- double mutant, 7134, which exhibits a marked impairment in its ability to replicate in the mouse eye and reactivate from latency.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo characterization of site-directed mutations in the promoter of the herpes simplex virus type 1 latency-associated transcripts.

Transient expression assays in PC12 cells showed that the cAMP response element (CRE) and the TATA box of the herpes simplex virus type 1 latency-associated transcripts (LATs) promoter are essential for basal expression. Recombinant viruses were generated containing site-specific mutations in these motifs. The abilities of these recombinants to replicate, express LATs and reactivate from latency were compared with wild-type and marker-rescued viruses in a murine ocular model. The acute replication of these TATA and CRE mutant viruses was at a level equivalent to their respective marker-rescued viruses. The reactivation of virus was unaffected by mutation in the TATA box as compared with wild-type or marker-rescued viruses. In situ hybridization of TATA box mutant virus-infected ganglia, however, showed threefold fewer LAT-positive neurons than wild-type virus-infected ganglia, with consistently weaker hybridization signals. Thus, this TATA box is required for normal expression of the LATs but not for efficient reactivation. The LATs CRE mutant reactivated with slightly but reproducibly reduced frequency and delayed kinetics relative to marker-rescued virus. By in situ hybridization, however, the percentage and intensity of LATs-positive neurons were found to be comparable for the CRE mutant- and wild-type virus-infected ganglia, suggesting that the CRE is dispensable for abundant LATs expression but that a reactivation function of the LATs may depend upon the presence of the CRE. Finally, using a modified assay for examining the timing of reactivation, we showed that the induction of viral reactivation by addition of exogenous cAMP can occur independently of the LATs.

The role of the immune system in establishment of herpes simplex virus latency--studies using CD4+ T-cell depleted mice.

The immunological mechanisms involved in establishment of herpes simplex virus (HSV) latency were studied in normal and CD4+ T-cell depleted C57BL/6J mice following intravaginal infection. During transition from acute to latent ganglionic infection two consecutive processes were observed: first, clearance of infectious virus from the ganglia, and second, reduction of the number of infected ganglia.

Investigation of the pathogenesis of varicella-zoster virus infection in guinea pigs by using polymerase chain reaction.

The polymerase chain reaction method (PCR) was used to investigate events in the pathogenesis of varicella-zoster virus (VZV) infection in strain 2, Hartley, and euthymic hairless guinea pigs. VZV was detected in peripheral blood mononuclear cells (PBMC) obtained 2-5 days after infection in 8 (50%) of 16 strain 2, 4 (40%) of 10 hairless, and 10 (34%) of 29 Hartley guinea pigs. The frequency of VZV-infected PBMC was estimated to be at least 1/200,000, which is comparable to that observed in human infection. When VZV PCR was used to test ganglia from hairless guinea pigs, samples from 6 of 8 animals were positive. Of 45 VZV-infected guinea pigs that were tested for cellular immunity by VZV T lymphocyte proliferation assay, 44 developed a stimulation index > 2.0. Control animals had no detectable virus by PCR and did not develop cellular immunity to VZV. These experiments showed that viremia was detectable by PCR during primary VZV infection of guinea pigs in about half of the animals regardless of the strain of guinea pig. Acquisition of cellular immunity provided a consistent marker of infection in all guinea pig strains. PCR was also useful for demonstrating VZV in guinea pig ganglia tissue, with VZV gene sequences being detectable for at least 80 days after infection. With the combination of PCR and immunologic assays, various guinea pig strains should be useful for studies of VZV pathogenesis and for the evaluation of antiviral agents and vaccine strategies.

Differential accumulation of herpes simplex virus type 1 latency-associated transcripts in sensory and autonomic ganglia.

We have analyzed the capacity of sensory and autonomic ganglia to demonstrate latency-associated transcripts (LATs) following inoculation of the anterior chamber of the mouse eye with Herpes simplex virus type 1 (HSV-1). In autonomic ganglia, the number of LAT-containing neurons decreased 50-fold or more from the acute to the latent phase, while in the trigeminal ganglion, the decrease was less than 2-fold. The decrease in autonomic ganglia could not be related to destruction of neurons expressing LATs, since these ganglia harbored substantial amounts of viral DNA. The data demonstrate that during the latent phase of the infection, accumulation of LATs varies depending on the type of infected neuron and suggest that some neurons may harbor a latent infection in the absence of LAT expression.

Biology and molecular aspects of herpes simplex and varicella-zoster virus infections.

The herpes simplex and varicella-zoster viruses are members of the subfamily alpha herpesviruses with specific properties of the virion and with the capacity to establish latent infections in humans. The genome of each of these viruses has been determined with an estimate of the number of genes and proteins encoded. The biology and molecular events of the herpes simplex virus productive and latent infection have been detailed with the use of both in vitro and in vivo model systems. The neuron is the site of latency in the ganglia with a limited transcription of genes expressed during the latent period. The specific molecular regulation of latency and reactivation are not well established. There are co-cultivation, electron microscopy, and biochemical studies that support the concept of corneal latency, although this has not been proven conclusively. Details about the varicella-zoster virus biology and molecular events are not as well advanced since animal models have been lacking. The biology of the productive infection (varicella) is different from herpes simplex virus infection since the portal of entry is the respiratory system. Data support the concept of the maintenance of latency within satellite cells in the ganglia rather than within neurons. There are multiple genes expressed during this latency. These features may explain the different clinical presentations and course of reactivation (zoster) compared with herpes simplex virus reactivation.

Prevalence and distribution of latent simian varicella virus DNA in monkey ganglia.

We used polymerase chain reaction to analyze the prevalence and distribution of latent simian varicella virus (SVV) in ganglionic and nonganglionic tissues from nine African green monkeys experimentally infected with SVV. Primers specific for three different regions of the SVV genome were used for amplification. SVV DNA sequences were detected in trigeminal ganglia from seven of nine monkeys and in thoracic ganglia from seven of nine monkeys. Analysis of DNA from nonneuronal tissues of three monkeys and from adrenal glands of nine monkeys revealed the presence of SVV-specific sequences in the adrenal gland of one monkey. The results indicate that, like human varicella, SVV becomes latent primarily in ganglia at multiple levels of the neuraxis, and more than one region of the SVV genome is present in latently infected ganglia. SVV latency in primates may be a useful model for varicella latency in humans.

Localization of herpes simplex virus and varicella zoster virus DNA in human ganglia.

Human dorsal root ganglia from 14 randomly autopsied adults and 1 infant (all seropositive for both herpes simplex virus [HSV] and varicella zoster virus [VZV]) were examined for latent HSV-1 and VZV DNA by polymerase chain reaction. Thoracic ganglionic DNA from all subjects and trigeminal ganglionic DNA from 11 adults were analyzed. HSV-1 DNA was detected in trigeminal ganglia from 8 of 11 (73%) adults and in thoracic ganglia from 2 of 14 (14%) adults. VZV DNA was detected in trigeminal ganglia from 10 of 11 (91%) adults and in thoracic ganglia from 12 of 14 (86%) adults. None of the DNA samples were positive with primers specific for HSV-2. These findings indicate the presence of latent HSV-1 and VZV DNA in trigeminal ganglia and latent VZV DNA in thoracic ganglia of most seropositive adults. Furthermore, although HSV-1 latency most commonly develops in trigeminal ganglia, we also show for the first time the presence of HSV-1 latency in thoracic ganglia. Finally, both viruses can become latent in the same trigeminal ganglion.

Rapid in vivo reactivation of herpes simplex virus in latently infected murine ganglionic neurons after transient hyperthermia.

A rapid and physiologically relevant hyperthermia-based induction procedure has been utilized to develop an in vivo model of induced herpes simplex virus (HSV) reactivation in outbred Swiss Webster mice. This procedure was found to efficiently reactivate latent virus from both trigeminal and lumbosacral ganglia. Examination of the time between hyperthermia and virus production demonstrated that detectable levels of infectious virus were present in ganglia as soon as 14 h posttreatment, with peak percent recoveries at 24 h. These data indicated that the switch from latent to active viral gene transcription occurred rapidly following treatment. Immunohistochemical staining for HSV type 1 antigens revealed rare antigen-positive ganglionic neurons 24 h postinduction. HSV antigens were not detected in any other cell type, and lateral spread of the infection was not observed. This is the first report of the detection of HSV antigens in vivo following induced reactivation in the intact nervous system and demonstrates that the neuron is the site of infectious virus production. In addition, our data strongly suggest that at least some neurons in which HSV antigens are detected during reactivation do not survive. Because the temporal and spatial characteristics of HSV reactivation have been clearly defined, this model is uniquely suited for the molecular dissection of the reactivation process.

Herpes simplex virus type 1 latency-associated transcription unit promotes anatomical site-dependent establishment and reactivation from latency.

Defined herpes simplex virus type 1 (HSV-1) mutants KOS/1 and KOS/62 (positive and negative, respectively, for latency-associated transcripts [LATs]) express the Escherichia coli beta-galactosidase (beta-Gal) gene during latency. These mutants were employed to assess the functions of the latency-associated transcription unit on establishment and maintenance of and reactivation from the latent state. It was found that in the trigeminal ganglia, the frequencies of hyperthermia-induced reactivation of KOS/62 and an additional LATs- mutant (KOS/29) were reduced by at least 80%. Quantification of latently infected neurons expressing the beta-Gal gene revealed that the LATs- mutant KOS/62 established approximately 80% fewer latent infections in the trigeminal ganglia than did KOS/1 (LATs+). This reduction in establishment which is evident in the trigeminal ganglia could account for the reduced frequency of reactivation from this site. In striking contrast, both LATs- mutants reactivated with wild-type frequencies from lumbosacral ganglia. Quantification of beta-Gal-positive neurons at this site revealed that KOS/62 established as many as or more latent infections than the LATs+ virus, KOS/1. Colocalization of HSV antigen and beta-Gal suggested that the decreased establishment by LATs- mutants in trigeminal ganglia was the result of inefficient viral shutoff. Thus, one function of the HSV-1 LATs transcription unit is to promote the establishment of latency in trigeminal but not lumbosacral ganglia. Such a function may be relevant to understanding the distinct clinical recurrent disease patterns of HSV-1 and HSV-2.

Herpes simplex types 1 and 2: latency in the genital tract of guinea pigs.

Guinea pigs were infected with herpes simplex virus (HSV) intravaginally and then sacrificed during latent infection. Virus was recovered from the ganglia, spinal cord and genital tissues by co-cultivation after 1-6 weeks in culture. The virus could not be recovered from the genital tract during the first week of co-cultivation, nor from homogenized genital tissue. Cultivation of genital tissues with acyclovir did not reduce the recovery of HSV. Thus, HSV appeared to establish a truly latent infection in the genital tract and not a persistent infection as previously described.