Our analysis of a large, diverse cohort of patients with HSV-2 infection provides, to our knowledge, the first detailed kinetic evaluation of mucosal HSV-2 infection in the healthy host. HSV-2 infection reactivations vary substantially in and among individuals according to duration and peak HSV DNA copy number, 2 measures that strongly correlate. Episodes with higher viral production are more commonly associated with genital lesion formation and nonmonotonic viral decay [1
]. Regardless of peak copy number, episodes expand extremely rapidly, with subsequent rapid deceleration, followed by sharp decay, leading to a stereotypical episode appearance with sharp peaks. Duration of the expansion phase varies among episodes, and some episodes are eliminated within hours [1
]. However, even during prolonged lesional episodes, the exponential rate of expansion invariably decelerates during the first 24 hours. Moreover, exponential decay rate increases dramatically from episode peak to termination. During the final 24 hours of an episode, exponential decay rate may actually decrease slightly, although decay rate remains sufficiently high to ensure termination of viral replication.
Although these observations most obviously pertain to viral kinetics, they indirectly highlight the importance of the mucosal host immune response in containment of viral shedding. The rapid cessation of expansion phase and accelerated decay phase of episodes suggest that the peripheral immune response must be continually primed to rapidly eliminate HSV-infected cells. In a previous study [11
], we used a mathematical model to indicate that the most likely mechanism to explain high frequency of annual genital episodes in HSV-2–infected persons is nearly constant release of low numbers of viral particles from sensory nerve endings at the dermal-epidermal junction in the genital tract. After a single epithelial cell is productively infected, viral production is extremely rapid (>10000 HSV DNA copies/d/cell), and in the absence of an immediate immune response, spread to contiguous epidermal cells occurs within hours. Thus, in 1 day, thousands of cells may harbor replicating HSV.
However, all episodes are cleared in immunocompetent persons despite explosive viral expansion. Peak episode copy numbers almost never exceed 108
/mL, and despite an abundance of skin epithelial cells, most HSV-2 infection recurrences lead to vesicles and ulcers that are only several millimeters in diameter [9
]. Therefore, in contrast to HIV-1 [19
], hepatitis B and C [20
], and influenza [22
] infections, target cell saturation is not hypothesized to play a role in determining peak viral load. The deceleration of expansion during the initial 24 hours suggests early immune influence on viral replication and spread, perhaps because of innate pressure from local interferon production [23
]. Our modeling and experimental data also suggest that an intense, localized acquired mucosal immune response limits the extent of each episode and may promote early containment. HSV-2–specific CD4+
T cells form dense infiltrates at recurrence sites in genital skin and persist for months at the dermal-epidermal junction where HSV-2 is released from neurons [24
]. Presumably, these lymphocytes participate in immunosurveillance, and their density in the genital mucosa at episode initiation is likely to play an important role in determining extent of viral production before clearance [26
]. For these reasons, we speculate that viral decay kinetics may be extremely different in the immunocompromised host.
Our data suggest a decreased rate of decay toward the completion of an episode, an observation that initially seems counterintuitive. Mathematical models of HIV-1 decay during antiretroviral therapy document differential decay kinetics of virus in different cellular compartments [19
]. In the case of HSV, the cytotoxic immune response effectively clears infected epidermal cells. If decay of remaining free virus is slower than that of infected cells, viral decay rate may decrease after infected cells are no longer present.
Episode prolongation is an important strategy used by HSV-2, presumably to enhance transmission, because likelihood of transmission is probably a function of shedding frequency and quantity. Prolonged episodes are associated with higher peak copy number [1
], and increased inoculum at time of coitus is likely to correlate with higher probability of transmission. It is therefore an important finding of our study that prolonged episodes often have nonmonotonic decay or re-expansion, of virus.
Mechanisms of viral re-expansion during an episode are not currently understood. One hypothesis for re-expansion proposed for acute influenza infection is that antiviral cytokines fail to clear all infected cells of a given plaque and have a short half-life in tissue, allowing for bimodal episodes [22
]. Similarly, with HSV-2 infection, local innate immune mechanisms may be inadequate, because acquired immune cells must arrive from external sites to achieve episode containment. Alternatively, the common appearance of secondary crops of vesicles during prolonged HSV-2 infection recurrences [9
] suggests that new plaque formation at spatially distinct sites (via cell-free particles or separate neuronal reactivations) may allow for evasion of the localized immune response. The recent findings that HSV-2 is commonly detected throughout the genital tract during focal genital lesions and that maximum genomic copy number is associated with wider spatial spread support the hypothesis that HSV-2 infects new sites to avoid an intense but highly localized immune response at the primary plaque of infection [30
An important limitation of our study is that sampling occurred every 24 hours. In studies using swabs every 6 hours, 2 of 3 HSV-2 infection episodes lasted <24 hours; median duration was 13 hours rather than 3 days [1
]. Brief episodes are largely asymptomatic and peak at low HSV DNA copy numbers. Therefore, our study overestimates proportion of episodes with high copy number, long duration, and associated lesions. Moreover, our data are from studies in which participants performed mixed swabs of the anogenital tract. We believe that episodes might be shorter and without re-expansion if we localized swabs to single vesicles in lesions. Finally, we measured viral quantity with use of genome equivalent copies, a more sensitive method than viral isolation; prior studies indicate that high DNA copy numbers correlate with presence of infectious viral particles [2
Detailed examination of HSV-2 shedding episodes reveals a remarkable compromise between virus and host immunity. Viral replication and spread is explosive during the initial hours after episode initiation because of infection of a single epithelial cell. Nevertheless, in immunocompetent hosts, immune cells clear all episodes and the majority of episodes are terminated in <5 days. Most shedding is asymptomatic, and most recurrences, although a clinical nuisance, are not harmful to the infected host. Nevertheless, the cumulative effect of these episodes is that virus is present for enough of the time to facilitate transmission after relatively few coital acts [31
], which in turn explains high worldwide seroprevalence [32
]. Therefore, frequent, highly dynamic shedding episodes are an extremely effective strategy for HSV-2.