Division of Virology, Dept of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK1
Author for correspondence: Stacey Efstathiou. Fax +44 1223 336926. e-mail se{at}mole.bio.cam.ac.uk
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Abstract |
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Introduction |
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To address this question we have used the mouse ear model of infection to study the establishment of latency in the CNS. The mouse ear is innervated by sensory nerves from the C2, C3 and C4 spinal levels, and motor neurones from the facial nucleus in the brainstem (Hill et al., 1975 ; Holstege et al., 1977
). By infecting these neurones, HSV-1 can access the CNS via both the spinal cord and brainstem and pass transneuronally along the sensory and motor nerve pathways connected to them. As this route of inoculation leads to the infection of multiple types of CNS neurones which vary in size, morphology and function, we have been able to study the ability of the LAP to function in a wide variety of neuronal types. Using a recombinant HSV-1 (SC16-C3b) which expresses the lacZ reporter gene under the control of the cytomegalovirus immediate early (CMV-IE) promoter (inserted at the LAT locus), we have mapped out in detail the distribution and time-course of the acute virus infection in the CNS. With a second virus (SC16-L
A), in which LacZ is expressed under LAP control, we have identified the sites in the CNS in which the LAP is active, and also studied the kinetics of LAP activity at these sites. Our results suggest that the LAP can function in many (and possibly all) types of neurones that become infected at early time-points. However, at later time-points (after 34 weeks) LAP-driven LacZ expression was predominantly detected in the facial, and sometimes in the hypoglossal, nuclei, suggesting that not all neurones support LAP transcription equivalently.
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Methods |
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Construction of recombinant viruses was as follows. SC16-C3b contains the lacZ gene driven by the CMV IE promoter inserted into a 168 bp HpaI deletion (nucleotides 120301120469) within the major LAT locus of HSV-1 strain SC16, as previously described (Lachmann et al., 1996 ). SC16-L
A contains an encephalomyocarditis virus IRES linked to lacZ inserted into the same locus within major LAT, as described previously (Lachmann et al., 1997
).
Mice.
Groups of 56-week-old BALB/c mice were infected with 2x106 p.f.u. virus, in 20 µl PBS, by subcutaneous injection into the left ear pinna.
The number of animals sampled at each time-point is shown in Table 1. For all three viruses (SC16 wild-type, SC16-C3b and SC16-L
A), 2030% of the animals showed obvious signs of neurological impairment (such as limb paralysis) following infection, and so were euthanased and excluded from the study.
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-Gal histochemistry.
The brain was embedded in 4% LGT agarose. Sections (100 µm) were cut with a vibratome and collected in detergent solution (0·01% sodium deoxycholate, 0·02% Nonidet P-40, 2 mM MgCl2 in PBS) in 24-well plates and incubated at 4 °C. After 2 h the detergent was replaced with X-Gal solution (1 mg X-Gal per ml of detergent solution, with 4·5 mM potassium ferricyanide, 4·5 mM potassium ferrocyanide) and incubated at 37 °C for 212 h. The sections were then rinsed in three changes of PBS, mounted on polylysine-coated slides and air-dried. The tissue was then defatted in xylene, rehydrated through graded ethanols and counter-stained with neutral red.
Immunohistochemical detection of
-Gal.
Following fixation, the brains and spinal cords were equilibrated in 25% sucrose for 24 h. Frozen sections (60 µm) were cut using a sledge microtome. Alternate sections were taken for -Gal histochemistry and immunohistochemistry, respectively. Sections taken for immunohistochemistry were immersed in 3% hydrogen peroxide, 10% methanol solution for 5 min to quench endogenous peroxidase activity, rinsed with three 10 min changes of TBS (0·05 M TrisHCl, 0·15 M NaCl, pH 7·4) and blocked for 1 h with 3% normal goat serum (NGS) in 0·2% Triton-X in TBS. They were then incubated overnight with a rabbit IgG to
-Gal (ICN/Cappel cat. no. 55976) diluted 1 in 6000 in 0·2% Triton-X in TBS, 1% NGS. The secondary antibody was a biotinylated goat anti-rabbit antibody diluted 1 in 200 in TBS, 1% NGS.
-Gal-positive cells were revealed using DAKO streptABComplex/HRP (Dako K 0377) with 0·6 mg/ml DAB containing 0·03% hydrogen peroxide.
In situ hybridization.
Brains and spinal cords were fixed for 24 h in 4% paraformaldehyde, embedded in paraffin, and 5 µm sections were cut using a microtome. Every 20th section was mounted onto a 3-aminopropyltriethoxysilane-coated glass slide. ISH was carried out using a LAT-specific probe as described previously (Arthur et al., 1993 ).
Plotting of labelled cells.
Tissue from an uninfected animal was sectioned, mounted and counterstained as described above. Drawings of the sections (made with a camera lucida) were transferred to CorelDraw 8.0 using a Wacom A3 digitizing plotter, and were labelled according to the atlas of Paxinos & Watson (1986) . Labelled cells were then plotted onto copies of each of the drawings with CorelDraw.
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Results |
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Spinal cord
5 Days.
In the spinal cord the majority of the labelling was found on the infected side (left), in and around the dorsal columns, Lissauers tract, and the dorsal horn grey matter, from the level of C3 upwards (Fig. 1). At this time, a column of labelled fibres and glial cells was seen in the left dorsal root entry zone and dorsal column, extending rostrally to the brainstem. In the spinal grey matter, infected neurones were found in the superficial (II, III, IV) and deeper Rexed laminae (V, VII and X). Longitudinal spinal cord sections showed that these labelled cells formed a continuous column in the dorsal horn, stretching from approximately the level of C4 to C2 (Fig. 1
).
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21 Days.
No cells were labelled by the CMV-IE promoter and the spinal cord contained only regions of inflammation where virus replication had occurred.
Brainstem
The distribution of labelling seen in the brainstem over time is presented in Fig. 2. For clarity the combined results of two animals each from the 5, 7 and 15 day time-points are shown, since at day 11 the distribution was very similar to that seen at day 15, and at day 21 only one or two labelled cells were detected in the brainstem of each animal.
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7 Days.
The distribution of labelling remained grossly the same, though the zones of infection were larger, especially contralaterally, and the virus had spread to a number of additional sites in the brainstem. The mean cell count per brainstem was 194 (range 71390, n=3). The distribution of 7-day labelling seen in the brainstem is indicated in red in Fig. 2, and shows that the virus had spread to the perihypoglossal nucleus, the vestibular nuclei, the nucleus tractus solitarius, the hypoglossal nucleus, the external cuneate nucleus, an extensive area of the reticular formation around the (left) facial nerve and the periaqueductal grey matter (PAG). In the PAG the labelled cells were mostly found in two columns on either side of the aqueduct.
1521 Days.
The distribution of labelled cells seen at 15 days was almost identical to that seen at 7 days, though there were fewer labelled cells overall. At 15 days the mean labelled cell count was 180 cells per brainstem (range 70280, n=3) and by 21 days it was only 1 or 2. In Fig. 2, the distribution of labelled cells seen at 15 days is shown in blue. By this time-point, in some animals, the virus had also spread to the dorsal reticular formation, the pedunculopontine nucleus and the inferior and superior colliculi. Significantly, the brainstems of animals sampled at 15 and 21 days all contained variable-sized zones of inflammation and vacuolation. The tissue destruction was most marked in regions where the labelling had been found to be especially dense in animals sampled at earlier time-points, particularly in the reticular formation around the left facial nerve and also in the medial part of the left facial nucleus, which was completely destroyed in some animals.
LAT promoter
To identify the sites of LAP activity in the brainstem and spinal cord, animals were inoculated with 2x106 p.f.u. of SC16-LA (in which LacZ expression is driven by the LAP) in the left ear pinna. Animals were sampled at 7, 11, 15, 21 and 34 days p.i. and a second group of seven animals was examined at 1 year p.i. In animals infected with SC16-L
A the labelled cells all appeared to be neuronal, although in brain regions where there had been obvious cellular destruction and inflammation, such as in the spinal cord dorsal columns and along the facial nerve course, non-neuronal cells around the sites of infection sometimes produced a weak pattern of staining. These cells were most likely phagocytic and stained positively because they had engulfed LacZ-positive neuronal debris.
Spinal cord LAT promoter.
The pattern of LAP labelling in the spinal cord from 7 to 21 days was identical to that seen acutely with the CMV-IE promoter (see Fig. 4A, B
). Labelled neurones were present in the dorsal horn, around the central canal, and in the ventral horn. Labelled fibres of latently infected DRG cells could be seen running in the dorsal roots, dorsal horn and left dorsal column. At 34 days, however, most of the labelled cells had disappeared and the only positive cells remaining were motorneurones in the ventral horn.
Brainstem LAT promoter.
Over the 34 days following infection, labelled cells were detected in the brainstem at all the same sites that were labelled by the CMV-IE promoter acutely, including the locus coeruleus, periaqueductal grey, the reticular formation, the perihypoglossal, trigeminal, facial and hypoglossal nuclei (Fig. 4). The anatomical distribution of labelling seen in the brainstem at successive time-points is presented in Fig. 3
. For the first 21 days the numbers of labelled cells increased in each brain region; at 7 days the mean labelled cell count per brainstem was 468 (range 409526, n=3), at 15 days 920 (range 3291885, n=3) and by 21 days (when cell labelling by the CMV-IE promoter had ceased) 1083 (range 10291136, n=3). However, by 34 days p.i. the numbers of labelled cells were greatly reduced. In most of the CNS regions that had contained labelled cells at earlier time-points (perihypoglossal nucleus, the locus coeruleus, periaqueductal grey), only a few weakly staining cells were present (two of three animals). The majority of the labelled cells were motorneurones in the facial (three of three animals) and hypoglossal nuclei (two of three animals), the trigeminal motor nucleus (Vm) and the trochlear nucleus (IV) (one of three animals). In all of these animals, even at this time-point, there were still clear signs of inflammation, suggesting a persistent immune response, perhaps to the LacZ expression driven by the LAP.
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Comparison of sites of CMV promoter and LAT promoter labelling
To demonstrate that the LAP is functional in the same regions of the brainstem as those that are infected acutely (as shown by CMV-IE activity) we superimposed plots of the distributions of CMV-IE-labelled and LAP-labelled cells. There is a very close correspondence between the regions showing CMV-IE activity and regions showing LAP activity for all the time-points studied. As an example, Fig. 5 shows the distribution of labelling seen with the CMV-IE promoter at 7 days (plotted in red) against the distribution of labelling seen with the LAP at 21 days (plotted in blue). However, it must be appreciated that there is some variability between cases which probably reflects the difference in the severity of the infection seen in different animals.
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Finally, to confirm that the pattern of spread through the CNS during acute infection is the same for all the viruses utilized, we used a polyclonal rabbit anti-HSV antiserum to study the distribution of viral antigens in the brains of two animals infected with each virus at 7 days p.i. The same pattern of labelling was seen with all three viruses, indicating that they behave similarly in the way that they spread through the CNS (data not shown).
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Discussion |
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In this paper we have addressed this issue by mapping the spread of HSV-1 from the ear through the spinal cord and brainstem, and comparing the sites of acute infection with those found to support transcriptionally active latency. The spread of virus was traced using SC16-C3b, which expresses the lacZ reporter gene under CMV-IE promoter control. The CMV-IE promoter is active in acutely infected cells, but is silenced during latency when the LAP is active. It is possible that there are areas in the CNS where latency is established without any prior expression from the CMV-IE promoter; however, since LAP activity can be detected in the same CNS regions which are labelled by CMV-IE promoter activity at early time-points, the CMV-IE promoter is probably an accurate marker of the acute phase distribution and spread of the virus. This result also shows that, as the virus spreads transneuronally, some of the infected cells in each brain region become lytically infected, whilst the rest establish latency, as has been observed by others using corneal routes of infection (Deatly et al., 1988 ; Stroop et al., 1984
). On the basis of our cell counts it would appear that far more infected neurones establish latency than enter a lytic round of replication.
Cells labelled by the CMV-IE promoter were detected in the brainstem and spinal cord at 5 days p.i. The infection was restricted to a few sites only, suggesting that the infection had only just reached the CNS at this time-point. This finding is in agreement with previous studies (Kristensson et al., 1982 ; Stroop et al., 1984
; LaVail et al., 1990
; Margolis et al., 1989
) which used corneal and snout routes of infection and in which the first signs of CNS infection were detected at 4 days p.i.
In the spinal cord at 5 days the infection was restricted to the dorsal horn and dorsal columns at the segmental level of entry of the virus. An interesting finding was that the virus had been transmitted to a column of cells in the dorsal horn (seen best in longitudinal section, Fig. 1), which corresponds to the sites of termination of sensory axon collaterals from the neurones innervating the ear pinna. This observation may help to explain the appearance of latently infected DRG cells at segmental levels above and below the level of the primary infection (Speck & Simmons, 1992
, 1991
; Lachmann et al., 1999
). The dorsal horn is organized into continuous overlapping cell columns which receive inputs from DRG cells over the entire long axis of the spinal cord (for review see Noback & Demarest, 1984
; Fitzgerald, 1994
). Interneuronal transmission of the virus rostrally and caudally through the cell column would lead to the infection of sensory nerve terminals of DRG cells which enter (and terminate) at other segmental levels, permitting the establishment of latency at rostral and caudal ganglionic sites. HSV clearly passes trans-synaptically in the spinal cord since, by 7 days p.i., CMV-IE promoter-labelled motorneurones were seen bilaterally in the ventral horn. As there is no direct connection between spinal motorneurones and the mouse ear, the virus must have reached these cells by trans-synaptic transmission via spinal interneurones. In the brainstem a similar hypothesis has been suggested to account for the latent infection of neurones in all three divisions of the trigeminal ganglion following corneal inoculation with HSV-1 (Tullo et al., 1982
).
Another way in which the virus might be spreading to ganglia at other segmental levels is by inter-axonal transmission in the dorsal columns. We frequently found that animals sampled after 7 days p.i. showed lytic damage stretching across the entire dorsal column on the infected side. It is possible that axons from ganglionic levels further down the cord could have become infected en passage from adjacent infected axons and glia. However, there is some question over the ability of HSV to spread past the glial cells which surround each of the axons, as glia are believed to be relatively resistant to such infection (Card et al., 1993 ), although it is known that neuronal cell bodies lying adjacent to infected axons can themselves become infected (Ugolini et al., 1987
).
The LAP was active in the spinal cord from 7 days p.i. Cells labelled by the LAP were found in the same anatomical sites as cells labelled by the CMV-IE promoter, and morphologically, appeared to be the same cell types. The only difference at the 7 day time-point was that the CMV-IE promoter labelled motorneurones in the ventral horn, and these were not found with the LAP until 15 days, consistent with a lag between infection, the establishment of latency, and activation of the LAP. By 21 days no cells were labelled by the CMV-IE promoter, but with the LAP, labelled cells were still present. These findings suggest that the LAP can function, at least until 21 days, in spinal cord interneurones and ventral horn motorneurones.
In the brainstem the CMV-IE promoter labelled cells at 5 days p.i., and showed the widest distribution of labelling at the 7 day time-point. The LAP was active in all of the same anatomical regions from 7 days p.i. In the facial nucleus, however, the acute infection involved only the medial part of the nucleus, yet latency was established in neurones distributed throughout the nucleus. The most likely explanation for this finding is that the virus had spread from lytically infected neuronal cell bodies in the medial part of the nucleus onto the dendrites of cells distributed throughout the nucleus, although an interaxonal model of spread in the facial nerve bundle (as described for the dorsal columns above) is also possible.
A significant finding was that by 34 days p.i. the majority of the labelled cells had disappeared and LAP-driven LacZ expression was only consistently detected in the facial, and occasionally the hypoglossal nuclei, and in a small number of spinal cord motorneurones. This result could be the consequence of immune clearance of neurones expressing the foreign transgene (-Gal). However a similar result was obtained when animals were infected with a wt virus and LAP activity was measured by ISH for LATs. Since LATs are not thought to encode a protein, immune clearance is an unlikely explanation and the evidence argues more strongly in favour of selective activity of the LAP in certain types of neurones, specifically motorneurones. The LAP may function more effectively in (lower) motorneurones as these represent a single class of cells within the CNS which send axons to the periphery and are therefore a potential route by which the virus might spread to other susceptible individuals following reactivation.
Another interesting finding in animals infected with wt SC16 was that we detected (by ISH) many (at least tenfold) more labelled cells in the facial nucleus than were -Gal positive with SC16-L
A. It is possible that this disparity occurs because the immune system is removing some of the SC16-L
A-infected cells because they are expressing a foreign transgene, whilst the wt virus makes only LATs, which do not encode any antigen and hence escape immune surveillance. However, to determine the impact of the immune system in this study would require the use of transgenic mice tolerized to
-Gal. Another explanation for the detection of more LAP-labelled cells in the facial nucleus following wt virus infection might be that the wt virus has an intact LAT region and hence can establish latency in the CNS more efficiently than SC16-L
A. Previous studies have shown that LAT-negative mutants exhibit a reduced frequency of latency establishment in the trigeminal ganglion following corneal inoculation (Sawtell & Thompson, 1992
; Thompson & Sawtell, 1997
), but it is not known whether the LATs aid in the establishment of latency in CNS neurones in the same way. Indeed, it has been shown that the deletion of LATs does not affect latency establishment in lumbrosacral ganglia following footpad infection (Sawtell & Thompson, 1992
), so the role of LATs in the establishment of CNS latency cannot be predicted.
The genome copy number in the latently infected neurone may also play a role in the duration of expression through a template effect. It is possible that the facial nucleus, being a first order site of infection, might have a greater copy number than other subsequently infected sites within the CNS, producing an apparent preferential persistence of expression in this site. To test this hypothesis would require the delivery of known amounts of virus particles to defined cell populations within the CNS, which is best done by stereotaxic injection of a replication-defective virus. We are currently carrying out such experiments to determine whether virus input has a significant consequence for long-term expression in the CNS.
Finally, it has previously been shown that LAP-driven -Gal expression resulted in tenfold more cells positive for
-Gal transcript than cells positive for
-Gal enzyme, suggesting that sequences at the 5' end of the
-Gal transcript influence translation of the
-Gal message (Huang et al., 1997
). Although the lacZ gene used in the current study is preceded by an IRES element to circumvent such translational difficulties, further studies are required to examine
-Gal gene expression both at the RNA and protein levels to determine the efficiency of
-Gal translation in our system.
In conclusion, we have shown that following ear inoculation with HSV-1 the virus spreads to a reproducible series of sites in the CNS. By using a virus (SC16-LA) in which lacZ is driven by the LAP we have detected
-Gal expression in all of the same sites in the CNS that were accessed by the virus acutely and also in which LATs were detected by ISH following wt virus infection. These data demonstrate that the LAP can function for at least 21 days in many, if not all, CNS neuronal cell types in the brainstem and spinal cord. With the passage of time, however, there was a reduction in the distribution of labelling, and LAP-driven reporter gene expression was detected predominantly in the facial nucleus and sometimes also in the hypoglossal nucleus and spinal motorneurones. A similar result was obtained following wt virus infection, in which LAP activity was detected by ISH for LATs. These findings suggest that the preferential long-term activity of the LAP in motorneurones is probably not a consequence of immune clearance but instead that the LAP may function selectively, or at least more efficiently in some CNS neurones than others.
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Acknowledgments |
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References |
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Received 29 September 1999;
accepted 29 November 1999.