Our study demonstrates several new concepts about HSV infection. HSV reactivation has both a more frequent onset and more rapid clearance than previously appreciated. Collection of samples 4 times daily revealed that approximately half of HSV mucosal reactivations last ≤12 h and that these short reactivation episodes are largely asymptomatic, characterized by rapid emergence of 103–104 copies/mL of HSV DNA in the skin or mucosa, and accompanied by rapid viral clearance by the host. Prolonged genital shedding was associated with a higher initial viral level and a greater likelihood of symptoms and lesions. Median genital HSV reactivation frequency was 18 episodes annually, 81% of genital mucosal HSV reactivations were subclinical, and half lasted <12 h. Only 19% of genital shedding episodes were associated with symptoms and 15% with overt genital lesions, illustrating the importance of subclinical HSV reactivation in the biology of HSV reactivation. Similarly, reported oral ulcerations accompanied only 2 of 33 oral HSV reactivations of known duration.
Our data raise several issues pertinent to the pathogenesis of HSV reactivation. Most samples were collected from a large surface area and placed into 1 mL of viral transport solution. Although this method of collection is consistent and reproducible, HSV reactivations are exquisitely anatomically localized [13
]. Hence, the in vivo number of HSV virions released from subclinical ulcerations is undoubtedly markedly higher then the 103
copies/mL we detected. This hypothesis is supported by the higher viral level found from swab specimens of identifiable lesions than samples collected at the same time from the entire anogenital area (105.9
copies/mL). Rapid HSV clearance within 6–12 h after shedding appearance illustrates prompt and effective immunocompetent host defense mechanisms. Prior work has shown that HSV-2 clearance from genital lesions is associated with HSV-2–specific cytotoxic T cell activity in the CD8+
fraction of T lymphocytes [19
]. HSV-specific CD8+
T cells have also been found in ganglia, suggesting that some control occurs in ganglia [22
]. However, when control at the ganglia fails and virus travels via anterograde transport to genital mucosa, the fact that mucosal replication is effectively eliminated within hours after appearance suggests that host T cells must either move extremely rapidly to the mucosal site of replication or remain in the genital mucosa between recurrences to rapidly control and eliminate HSV mucosal replication when virus first appears from peripheral nerves. Recent immunohistologic studies suggest that HSV-2–specific CD8+
T cells can persist in genital skin for extended periods and are associated with localized clearance of subclinical HSV-2 reactivation [13
]. The rapid host elimination of HSV-2 suggests that much HSV-2 control rests within the peripheral mucosal immune system.
Our study also helps explain the large body of data implicating HSV infections as an important factor in HIV acquisition. The rapid reactivation, release, and resolution of relatively high copy numbers of HSV DNA in genital mucosa may help explain the high rate of sexual transmission of HSV-2 and the increased risk that HSV-2 confers in HIV acquisition [27–29]. Frequent, short, subclinical genital mucosal reactivations place large numbers of activated CD4+
T lymphocytes at risk for HIV infection at the genital mucosa [20
]. If subclinical ulcerations are also present during these reactivations, then they would provide portals of entry for HIV, further enhancing the risk owing to increased numbers of CD4+
T lymphocytes at the mucosa. Our findings also put into perspective the use of episodic therapy for mucosal HSV infection. Although effective in relieving the discomfort of individual episodes [30
], such an approach treats only a small fraction of reactivations. Daily suppressive antiviral therapy can reduce genital HSV-2 shedding, as detected by once daily sampling, by 70%–85% of days but reduces HSV-2 sexual transmission by only 48% [33
]. This disparity between antiviral effects on shedding and transmission may suggest that once daily antiviral therapy does not eliminate short reactivations.
Participants included in these studies were persons well versed in the signs and symptoms of genital and oral herpes. Hence, the ratio of subclinical to clinical ulcerations among HSV-2–infected persons in the general population is probably even higher than the 85% ratio found in our study, as recognition of lesions is likely lower. Frequent sampling is difficult for participants and results in a large number of samples for the laboratory. We collected and performed PCR analysis on >8300 separate samples from 43 participants. The genital shedding rate we found among our 25 HSV-2–infected participants is comparable to historical shedding rates among our previously studied HSV-2–infected patients; among 352 participants (145 men and 207 women) in previous genital HSV-2 shedding studies, once-daily samples were PCR positive on 2740 (23.1%) of 11,838 days, with samples collected on a median of 57 days per person. Thus, data from the present study are likely representative of immunocompetent persons with oral and genital HSV infections in the studied age range. Significant individual variability in HSV shedding frequency is seen, and age, sex, immune status, host genetics, and number and density of recurrences in dorsal root ganglia appear to affect the severity of HSV reactivation [10
]. Whether rapidly cleared episodes of mucosal HSV are seen in immune-suppressed patients remains to be determined. The high combined rate of HSV-1 and HSV-2 reactivation we observed among immunocompetent persons, if subsequently demonstrated among HIV-infected persons, could provide an explanation for how HSV increases plasma HIV level [38
], as most HIV-infected persons worldwide have HSV-1 and HSV-2 infections.
In summary, our data indicate that the frequency of mucosal HSV reactivation and the pace of clearance are much faster than previously appreciated. The most common form of HSV reactivation is an asymptomatic reactivation associated with rapid onset and clearance of virus within 12 h, illustrating a dynamic interaction between virus and host in the peripheral skin and mucosa. These short subclinical reactivations help explain the observations that most HSV-2 transmission events occur during subclinical reactivations and that clinical disease manifestations predict neither mother-to-child nor sexual transmission. The frequency of these reactivations also provide a possible explanation for how incident and prevalent HSV-2 infections increase both the risk of HIV acquisition and the HIV level in coinfected persons.