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Natalizumab-associated progressive multifocal leukoencephalopathy in multiple sclerosis (MS) occurred in two individuals also treated with interferon β1a, raising concerns about the interaction of these disease-modifying agents and leading to the recommendation to avoid their concomitant administration. However, type I interferons are antiviral. Using a real-time quantitative polymerase chain reaction for the detection and quantification of the John Cunningham virus (JCV), DNA in peripheral blood mononuclear cells (PBMCs), and urine in MS patients, we tested the hypothesis that MS disease-modifying drugs (DMD) qualitatively and quantitatively alter JCV prevalence and viral copy numbers. Two hundred thirty-nine patients were enrolled in a cross-sectional study in which blood and urine specimens were collected at a single time and 37 newly diagnosed, treatment-naïve MS patients were enrolled in a longitudinal study in which specimens were obtained at diagnosis and 6 months after treatment initiation. JCV DNA was detected in PBMCs of only two patients (0.07 %), but was commonly detected in the urine (46.8 %) in this population. There was no effect of DMDs on blood or urinary JCV prevalence or viral copy numbers with either glatiramer acetate (Copaxone®) or interferon-β therapy (Avonex®, Betaseron®, or Rebif®). The small number of patients on other therapies precluded meaningful comment about their effects. No obvious effect of the platform DMDs on JCV prevalence was observed even for the interferon-βs.

Strain-specific factors contribute in significant but undefined ways to the variable incidence of herpes simplex virus (HSV) recrudescence. Studies that investigate these strain-specific factors are needed. Here, we used qPCR, in vitro assays, and genomic sequencing to identify important relationships between in vitro and clinical phenotypes of unique HSV-1 clinical isolates. Nine HSV-1 isolates from individuals displaying varying reactivation patterns were studied. Isolates associated with frequent recurrent herpes labialis (RHL) (1) displayed higher rates of viral shedding in the oral cavity than those associated with rare RHL and (2) tended to replicate more efficiently at 33 °C than 39 °C. HSV-1 isolates also displayed a more stable phenotype during propagation in U2OS cells than in Vero cells. Draft genome sequences of four isolates and one variant spanning 95.6 to 97.2 % of the genome were achieved, and whole-genome alignment demonstrated that the majority of these isolates clustered with known North American/European isolates. These findings revealed procedures that could help identify unique genotypes and phenotypes associated with HSV-1 isolates, which can be important for determining viral factors critical for regulating HSV-1 reactivation.

Histone acetylation is implicated in the regulation of herpes simplex virus type 1 (HSV-1) latency. However, the role of histone acetylation in HSV-1 reactivation is less clear. In this study, the well-established model system, quiescently infected, neuronally differentiated PC12 (QIF-PC12) cells, was used to address the participation of histone acetylation in HSV-1 reactivation. In this model, sodium butyrate and trichostatin A (TSA), two histone deacetylase inhibitors, stimulated production of infectious HSV-1 progeny from a quiescent state. To identify viral genes responsive to TSA, the authors analyzed representative alpha, beta, and gamma viral genes using quantitative real-time polymerase chain reaction. Only the latency-associated transcript (LAT) accumulated in response to TSA treatment, under culture conditions that restricted virus replication and spread. This led the authors to evaluate the importance of LAT expression on TSA-induced reactivation. In QIF-PC12 cells, the LAT deletion mutant virus dLAT2903 reactivated equivalently with its wild-type parental strain (McKrae) after TSA treatment, as well as forskolin and heat stress treatment. Both viruses also reactivated equivalently from latently infected trigeminal ganglia explants from rabbits. In contrast, there was a marked reduction in the recovery of dLAT2903, as compared to wild-type virus, from the eyes of latently infected rabbits following epinephrine iontophoresis. These combined in vitro, ex vivo, and in vivo data suggest that LAT is not required for reactivation from latently infected neuronal cells per se, but may enhance processes that allow for the arrival of virus at, or close to, the site of original inoculation (i.e., recrudescence).

The herpes simplex virus (HSV)-1 alpha0 promoter contains a putative cAMP response element (CRE) located at positions -68 to -60 with respect to the initiation of transcription. In this report, the authors examined the functionality of this element using (1) luciferase reporter gene assays in nerve growth factor-differentiated (ND)-PC12 cells and (2) virus-induced activation from quiescently infected (QIF)-PC12 cells. The putative alpha0 CRE was completely eliminated by digestion with the restriction enzyme Tsp45I followed by mung bean nuclease treatment. The mutated region was verified by DNA sequencing and was inserted into the alpha0-luciferase reporter plasmid (pRDalpha0-LUC) creating (pRDalpha0deltaCRE-LUC), and into the HSV-1 genome of strain 17(+)(alpha0deltaCRE). Insertion into both copies of the alpha0 promoter was verified by Southern blot analysis. ND-PC12 cells transfected with pRDalpha0-LUC and pRDalpha0deltaCRE-LUC plasmids responded similarly to forskolin (50 microM), with approximately 250% increases in luciferase activity compared to mock-treated cultures as measured 3 days following treatment. When QIF-PC12 cultures established with HSV-1 strain 17(+) and alpha0deltaCRE were treated with forskolin (50 microM) 17 days post infection, virus was detected in 9/24 (37.5%) and 13/24 (54.2%) of induced cultures by day 8 post treatment, respectively. In contrast, virus was detected in 0/23 and 1/24 (4.2%) of mock-treated cultures by day 8 post treatment for wild-type and mutant viruses, respectively. These findings indicate that the alpha0 promoter is forskolin responsive, the purported CRE of the alpha0 promoter does not confer forskolin responsiveness in ND-PC12 cells, and this element is not required for reactivation of HSV-1 from QIF-PC12 cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring peptide found in the central nervous system that plays a role in somatosensory processing and activation of protein kinase A (PKA) and protein kinase C (PKC). Because activation of PKA or PKC results in reactivation of HSV-1 from latently infected embryonic neuronal cells, PACAP was used to evaluate HSV-1 activation from quiescently infected (QIF)-PC12 cells. Our studies demonstrate that physiologically relevant concentrations of PACAP38 and PACAP27 induce HSV-1 activation from QIF-PC12 cell cultures in a dose-dependent fashion. PACAP-induced activation of virus was significantly impaired by the PKA-inhibitor, H-89 (20 microM), whereas treatment with the PKC-inhibitor, GF109203X (1 microM), was without affect. Additionally, direct activation of PKA with cAMP analogs, 8-(4-chlorophenylthio)- and dibutyryl-cAMP, only partially mimicked the effect of PACAP on virus activation. Taken together, PACAP induced HSV-1 activation from QIF-PC12 cells involves the PKA and possibly cAMP-independent pathways. This report is the first to demonstrate that PACAP induces HSV-1 activation from a quiescent state and that this in vitro cell model is useful for studying early inductive events that lead to virus production from quiescence.

We have previously described a novel in vitro model of a non-productive herpes simplex virus type 1 (HSV-1) infection in neurally differentiated (Nd)-PC12 cells that allows for inducible virus replication upon forskolin treatment. In this study, we further characterized the quiescent state of infection and examined the ability of heat stress (HS) to induce virus from this non-productive state. These studies demonstrated that (i) the quiescent state is characterized by the absence of cell-associated virus, capsids, and viral antigens; (ii) HS (43 degrees C, 3 h) efficiently activated virus from quiescently infected Nd-PC12 (QIF-PC12) cells; (iii) the rate of virus production was significantly greater following HS than forskolin treatment, and the rates of both were dependent on MOI; (iv) forskolin and HS appeared to affect pathways of viral activation from a quiescent state as they did not enhance viral growth in Nd-PC12 cells; (v) viral alpha4 gene and host HSP72 gene transcription were rapidly induced in QIF-PC12 as soon as 3 h post-HS initiation; (vi) induction of the viral alpha27 gene followed that of representative beta and gamma genes, U(L)30 and U(L)18, respectively, and (vii) HS induced asynchronous HSV-1 replication from QIF-PC12 cells with 1:400 to 1:22000 positive foci detected as rapid as 24 h post-induction when established at MOIs of 30 and 3, respectively. These findings provide evidence that alpha4 may be involved in the switch from quiescence to productive infection. Furthermore, this model has the potential to advance our understanding of how HS initiates the HSV-1 productive cycle from a cryptic viral genome.

We previously described a novel in vitro model of a non-productive herpes simplex virus type 1 (HSV-1) infection in neurally differentiated (ND)-PC12 cells that allows for inducible virus replication upon forskolin and heat stress (HS) treatment. In this research, we further characterized the model with respect to HSV-2 strain 333. We found that: (i) ND-PC12 cells are non-permissive to HSV-2 replication; (ii) HSV-2 can establish a quiescent infection, like HSV-1, in ND-PC12 cells with the transient use of acycloguanosine (ACV); however unlike HSV-1, anti-viral conditions are not obligatory to establish and maintain a quiescent state; (iii) the quiescent state is maintained in the presence of Vero cell cocultivation indicating that such cultures are free of infectious virus; and (iv) a high percentage of quiescently infected (QIF)-PC12 cell cultures (80 - 100%) produce HSV-2 in response to forskolin and HS (43 degrees C, 3 h) treatment for as long as 4 weeks post infection. These findings indicate that ND-PC12 cells can harbor HSV-2 in a cryptic and non-productive state that is reversible. This model has appealing features for studying gene expression during the establishment, maintenance and reactivation phases of the HSV-2 quiescent state in cell culture.

Rat pheochromocytoma (PC12) cells differentiated with nerve growth factor (Nd-PC12) were used to investigate the establishment of a non-productive herpes simplex virus type 1 (HSV-1) infection that is reversible. The results of this work are as follows: (i) Nd-PC12 cultures could be maintained as long term (>7 weeks) non-dividing cultures only when plated on collagen-coated dishes in the absence of serum; (ii) Infection of Nd-PC12 with HSV-1 strains KOS and 17 in the transient presence of acycloguanosine (ACV) resulted in all cultures free of detectable levels of infectious virus at the time of ACV removal and ACV was not needed to maintain the non-productive quiescent state in the subsequent 8 weeks; (iii) These persistently infected and quiescent (QIF)-PC12 cultures demonstrated both spontaneous and forskolin-inducible virus production, at low (5%) and high frequencies (92-100%), respectively during the first 2 weeks post-ACV withdrawal. (iv) In contrast to other in vitro models, HSV-1 failed to reactivate following removal of nerve growth factor. (v) A high percentage of QIF-PC12 cultures (50-100%) produced virus in response to forskolin treatment as long as 7 weeks post-ACV withdrawal. (vi) Expression of HSV-1 productive genes (i.e. alpha0, alpha4, alpha27, UL30 and UL18) dropped precipitously in the presence of ACV and remained undetectable or continued to decline following its removal, whereas the levels of LAT and the host gene G3PDH remained relatively constant throughout the 31 day study period as measured by RT-PCR. These results indicate that QIF-PC12 cells offer a novel, neuronal cell culture system that may enhance our ability to study HSV-1 reactivation from a cryptic, latent-like, non-productive state in the absence of replication inhibitors.