Persistent Early Knee Osteoarthritis Symptoms in the First Two Years After ACL Reconstruction.

CONTEXT: Early identification of knee osteoarthritis (OA) symptoms after anterior cruciate ligament reconstruction (ACLR) could enable timely interventions to improve long-term outcomes. However, little is known about the change in early OA symptoms from 6 to 12 months following ACLR. OBJECTIVE: To evaluate the change over time in meeting classification criteria for early knee OA symptoms from 6 to 12 months following ACLR. DESIGN: Prospective Cohort Study. SETTING: Research laboratory. PATIENTS OR OTHER PARTICIPANTS: 82 participants aged 13-35 years who underwent unilateral primary ACLR. On average, participants' 1st and 2nd visits were 6.2 and 12.1 months post-ACLR. MAIN OUTCOME MEASURES: Early OA symptoms were classified using generic (Luyten Original) and patient population-specific (Luyten PASS) thresholds on Knee Injury and Osteoarthritis Outcome Score (KOOS) subscales. Changes in meeting early OA criteria were compared between an initial and follow-up visit at an average of 6 and 12 months post-ACLR, respectively. RESULTS: Twenty-two percent of participants exhibited persistent early OA symptoms across both visits using both the Luyten Original and PASS criteria. From initial to follow-up visit, 18-27% had resolution of early OA symptoms while 4-9% developed incident symptoms. In total, 48-51% had no early OA symptoms at either visit. There were no differences between change in early OA status between adults and adolescents. CONCLUSIONS: Nearly one quarter of participants exhibited persistent early knee OA symptoms based on KOOS thresholds from 6 to 12 months post-ACLR. Determining if this symptom persistence predicts worse long-term outcomes could inform the need for timely interventions after ACLR. Future research should examine if resolving persistent symptoms in this critical window improves later outcomes. Tracking early OA symptoms over time may identify high-risk patients who could benefit from early treatment.

Lack of effect of treatment with penciclovir or acyclovir on the establishment of latent HSV-1 in primary sensory neurons in culture.

Recent studies suggest reductions in establishment of herpes simplex virus, type 1 (HSV-1) latency using the nucleoside analog penciclovir compared with acyclovir in the murine model. These observations raise the possibility that the new analogs may have novel activities that directly interfere with the establishment of the latent infection, suggesting a mechanism other than simply blocking the productive infection. To determine if penciclovir has a direct action on the establishment of latency, we compared the effects of penciclovir versus acyclovir in an in vitro model of HSV-1 latency in rat dorsal root ganglia neurons in culture. In neurons in culture, both penciclovir and acyclovir were highly effective in blocking the productive infection. However, neither penciclovir nor acyclovir blocked establishment of latency as demonstrated by similar percentages of neurons expressing the latency-associated transcript (LAT). Following removal of the respective nucleoside analog, latency was maintained until reactivation was induced by nerve growth factor deprivation. Similar virus titers were recovered after induction of reactivation of latent infections, which were established in the presence of either penciclovir or acyclovir. These results indicate that neither penciclovir nor acyclovir treatment directly prevents the establishment of latent HSV-1 infections in primary sensory neurons in culture.

Inducible cyclic AMP early repressor produces reactivation of latent herpes simplex virus type 1 in neurons in vitro.

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of the peripheral nervous system. During latent HSV-1 infection, viral gene expression is limited to latency-associated transcripts (LAT). HSV-1 remains latent until an unknown mechanism induces reactivation. The ability of the latent virus to periodically reactivate and be shed is essential to the transmission of disease. In vivo, the stimuli that induce reactivation of latent HSV-1 include stress, fever, and UV damage to the skin at the site of initial infection. In vitro, in primary neurons harboring latent HSV-1, nerve growth factor (NGF) deprivation or forskolin treatment induces reactivation. However, the mechanism involved in the induction of reactivation remains poorly understood. An in vitro neuronal model of HSV-1 latency was used to investigate potential mechanisms involved in the induction of reactivation of latent HSV-1. In situ hybridization analysis of neuronal cultures harboring latent HSV-1 showed a marked, rapid decrease in the percentage of LAT-positive neurons following induction of reactivation by NGF deprivation or forskolin treatment. Western blot analysis showed a corresponding increase in expression of the cellular transcription factor inducible cyclic AMP early repressor (ICER) during reactivation. In transient-transfection assays, ICER downregulated LAT promoter activity. Expression of ICER from a recombinant adenoviral vector induced reactivation and decreased the percentage of LAT-positive neurons in neuronal cultures harboring latent HSV-1. These results indicate that ICER represses LAT expression and induces reactivation of latent HSV-1.

Characterization of promoter function and cell-type-specific expression from viral vectors in the nervous system.

Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.

Pseudorabies virus expressing enhanced green fluorescent protein: A tool for in vitro electrophysiological analysis of transsynaptically labeled neurons in identified central nervous system circuits.

Physiological properties of central nervous system neurons infected with a pseudorabies virus were examined in vitro by using whole-cell patch-clamp techniques. A strain of pseudorabies virus (PRV 152) isogenic with the Bartha strain of PRV was constructed to express an enhanced green fluorescent protein (EGFP) from the human cytomegalovirus immediate early promoter. Unilateral PRV 152 injections into the vitreous body of the hamster eye transsynaptically infected a restricted set of retinorecipient neurons including neurons in the hypothalamic suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) of the thalamus. Retinorecipient SCN neurons were identified in tissue slices prepared for in vitro electrophysiological analysis by their expression of EGFP. At longer postinjection times, retinal ganglion cells in the contralateral eye also expressed EGFP, becoming infected after transsynaptic uptake and retrograde transport from infected retinorecipient neurons. Retinal ganglion cells that expressed EGFP were easily identified in retinal whole mounts viewed under epifluorescence. Whole-cell patch-clamp recordings revealed that the physiological properties of PRV 152-infected SCN neurons were within the range of properties observed in noninfected SCN neurons. Physiological properties of retinal ganglion cells also appeared normal. The results suggest that PRV 152 is a powerful tool for the transsynaptic labeling of neurons in defined central nervous system circuits that allows neurons to be identified in vitro by their expression of EGFP, analyzed electrophysiologically, and described in morphological detail.

Myelin and collapsin-1 induce motor neuron growth cone collapse through different pathways: inhibition of collapse by opposing mutants of rac1.

Precise growth cone guidance is the consequence of a continuous reorganization of actin filament structures within filopodia and lamellipodia in response to inhibitory and promoting cues. The small GTPases rac1, cdc42, and rhoA are critical for regulating distinct actin structures in non-neuronal cells and presumably in growth cones. Collapse, a retraction of filopodia and lamellipodia, is a typical growth cone behavior on contact with inhibitory cues and is associated with depolymerization and redistribution of actin filaments. We examined whether small GTPases mediate the inhibitory properties of CNS myelin or collapsin-1, a soluble semaphorin, in chick embryonic motor neuron cultures. As demonstrated for collapsin-1, CNS myelin-evoked growth cone collapse was accompanied by a reduction of rhodamine-phalloidin staining most prominent in the growth cone periphery, suggesting actin filament disassembly. Specific mutants of small GTPases were capable of desensitizing growth cones to CNS myelin or collapsin-1. Adenoviral-mediated expression of constitutively active rac1 or rhoA abolished CNS myelin-induced collapse and allowed remarkable neurite extension on a CNS myelin substrate. In contrast, expression of dominant negative rac1 or cdc42 negated collapsin-1-induced growth cone collapse and promoted neurite outgrowth on a collapsin-1 substrate. These findings suggest that small GTPases can modulate the signaling pathways of inhibitory stimuli and, consequently, allow the manipulation of growth cone behavior. However, the fact that opposite mutants of rac1 were effective against different inhibitory stimuli speaks against a universal signaling pathway underlying growth cone collapse.

Developmental expression of C1C-2 in the rat nervous system.

Regulation of expression of the voltage-gated chloride channel, C1C-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of C1C-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated C1C-2 expression in deep brain nuclei and scattered cells within the neuroepithelial layers, but not in the regions of subventricular zone that primarily give rise to glial populations. By E18 all neurons within the emerging cortical plate and its equivalent in other areas of the CNS were heavily labeled. During the first postnatal week, C1C-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of C1C-2 mRNA in many neuronal populations. In adult rat brain, C1C-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammon's Horn of hippocampus, or mitral cells of the olfactory bulb and Purkinje cells within the cerebellum. Many smaller neurons within the diencephalon maintained significant levels of expression. A functional conductance was readily detected in hippocampal neurons during the first postnatal week, which had the same characteristic properties as the conductance observed in adult neurons. The observed expression and functional presence of C1C-2 suggest a widespread role in neuronal chloride homeostasis in early postnatal life, and demonstrated that cell specific shut-down resulted in the adult pattern of expression.

HSV Latency In Vitro : In Situ Hybridization Methods.

We have developed an in vitro model of herpes simplex virus (HSV) latency in primary neurons that mimics many aspects of HSV latency in animal models and the human disease (1-3). Using this model, we demonstrated that HSV-1 and HSV-2 establish latent infections in vitro in the same neuronal cell types that are shown to harbor latent HSV in humans (3). Latent HSV infections can be produced in neuronal cultures from ganglia of rodents and primates with similar results (3). In all cases examined, the neurotrophin, nerve growth factor (NGF), is required to maintain the latent infections. Depletion of NGF results in the reactivation of latent virus (1-3). Depending upon the conditions and the use of a high multiplicity of infection, latent HSV-1 infections are established in the majority of primary sensory or sympathetic neurons in tissue culture (2,4). To achieve high efficiency of establishment of latency with little or no evidence of lytic infection, an antiviral agent (e.g., acyclovir) is added to the neuronal cultures during the first week after inoculation with virus. However, latency can be established in the absence of antiviral treatment provided that the multiplicity of infection (MOI) is very low (1,2). At least one of the actions of the antiviral treatment is to prevent amplification of the input virus in the nonneuronal cells that are present in the culture at the outset of the infection. These nonneuronal cells are destroyed in the presence of acyclovir and virus (4). Latency is maintained in neurons in culture for as long as 10 wk in the presence of NGF. Viral transcripts and antigens associated with the productive infection are not detected during the latent infection (2,3,5). Viral transcription is restricted to the latency-associated transcripts (LAT) during the latent infection and is present in the nuclei of 80-90% of the neurons by 3 wk postinfection (4,5) Upon removal of NGF from the culture medium, for as brief as 1 h, reactivation of latent virus is induced (3), and viral antigens associated with the productive infection and infectious virus are detected between 48-72 h after NGF deprivation.

The herpes simplex virus type 1 immediate-early protein ICP0 is necessary for the efficient establishment of latent infection.

The immediate-early protein ICP0 of herpes simplex virus type 1 (HSV-1) is not essential for viral replication. However, ICP0 is important for efficient viral replication during the productive infection and for reactivation of latent HSV-1 in vivo. The in vitro model of HSV-1 latency in dorsal root ganglia neurons was used to examine the role of ICP0 in the individual steps that could lead to the appearance of a decreased reactivation phenotype of ICP0 mutant viruses. After establishment of latent infections in the neuronal cultures, induction of reactivation by nerve growth factor (NGF) deprivation resulted in the production of infectious virus with delayed kinetics and a burst size that was significantly decreased for the ICP0 mutants compared with wild-type HSV-1. The efficiency of establishment of latency with the ICP0 mutants was similarly decreased at least 10-fold, as measured by three criteria: (i) the percentage of neurons expressing the major latency-associated transcript during the latent infection, (ii) the amount of viral DNA detected in the neuronal cultures, and (iii) the percentage of neurons expressing ICP4 immunoreactivity after the induction of reactivation. The most striking finding was that ICP0 supplied by an adenovirus vector significantly restored the ability of an ICP0 mutant to establish latency and reactivation. These results strongly indicate a critical role for ICP0 in the establishment of the latent HSV-1 infection in the in vitro neuronal model.

Green fluorescent protein expressed in living mosquitoes--without the requirement of transformation.

Mosquitoes transmit viruses, protozoa and nematodes that are major causes of morbidity and mortality in humans. Details of arthropod anatomy and development, and the replication and development of pathogens in the arthropod vector, have relied upon examination of dissected or histologically processed material. We constructed a double-subgenomic Sindbis (dsSIN) virus expressing green fluorescent protein to demonstrate the potential of this protein for studying pathogen development in living arthropods. We were able to observe dissemination of virus, and furthermore, it was possible to observe components of the nervous system of mosquito larvae in extraordinary detail and record this on video tape. Although green fluorescent protein has been used as a reporter gene in a number of organisms, expression has relied upon transformation of cells or embryos. Transformation technology has limited applicability, thus we have described an alternative system that, due to the broad host range and viral tropisms of dsSIN viruses, may be useful to scientists in a range of disciplines. Green fluorescent protein may also provide a non-lethal selection method for use in transgenic arthropod research.

Efficiency of transduction by recombinant Sindbis replicon virus varies among cell lines, including mosquito cells and rat sensory neurons.

Recombinant alphaviruses have been used as vehicles for delivery and expression of heterologous genes in mammalian, avian and insect cell lines. We have used a Sindbis replicon virus (Sinreplac) able to express the E. coli lacZ gene to compare the efficiency of transduction in one insect, six mammalian cell lines and cultured rat dorsal neurons which apparently express beta-galactosidase over a 30-day time period. Results show that different cell lines were transduced with varying degrees of efficiency and that this efficiency could be improved in some cell lines by packaging the replicon with a helper derived from a more neurovirulent strain of Sindbis.

Long-term expression in sensory neurons in tissue culture from herpes simplex virus type 1 (HSV-1) promoters in an HSV-1-derived vector.

Amplicons, defective herpes simplex virus type 1 (HSV-1) vectors, were constructed to use four HSV-1 promoters, from the immediate-early (IE) 1 IE 3, IE 4/5, and late glycoprotein C (gC) genes, to regulate expression of the Escherichia coli lacZ gene, encoding beta-galactosidase, and packaged into infectious particles. Infection of sensory neurons in vitro with amplicons containing the IE 1, IE 3, or IE 4/5 promoter resulted in stable long-term expression of beta-galactosidase from 2 to 10 weeks after gene transfer. The number of neurons expressing beta-galactosidase was not changed by treatments previously shown to produce reactivation of latent HSV-1. In addition, the latency-associated transcript was detected in many of the same neurons that expressed beta-galactosidase, indicating that the viral IE promoters in the amplicons can function in the same neurons that harbor latent virus. Delivery of beta-galactosidase protein directly into neurons by microinjection indicated that the half-life for histochemical detection of beta-galactosidase was between 24 and 48 h, suggesting that the persistence of beta-galactosidase histochemical staining cannot be explained by the stability of the reporter protein alone. In contrast to the IE promoters, the gC promoter of the late gene class did not support long-term expression of beta-galactosidase; instead, beta-galactosidase was detected in only a few neurons per culture at 2 weeks after infection, and superinfection with wild-type HSV-1 did not increase the level of expression from the gC promoter. These results suggest that the HSV-1 IE promoters in the amplicons are not subject to the promoter inactivation that occurs with many types of virus vectors and that the IE promoters in the context of the amplicon avoid the promoter inactivation observed from the same promoters in the HSV-1 genome during latency.

Differential expression of an inwardly rectifying chloride conductance in rat brain neurons: a potential mechanism for cell-specific modulation of postsynaptic inhibition.

A voltage-sensitive inwardly rectifying chloride (Cl-) conductance (GCl(V) is present in hippocampal pyramidal but not dentate gyrus neurons and has a significant role in modulation of neuronal inhibition by GABA. GCl(V) has the same activation properties as the cloned and expressed Cl- channel CIC-2. In brain, CIC-2 was detected selectively in neurons, and in hippocampus was detected in the same populations of neurons that demonstrate GCl(V). CIC-2 mRNA expression varied widely in different neuronal populations in brain but was greatest in pyramidal and other large neurons and least in interneurons. The observed differential expression of CIC-2 provides a potential molecular basis for the paradoxical excitation produced by GABAA receptor activation in selected neuronal populations.

Role of extracellular and intracellular calcium on proctolin-induced contractions in an insect visceral muscle.

The pentapeptide proctolin requires extracellular calcium to produce an increase in frequency and amplitude of myogenic contractions and an increase in basal tonus of the oviducts of the locust, Locusta migratoria. Decreasing saline calcium concentrations reduced myogenic contractions, the delay to reach maximum basal tonus and the maximum basal tonus achieved. Proctolin (5.10(-9) M) resulted in an immediate influx of extracellular calcium into locust oviduct muscle during the first 15 s of stimulation. This was followed by an increase in calcium efflux which was maintained over the time of proctolin stimulation. A Na+/Ca2+ exchanger is suggested to occur in the oviducts and may play a role in Ca2+ extrusion following proctolin stimulation.

Regulation of the herpes simplex virus latency-associated transcripts during establishment of latency in sensory neurons in vitro.

The temporal appearance of the major latency-associated transcript (LAT) of herpes simplex virus, type-1 (HSV-1) was examined in sensory neuronal cultures during the establishment of either a latent or a lytic infection. Under conditions that result in the establishment of a latent infection, a significant delay in LAT accumulation was observed. The delay in the appearance of LAT was reflected in both a reduced number of LAT-positive neurons detected by in situ hybridizations and by low levels of the major 2-kb LAT detected by Northern blot analysis at early times compared to later in the latent infection. The percentage of LAT-positive neurons shown by in situ hybridizations and the relative abundance of the major LAT by Northern blot analysis increased markedly by 14 days after inoculation with virus. In addition to the major LAT, a spliced 1.5-kb LAT species was detected in Northern blot analysis after establishment of latency in the neuronal cultures, similar to observations in vivo. In contrast to the latent infection, under conditions that produced lytic infections in the neuronal cultures, LAT and HSV-1-specific antigens were detected in the majority of neurons 24 hr after inoculation with virus. These results indicate that LAT expression during the establishment of latency is regulated differently than during the lytic infection: LAT expression appears to be inhibited initially during the establishment of latency, whereas LAT is readily expressed during the lytic infection.

Latency in vitro of varicella-zoster virus in cells derived from human fetal dorsal root ganglia.

A potential in vitro model of varicella-zoster virus (VZV) latency was developed. Dissociated human dorsal root ganglion cultures were infected with VZV and maintained for 1 wk in the presence of bromovinyl arabinosyl uracil, a potent inhibitor of VZV. Seven to 21 d after removing the inhibitor (> or = 14 d after infection), the cells were trypsinized, passed to monolayers of human embryonic lung fibroblasts, and observed for VZV reactivation as indicated by typical cytopathic effects and the appearance of VZV antigens. VZV reactivated from 56% of the cultures containing both neurons and satellite cells but not from cultures specifically enriched for either neurons, satellite cells, or ganglion-derived fibroblasts. The failure to isolate VZV from cell suspensions that were sonicated before cocultivation with fibroblasts indicated that infectious VZV was not present before reactivation. Moreover, immunohistochemical and immunoprecipitation studies revealed no VZV-specific antigens in any cultures before the reactivation stimulus. VZV antigens were detected after trypsinization and cocultivation. These findings suggest that cultures containing both neurons and satellite cells provide a model system for VZV persistence that possesses many properties of a latent infection.

Activation of second-messenger pathways reactivates latent herpes simplex virus in neuronal cultures.

Herpes simplex virus type 1 (HSV-1) establishes latent infections in neurons of sympathetic and sensory ganglia in humans, and reactivation of latent virus results in recurrent disease. Previously, we reported establishment of latent HSV-1 infections in neuronal cultures derived from rats, monkeys, and humans; reactivation occurs following nerve growth factor (NGF) deprivation. The processes controlling HSV latency are not understood. Using the in vitro neuronal latency system, we have shown that latent HSV-1 reactivated in response to stimulation of at least two second-messenger pathways. Stimulation of cAMP-dependent pathways by several mechanisms or activation of protein kinase C by phorbol myristate acetate (PMA) resulted in reactivation of latent HSV-1. The reactivation kinetics following treatment with activators of protein kinase A and C were accelerated compared with those following NGF deprivation. 2-Aminopurine, which inhibits NGF-stimulated protein kinases and other classes of protein kinases, but does not effect protein kinase A or C, blocked reactivation produced by NGF deprivation or treatment with a cAMP analog, but not reactivation by PMA treatment. These results demonstrate that latent HSV-1 reactivates in neurons in vitro in response to activation of second-messenger pathways.

Replication, latent infection, and reactivation in neuronal culture with a herpes simplex virus thymidine kinase-negative mutant.

Herpes simplex virus type 1 (HSV-1) mutant viruses lacking functional viral thymidine kinase activity are reported to be incapable of replication in neurons. To investigate the role of viral thymidine kinase (TK) activity in the HSV-1 infection of the neuron, we studied a thymidine kinase-negative (TK-) mutant virus engineered to eliminate TK function without affecting the other known transcripts encoded in this region of the genome. Studies using the mouse eye model demonstrated that the mutant behaved as is reported for other TK- viruses: DNA of the mutant virus was detected in the ganglia during the latent infection by polymerase chain reaction, but virus did not reactivate after explantation of the ganglia. Utilizing the neuronal cultures, we investigated the ability of the mutant virus to replicate in neurons and the capacity of the mutant virus to establish latency and reactivate. With a low multiplicity of infection (m.o.i.), replication of the TK- mutant virus in sensory neurons in culture was significantly delayed compared to that of the wild-type virus. However, when a high m.o.i. was used, the mutant and the wild-type viruses replicated with similar kinetics. The TK- mutant virus was capable of establishment of latency and reactivation from the latent infection in sensory neurons in culture. These data suggest that HSV-1 thymidine kinase activity facilitates viral replication, but that TK activity is not essential for either replication or reactivation from latent infections in neurons in vitro.

Development of a lyophilized formulation of interleukin-2.

Native human interleukin-2 (IL-2) comprises a group of glycoproteins of MW 13,000-17,500. Recombinant human IL-2 (rhIL-2) (Cetus) is derived from E. coli and is not glycosylated. We have evaluated several processes for manufacturing rhIL-2, based on different chaotropic agents for solubilization of insoluble protein pastes. Formulation work carried out with material purified by one of these processes is reported here. Our studies have indicated that the presence of a stabilizer in the form of an amorphous excipient, such as amino acids, a non-ionic surfactant (polysorbate 80), hydroxypropyl-beta-cyclodextrin or human serum albumin was essential for preservation of rhIL-2 during lyophilization. Each of these formulations exhibited its own unique problems. We have overcome these problems through a systematic formulation development program and have been successful in developing several lyophilized formulations of rhIL-2 with optimum properties and performance.

Detection of the latency-associated transcript in neuronal cultures during the latent infection with herpes simplex virus type 1.

The transcriptional studies reported in this paper indicate that the latency-associated transcript (LAT) is present in neuronal cultures during the latent infection with herpes simplex virus type 1 (HSV-1). During the latent infection glycoprotein D (gD) mRNA, a mRNA characteristic of the productive infection, is not detected. However, following reactivation by nerve growth factor (NGF) deprivation, gD mRNA is detected in the neuronal cultures. Thus, the restricted viral gene expression in the in vitro neuronal model indicates that the latent infection in culture is analogous to that observed in vivo.