(See the article by Schiffer et al, on pages 554–561.)
Utilization of new technological advances in molecular biology have enabled investigators to accurately measure the quantitative amount of viral nucleic acids produced within a host over time, thereby enabling investigations into host-viral interactions. The initial development and use of quantitative viral load assays for human immunodeficiency virus type 1 (HIV-1) provided key insights into the kinetics and pathogenesis of viral replication, host immune responses responsible for the initial decline in viral replication, the establishment of viral set points, and clinical correlates associated with the natural history of HIV-1 infection [1–7]. Similarly, the effectiveness of antiretroviral therapy was first measured in humans by assessing the impact of these drugs on HIV-1 viral kinetics [8–10]. Viral kinetic studies have also elucidated our understanding of host-viral interactions in influenza A, hepatitis B and C, and Epstein-Barr virus and cytomegalovirus infections [11–15]. These detailed quantitative viral studies have enabled investigators to examine the efficacy of antiviral compounds in limiting viral replication while assessing the development of resistance with resurgence in viral replication. Viral kinetic studies have also provided a deeper understanding of the natural history of viral infections in humans with establishment of latent reservoirs, viral shedding, and viral clearance [16–20]. In addition, studies on viral load have shed light on the probability of viral transmission from mother to infant [21, 22] and sexual transmission from one person to another [23, 24]. More recently, these viral kinetic studies have stimulated a newly emerging field regarding the effectiveness of antiviral agents on viral replication as a surrogate in modeling their population transmission effects. For example, reductions in HIV-1 loads in a community as a consequence of scaling up treatment are being found to be associated with subsequent reductions in HIV-1 transmission and incidence .
In this issue of the Journal, Schiffer and colleagues  apply this same molecular technology to carefully dissect the viral kinetics of mucosal herpes simplex virus type 2 (HSV-2) infection in immunocompetent individuals. Their studies provide a unique insight into the viral replication capacity of HSV-2 and the subsequent host immune reactions to the virus responsible for limiting viral shedding. Genital herpes is an extremely common infection that establishes latency in nerve root ganglia for the life of an infected individual. More than 50 million people in the United States are estimated to be infected, and serologic surveys for HSV-2 estimate that 16.2% of 14–49-year-old individuals in the United States are infected [27, 28]. In 2009, >300000 individuals sought medical care for genital herpes, a steady increase in comparison with previous years . Worldwide, the frequency of infection is even higher, with prevalence rates of HSV-2 infection in Africa, Latin America, and Asia of 30%–60% in similarly aged groups . This high frequency of infection within such diverse populations suggests rather high efficiency in sexual transmission.
Genital herpes includes an initial infection that lasts several weeks followed by recurrent episodes of viral shedding, which are highly variable in frequency, and signs and symptoms. More than 80% of seropositive individuals are unaware of any signs or symptoms consistent with genital herpes [30–32]. However, nearly all seropositive individuals, regardless of symptoms, will intermittently shed herpes virus from dermatologic or mucosal sites [33–36]. Symptomatic individuals develop recurrences that are characterized by painful, small genital ulcers that usually resolve promptly within a week in immunocompetent individuals, only to recur intermittently over a subsequent period that varies among individuals. In asymptomatic individuals, serial sampling demonstrates shedding of HSV-2 DNA in intermittent bursts of replication at frequent intervals both during and between recurrences [33–35]. Previous detailed studies have documented the high frequency of viral shedding, which lasts for very brief periods (hours to days) [33–38]. It is this unpredictable intermittent shedding of HSV-2 that is responsible for subsequent transmission to sexual partners and that poses a continual threat of perinatal transmission [30, 31, 39, 40].
Although it has been postulated that the higher frequency of shedding is most likely associated with greater probabilities of transmission, the actual quantitative level of viral replication associated with these shedding episodes had not been thoroughly documented. Little is known about how much virus is produced locally at these genital sites either during or between episodes of recurrence, how long the shedding lasts, and what the characteristics of the host immune response are that limit these viral kinetics. Schiffer et al  addressed several of these questions by quantifying the amount of HSV-2 shedding on a daily basis in 531 immunocompetent HSV-2–seropositive persons. They systematically collected >14600 genital swabs over several years. By measuring the quantity of virus in these swabs, the investigators were able to fully document the duration, peak copy number, and expansion and decay rates of HSV-2 in >1800 symptomatic and asymptomatic viral shedding episodes. This is the first study to document quantitatively the amount of virus actively being produced and shed locally during these recurrent herpetic episodes.
Consistent with the results of previous studies on shedding frequency, Schiffer et al  found significant heterogeneity in the duration of episodic viral shedding, with a median duration of 3 days and with slightly more than one-quarter of episodes lasting only 1 day and 20% lasting >9 days. Similarly, there was a wide range of peak genomic production levels, with a positive association between episode duration and peak copy number. The investigators were also able to calculate an extremely rapid expansion rate of viral replication of 7.5 log copies/mL of viral DNA produced during the first 12 hours of an episode. Because only 1 swab was collected per 24 hours, it remained uncertain as to whether the rate of replication was constant, whether there was a single burst of viral replication, or whether it was slower during the initial hours of the episode with a subsequent burst after HSV-2 DNA copy numbers surpassed 103 copies/mL. Only more frequent sampling just before and during an episode will enable us to address the initial kinetics of viral replication.
Following this initial burst of replication, the host’s immune response is rapidly activated and limits viral production, producing a unimodal curve in the majority of people sampled. Schiffer et al  calculated that the average rate of viral decay following the peak viral replication was −6.2 log copies/mL of viral DNA during the final 12 hours of the episode, ultimately resulting in final termination of each episode. However, as with all biological responses to infection, there is heterogeneity in the observed responses to herpes virus production. One-fifth of the subjects with a detectable viral level demonstrated a nonmonotonic decrease, in which there were intermittent episodes of viral replication interspersed within the viral decay curves, resulting in biphasic or multiphasic decays. This observation was more characteristic of individuals who had prolonged episodes of >7 days. These nonmonotonic decreases in viral replication levels suggest that the host immune response was only partially effective in its initial response, but improved over time to finally suppress viral replication. Similar variability within viral kinetics was observed in those individuals who developed genital ulcers (higher peak viral copy numbers and prolonged decay), compared with those who remained asymptomatic. These findings also suggest that the host’s ability to limit viral replication may directly influence the clinical variability observed in infections in humans.
These observed viral dynamics provided additional support for a model of viral-host immune interactions previously postulated by these investigators based on the frequency of viral shedding [30, 41–43]. Following primary infection, viral latency is established in the sacral ganglia. Viral reactivation in the ganglia may be self-limited, but it results in centrifugal migration of HSV-2 along sensory nerves. Subsequent viral release from the sensory neurons into the genital tract leads to subclinical shedding of virus or development of a herpetic lesion with even higher viral production. These prolonged episodes are associated with higher peak copy numbers and, consequently, higher inocula at time of coitus, which correlates with the higher probability of transmission.
The enormous amount of viral replication stimulates a host immune response that is characterized by infiltration of the dermis with both CD4+ and CD8+ lymphocytes . This response appears to be critical in the clearance of a genital lesion and ultimately leads to cessation of viral shedding. Serial biopsies of genital sites and examination of excised foreskins from HSV-2–seropositive individuals demonstrate persistence of specific immune CD4+ and CD8+ T cells for months at the dermal-epidermal junction where HSV-2 is released from the neurons [44–46]. It is, therefore, likely that the persistence and subsequent activation of these immune cells during viral replication is ultimately responsible for the very rapid clearance of the virus from these sites. The high frequency of viral shedding may contribute to the persistence of these immune cells locally. Thus, the response to intermittent episodes of peak viral production is likely to be dependent on the density and effectiveness of HSV-2 immune primed CD8+ lymphocytes.
Although the study by Schiffer et al  provides insight into HSV-2 viral kinetics in immunocompetent individuals, it raises the specter of how an immunocompromised host would alter HSV-2 viral kinetics. HIV-1/HSV-2 dually infected individuals are known to have greater persistence of genital ulcers, and it is likely that peak viral production at genital sites will be much higher and prolonged due to slower decay rates than that observed in this study, thereby complicating the healing of any ulcers and enhancing the transmission of both viruses [47–49]. Another question raised by this study is what the effect of antivirals will be on the peak and duration of viral replication. Although HSV-2 antivirals are effective in suppressing clinical episodes and partially limiting episodes of shedding, they are only partially effective in preventing sexual transmission , once again emphasizing the importance of a very effective immune response in addition to therapy. Finally, these studies raise the question of what host or viral signals activate virus replication, and why and when they occur. What role do cytokines actively play in promoting activation or cessation of virus production? These questions are clinically important and might translate ultimately into a better understanding of this viral infection, leading to better therapy, prevention in transmission, and development of an effective vaccine.