We present a stochastic model of mucosal HSV-2 pathogenesis that closely reproduces the pattern of actual HSV-2 genital shedding and measurement-based estimates of shedding frequency (6
), annual clinical recurrences (8
), peak copy number during episodes (6
), duration of shedding episodes (8
), and lesion diameter (20
). The output from our stochastic model mirrors the substantial variability in HSV-2 shedding frequency and clinical recurrence among patients and within the same patient over time (7
). Although HSV-2 is detectable in the genital tract on a median of 20% of days, the range within the 95% of seropositive patients who shed HSV-2 during 6–12 weeks of observation is 0–78% of days (6
). Our model generates similar heterogeneity within patients by virtue of its stochastic nature, and among patients as a result of patient-to-patient differences in parameters that determine viral replication, viral spread and cytolytic immune response.
Our simulations suggest that genital HSV-2 infection represents a highly dynamic system in which viral particles are frequently but slowly introduced into the genital area in very low numbers by sensory neurons, and periodically released in an explosive fashion by infected epithelial cells. Once an epithelial cell produces a burst of viral particles, these particles have a few hours to infect contiguous cells before losing infectivity. Whether any of the hundreds of particles released from the first infected cells are able to infect surrounding cells and initiate a chain reaction of infection determines which of three distinct shedding episodes will ensue: (i) a brief low-copy episode, (ii) a more prolonged subclinical, medium-copy episode, or (iii) a high-copy episode associated with a clinically apparent genital lesion.
In our model, no more than a single epithelial cell is infected at any point in time during many low-copy shedding episodes. These smallest reactivations are the most likely to be missed with current methods of detecting shedding. First, if a tiny number of nucleated epithelial cells are infected during an episode, then only cells at the dermal epidermal junction will produce HSV: viruses may never reach the skin surface, and will not be detected, even with well-timed sampling. Second, because most low-copy shedding episodes are brief, many are missed with every six-hour swabbing protocols. Our model’s output reinforces our observation that current estimates of shedding frequency with every 6-hour sampling are accurate (7
), but implies that measures of number of detectable shedding episodes per year are likely to be underestimates.
Medium-copy episodes may be responsible for person-to-person transmission because, although not enough epithelial cells die to generate a visible lesion, shedding is prolonged. In addition, there is a much higher inoculum of HSV-2 during a medium than a low-copy episode. High-copy episodes create enough infected cells to generate a detectable lesion. Lesions used for our curve fitting all were associated peak copy numbers greater than 106. Fitting results were equivalent whether we used anogenital or lesion only swabs. This does not rule out concurrent HSV shedding at other anogenital sites during a genital lesion. Rather, the amount of virus produced at the lesion site so far exceeds that from other subclinical shedding sites that curve fitting is not affected, and swabs of the entire anogenital region are adequate for this study.
In order to initiate shedding episodes in epithelial cells of the genital mucosa, our model suggests that miniscule numbers of viruses must be introduced from the neuronal tissue per day. Moreover, the vast majority of detectable viral DNA in the genital tract appears to be of epithelial cell origin, rather than from neurons. Several clinical phenomena are potentially explained by a slow but steady release of HSV from genital tract neurons. The low amount of neuronal virus required for disease and transmission might explain why neurons survive HSV-2 infection, and why peripheral neuropathy is not a disease manifestation (22
). The prodromes that often precede ulcer formation are conventionally thought to result from travel of viral particles down the neuron (20
). However, asymptomatic episodes, which lack prodromes, also occur after neuronal reactivation. Our model suggests that prodromes could possibly be from high levels of viral replication among epithelial cells early during recurrence and prior to lesion detection. The frequent pattern of neuronal viral release might also explain why currently available DNA nucleoside analogues prevent recurrences more than sub-clinical shedding episodes and transmissions (6
). Recurrences occur over several days. Therefore, high levels of an antiviral medication that is dosed on a daily basis are certain to coincide with early lesion formation. However, because viral release from neurons is so frequent, even brief periods with sub-therapeutic drug levels might be sufficient to allow for shedding episode initiation.
Even small, modeled variations in neuronal virus introduction rate have a dramatic impact on detectable shedding frequency. Determinants of neuronal viral production remain unclear but may include ganglionic lymphocyte infiltration, cytokine production, and immune response to primary HSV-2 infection (10
). These mechanisms likely determine the rate of virus release into the genital tract, which in turn predicts shedding frequency in our model. If the number of viruses produced from the neurons is below a certain threshold, then shedding episodes are rare. This scenario may explain findings in six patients with HSV-2 directed antibodies who appear to never shed virus (26
). If over 200 copies of HSV are shed from neurons per day in our simulations, then detectable shedding from infected epithelial cells is present over 50% of the time. This result suggests a mechanism for persistent shedding described in immunocompromised patients, as well as a subset of immunocompetent patients (6
It was once common wisdom that infectious HSV-2 particles are only periodically introduced into the mucosa from neuronal tissue, and that these reactivations almost always caused genital lesions (5
). Because initiation of an HSV-2 shedding episode implies that ganglionic reactivation recently occurred, reactivation must be at least as common as episode frequency. Recent work documenting sub-clinical detection of HSV-2 in the genital mucosa on 20% of days suggests that reactivation occurs at least this often (7
). Our model is consistent with another study that concluded that HSV-2 release from sacral ganglia is likely to occur much more frequently than actual HSV-2 detection (17
). Our findings also suggest that a constant, extremely slow release of infectious viruses from sensory neurons can explain localized HSV-2 shedding episodes in the genital mucosa. The same net amount of virus, whether introduced on a continuous, daily, weekly or bi-weekly basis in our model, causes the same frequency of detectable virus. The key periodic event in HSV-2 shedding may not be release of HSV-2 from the neurons, which is possibly a constant, low-grade process, but rather infection of a first epithelial cell that denotes episode initiation.
Our model has important limitations. First, the mucosal immune compartment involves innate and humoral processes not included in our current model for lack of adequate in vivo
data. Since our model accurately reproduced true clinical outcomes, these immunologic phenomena are at least partially captured within included model parameters such as viral lifespan, infectivity, and burst phase. Second, our model oversimplifies kinetics of the infected cell in its progress towards death. There is more viral production over time as transcriptional activity is up-regulated (28
), and HSV-2 may inactivate the cytolytic immune response against the infected cell as infection progresses (29
). With accrual of more precise laboratory and clinical data, we hope to incorporate these relevant concepts of viral replication into the model.
Despite the model’s oversimplification of viral pathogenesis, we include a high number of parameters and we may have arrived at local optima for estimates of certain parameter values. Nevertheless, we incorporated the minimum possible number of parameters to describe HSV-2 shedding in the genital tract: exclusion of basic CD8+ T-cell parameters resulted in poor model fit to empirical data. In addition, our parameter estimates are from several sources and are not all disease or host specific. Increasing the complexity of the model and detail of empirical data for model fitting will inevitably change certain parameter estimates, although the fundamental concepts described here will likely remain intact. Our conclusions are model-derived and represent hypothesis generation. Future studies should focus on validating our findings experimentally and clinically, and will inform more detailed permutations of our model.
The findings of our model suggest a formidable challenge in controlling HSV shedding and person-to-person transmission. In persons infected with HSV, successful antiviral medication or immunotherapy will need to completely eliminate the frequent trickle of HSV from the neurons into the genital tract. A vaccine that is effective in promoting mucosal immunity may limit recurrences only, while elimination of both asymptomatic shedding and recurrences may require immune activation in both neurons and genital mucosa.