Intermittent viral reactivation from latency is a key feature of human infection with HSV-1 and is responsible for most of the morbidity caused by this virus, as well its epidemic spread. For this reason a significant research effort has focused on the mechanisms responsible for the regulation of HSV-1 latency in neurons, primarily in mouse models of viral infection. An unfortunate drawback to studying this process in the mouse has been the apparent lack of spontaneous viral reactivation and shedding. We recently published data describing productive cycle viral gene transcripts and viral protein in rare neurons of latently infected murine ganglia, thus providing evidence for spontaneous molecular reactivation of latent HSV-1 (6
). In the present study we confirm and expand upon this observation, providing strong evidence for spontaneous reactivation of infectious HSV-1 in latently infected murine TG.
The amount of infectious virus that we detected in latently infected TG was at the lower limit of the sensitivity of our viral plaque assay but is unlikely to represent contamination. Not only did we fail to detect infectious virus in control ganglia from uninfected and sham-inoculated mice but the frequency that we observed infectious virus in latently infected TG closely matched the frequency with which we detected viral antigen-positive neurons in these ganglia. Furthermore, the amount of infectious virus that we detected in “positive” latently infected TG (2 PFU) correlated closely with the number of PFU generated per HSV-antigen positive neuron in both (i) acutely infected murine ganglia and (ii) latently infected murine ganglia undergoing induced reactivation (20
). Taken together, these data indicate that infectious virus is present in latently infected murine TG and is most likely the result of spontaneous reactivation.
The relatively low level of infectious virus that we detected in “positive” ganglia may have several different explanations. First, ganglionic neurons may simply generate very little infectious virus in their cell bodies since viral capsid and membrane proteins are axonally transported out of the cell bodies separately (32
) and thus not as an infectious unit. Second, a rapid immune response may severely limit the amount of time that reactivated infectious virus can persist in the ganglion, thereby decreasing the amount of infectious virus found at a single experimental time point. Third, ganglionic homogenates may contain substances that bind, neutralize, or inactivate infectious virus. Regardless of the explanation, since we were operating at the lower limit of sensitivity of our assay, it is likely that we underestimated both the percentage of latently infected ganglia that harbor infectious virus and the amount of infectious virus generated by spontaneous reactivation in these ganglia.
A key feature of spontaneous reactivation of HSV-1 in our model is that it is not a rare event. The data presented in our present and our prior study suggest a rate of spontaneous reactivation of at least once every 10 days, which in our model represents at least 15% of all latently infected TG neurons over the average 2-year life span of the mouse. This is similar to the rate of spontaneous shedding of HSV-1 onto the ocular surface of rabbits (22
) and more frequent than the average rate of spontaneous shedding of HSV DNA in the human eye, mouth, or genital tract (11
). Furthermore, the rate of spontaneous viral reactivation in our model is only about half of that reported for hyperthermia-induced reactivation of HSV-1 (KOS) from the mouse TG (27
The data and conclusions presented in the present study help to reconcile a number of previously published observations about latent infection of murine ganglia with HSV-1. These include reports of (i) rare spontaneous shedding of virus onto the ocular surface of latently infected mice (30
), (ii) persistent inflammatory cell infiltrates (3
) and cytokine expression (3
) in latently infected murine TG, (iii) PCR-detectable immediate-early and early viral gene transcripts in latently infected TG (12
), (iv) rare HSV antigen-positive neurons in latently infected ganglia (6
), (v) reduced cytokine expression in latently infected TG under conditions that limit viral DNA replication (4
), and (vi) rare viral PFU in latently infected ganglia serving as negative controls for studies of HSV reactivation (25
). All of these observations can be explained by spontaneous reactivation of latent virus from TG neurons.
Spontaneous viral reactivation in neurons of the murine TG may prove to be a more accurate model of HSV-1 reactivation in humans than other commonly used murine and rabbit models. Although trauma, fever, UV exposure, and hyperthermia have all been reported as triggers of reactivation of HSV-1 from the human TG, the vast majority of episodes of recurrent HSV cannot be tied to a specific trigger (37
). Thus, most cases of recurrent human HSV-1 are either spontaneous events or a consequence of the uneventful stresses of day-to-day living, making spontaneous viral reactivation in the mouse an attractive model for human disease.
In considering our results it is important to entertain the possibility that the very small amount of virus that we found in “latently” infected TG might represent residual virus from the initial productive infection of the TG rather than viral reactivation from a latently infected ganglionic neuron. Although an interesting alternative, this hypothesis does not explain our in situ hybridization (6
) and immunocytochemical data or the tight correlation between the percentage of ganglia with infectious virus and those with neurons expressing productive cycle viral genes. It also does not explain why the amount of infectious virus that we detected in positive ganglia closely matched the amount of infectious virus generated per neuron during both acute ganglionic infection and induced reactivation (20
). Finally, this hypothesis implies that the virus has evolved mechanisms that allow it to persist as an infectious particle in an immune activated ganglion for greater than a month, mechanisms distinct and in addition to the very successful strategy of viral latency and reactivation.
In contrast to the role that LAT has been reported to play in facilitating induced viral reactivation in the mouse and rabbit and spontaneous shedding of virus on the ocular surface of the rabbit (2
), our data do not support a role for LAT in facilitating spontaneous reactivation in the mouse. This cannot simply be attributed to the use of a different viral construct, mouse strain, or means of ocular inoculation than previously studied, since under virtually identical conditions Leib et al. (16
) reported that the rate of explant reactivation of KOS dl
LAT1.8 from latently infected TG is significantly less than that with either wild-type KOS or an appropriate viral rescuant. It is more likely that the region of the virus deleted in dl
LAT1.8 plays little, if any, role in spontaneous viral reactivation, but this can only be ascertained if one studies this process directly at the site of reactivation rather than at a downstream surrogate event such as ocular viral shedding; an outcome that reflects not only viral reactivation at the neuronal cell body but multiple downstream events. Although it is probably not wise to rely on a single experimental model of human HSV disease, given the logic of direct observation of a spontaneous event (rather than a downstream surrogate event), we feel that it is important to consider the relevance of studying spontaneous reactivation in the mouse TG to gain a broad and accurate understanding of the mechanisms involved in latency and reactivation. This is particularly important in light of our rapidly evolving understanding of the multifunctional nature of the LAT region of the viral genome, which appears to code for transcripts in both the sense and the antisense directions (24
), including functions that inhibit apoptosis (7
), interfere with interferon expression (21
), inhibit transactivation by ICP0 (5
), and stimulate the expression of heat shock proteins (1
); functions that should all be altered by the deletion in KOS dl
It is important to point out that our studies with KOS dl
LAT1.8 were carried out by assaying latently infected ganglia by immunocytochemistry for HSV antigen-positive neurons rather than for infectious virus. We felt comfortable with this approach since data from the present study as well as that published previously (6
) demonstrate a tight correlation between the frequency that antigen-positive neurons and infectious virus are found in latently infected TG. However, in the absence of studies of infectious virus in ganglia infected with KOS dl
LAT1.8 it could be argued that it would be more appropriate to conclude that the region of the virus deleted in KOS dl
LAT1.8 plays little, if any, role in spontaneous molecular reactivation, more restrictive terminology that we have used previously (6
In conclusion, the data presented above provide clear evidence of frequent spontaneous reactivation of HSV-1 from latently infected neurons of the mouse TG. This process is relatively easy to study and can be carried out in a relatively efficient manner by batching ganglia and assaying relatively thick (35-μm) tissue sections. In theory this process could even be carried out with whole mounts, in a manner similar to that used by Sawtell (26
). Studying spontaneous viral reactivation in this manner allows direct analysis of viral reactivation from neurons in vivo, thus avoiding a number of potential experimental confounders inherent to both explant reactivation and induced viral shedding on the ocular surface.