VIROLOGY

181,

793-797 (1991)

Detection and Characterization of Latent HSV RNA by in Situ and Northern Blot Hybridization in Guinea Pigs RAE LYN BURKE, * " KARIN HARTOG, * KENNETH D . OROEN,t ,2 AND JEFFREY M . OSTROVEt' 3 *The

Chiron

Corporation, Emeryville, California ; the

IMedical Virology

Received October 8,

1990;

Section, N/AID, National Institutes of Health, Bethesda, Maryland

accepted December 21,

1990

Following intravaginal infection of guinea pigs, herpes simplex virus establishes a latent infection in the sensory lumbosacral ganglia . Using the techniques of in situ and Northern blot hybridization, we have characterized this latent HSV-2 virus and compared it to latent HSV-1 at the same anatomical site . For HSV-2, a single 1 .8-kb latency-associated transcript (LAT) was detected . In contrast, as described for latent HSV-1 in the trigeminal ganglia of rabbits and mice, two HSV-1 LAT species were detected in the lumbosacral ganglia, an abundant transcript of 1 .8 kb and a less abundant transcript of 1 .55 kb . Despite these differences in LAT expression, the clinical course of the acute and recurrent genital disease was similar for both viruses. LAT was detected in 0 .3-6 .0% of the sensory neurons of sacral but not in lumbar ganglia . The abundance of LAT correlated with the severity of the initial infection, but not with the frequency of recurrent disease . Thus, vaccination strategies that substantially reduced or eliminated symptomatic disease following challenge infection appeared to block the establishment of a latent infection ®1991 Academic Press, Inc . Numerous animal models have been developed to study the immune and biologic effects of herpes simplex virus infections . While none of these models truly mimic the recurrent infections seen in man, the guinea pig may come the closest in that animals inoculated intravaginally develop a severe primary infection of the genital skin . Following resolution of the acute disease, animals develop spontaneously recurrent vesicular lesions of more moderate severity (1, 2) . To determine if latent HSV-2 infection of guinea pigs involves the expression of the latency-associated transcripts (LATs) described previously for HSV-1 from the trigeminal ganglia of mice (3-5), rabbits (6), and humans (7) we used the techniques of in situ and Northern blot hybridization . A single region of the genome is transcribed during latent HSV-1 infection (4-7) producing poly (A) transcripts of 1 .8 and 1 .4 kb localized to the nucleus of the ganglionic cell (4, 8, 9) . The smaller transcript is spliced from the larger (9) and their relative abundance varies with virus strain and animal species (9) . For HSV-2 in the trigeminal ganglia of mice, a single LAT transcript of 2 .3 kb was detected (10) . Although these two viruses have substantial sequence homology, there are marked differences in the recurrence pattern of human infection according to the latent virus type and the anatomical site of latency (11) . Thus, it is important to characterize each latent virus isolated from its predominant latent lair .

'

Northern blot analysis of HS V-2 LA T RNA from vaccinated guinea pigs . Fifteen guinea pigs immunized with recombinant HSV subunit vaccines (12) were infected intravaginally with HSV-2 strain MS . The severity of the acute viral infection and the frequency of recurrent disease following resolution of the primary infection was recorded for each animal (2) . Twenty weeks post infection, the seven pairs of lumbosacral ganglia (L4-L7 and S1-S3) were removed from each animal . Total RNA was prepared from the pooled ganglia of individual animals (13) for Northern blot analysis . The blot was probed with the 3 .9-kb BamHl HSV-2 fragment of plasmid pG-Bam5 encompassing both the LAT and ICPO genes (14) . As shown in Fig . 1 A, a major 1 .85-kb RNA species was detected in latently infected guinea pigs (lanes S, F, and M) but not in asymptomatic (lanes A) or uninfected guinea pigs (lanes U) . Hybridization of RNA prepared from the HSV-1 -infected cells (lane I) revealed two HSV-specific bands, the ICPO transcript at 2 .7 kb and the LAT transcript at 1 .85 kb . The hybridization signal for HSV-1 LAT from lytically infected cells was much weaker than the ICPO band due to the lower intertypic homology of LAT compared to ICPO . No smaller LAT was detected in HSV-2 guinea pig ganglionic RNA (or in HSV-1 lytically infected Vero cells) even with very long exposure times (data not shown) . HSV-2 LAT transcripts differ from those of Hi 1 . To determine if the absence of the smaller 1 .4-kb spliced latent transcript in HSV-2-infected guinea pigs is characteristic of HSV-2, or of the guinea pig animal model or the anatomical site of latency, four guinea pigs were infected intravaginally with HSV-1 strain 17 syn` . The clinical course of HSV-1 disease was indistinguishable from that of HSV-2 with acute infection followed by

reprint requests should be addressed . Current address : Division of Infectious Diseases, University of Cincinnati, Cincinnati, OH . 3 Current address : Microbiological Associates, 9900 Blackwell Road, Rockville, MD 20850 . To whom

s

793

0042-6822/91 $3 .00 Copyright® 1991 by Academic Press, Inc . Alt rights of reproduction in any form reserved.



794

A

SHORT COMMUNICATIONS

1 S

2 3 4 5 6 F M A U U

7 I

Lane Class

- 28S ICP0 1 8S LAT

FIG . 1A . Northern blot of RNA prepared from the lumbcsacral ganglia of individual guinea pigs latently infected with HSV-2 . Total RNA was prepared from ganglia of individual animals with different clinical disease profiles (Table 1) as follows : S, animals with a severe acute HSV disease ; F, animals with a high frequency of recurrent disease ; M, animals with moderate frequency of recurrent disease ; A, animals with an asymptomatic infection ; U, uninfected animals ; I, 5 µg total RNA from HSV-1 lytically infected Vero cells . The blot was probed with the 3 .9-kb BamHl fragment of plasmid pG-BamS (B5) from the IR region of HSV-2 (map units 0 .773-0 .800).

more mild recurrent disease . Eight weeks postinfection seven pairs of lumbosacral ganglia were removed for the preparation of RNA . Northern blot analysis of these RNA preparations revealed two distinct LAT transcripts at a ratio of about 1 :50 (Fig . 1 B), an abundant 1 .85-kb transcript and a second 1 .55-kb transcript as described for latent HSV-1 in the trigeminal ganglia of mice (3-5), rabbits (6), and humans (7) . Thus, neither the choice of animal model nor differences between trigeminal and sacral ganglia seem to change the HSV-1 LAT expression pattern, suggesting that the virus type dictates LAT expression . Also, it is apparent that both HSV-1 and HSV-2 cause similar patterns of spontaneous recurrent genital disease in guinea pigs despite the differences in their patterns of LAT expression . Correlation of LA T with disease expression . To determine if there was any correlation between the abundance of LAT and the severity of the initial infection or the frequency of recurrent disease, animals were divided into two categories, those that experienced a very severe primary infection (S, lesion score = 2 .43 ± 0 .24, N = 4) and those that were clearly infected but had little primary disease (L, lesion score = 0 .29

± 0 .54, N = 9) . As shown in Table 1, there was a trend such that animals with more severe disease had more LAT RNA . For the second comparison, those animals with frequent recurrent disease (12 .6 ± 2 .7 lesion days, N = 5) versus those with less frequent recurrences (3 .5 ± 1 .3 lesion days, N = 4), there was no apparent difference in the abundance of LAT . No LAT was detected in 2/3 animals that were asymptomatic during the entire observation period (data shown for 1 animal in Fig . 1 A, lane A), and also in 4/4 uninfected, control animals (data shown for 2 animals in Fig . 1A, lanes U) . Detection and characterization of latent HSVin vaccinated guinea pigs by in situ hybridization . In a second experiment, a series of 19 guinea pigs immunized with HSV vaccines (12) and 5 control, unimmunized animals were infected with HSV-2 as for Experiment 1 . In situ hybridization was performed as described (7, 15) on lumbosacral ganglia removed 10 weeks after viral infection from all animals listed in Table 2 . The HSV-1 and HSV-2 strand-specific RNA probes [the HSV-1 786-bp Sphl fragment of BamHI E and the HSV-2 BamHl subclone of pGR90, pG-BamS cloned in pGEM4Z transcription vectors (15)] were used to detect both the immediate early gene transcript ICPO (a marker for active infection) or LAT (a marker for latent virus) . LAT was detected in the lumbosacral ganglia of 4/19 immunized animals, 5/5 untreated control ani-

B 1

2 3 4 5 6

Lane

FIG . 1 B . Northern blot of RNA prepared from the lumbosacral ganglia of individual guinea pigs latently infected with HSV-1 . Lanes 1, 2, 3 : total RNA from ganglia of individual animals latently infected ; lane 4 : total RNA from an uninfected guinea pig ; lane 5 : 10 µg total RNA from HSV-1 lytically infected Vero cells ; lane 6 : 5 µg total RNA from uninfected Vero cells . The blot was probed with the 786-top Sphl fragment from the BamHl E fragment from the IR region of HSV-1 .



795

SHORT COMMUNICATIONS TABLE 1 EXPERIMENT 1 : CLINICAL COURSE OF ACUTE AND RECURRENT HSV-2 DISEASE IN GUINEA PIGS AND CORRELATION WITH LAT EXPRESSION

Acute disease mean lesion Animal

Severity`

Score'

Recurrent disease lesion days`

LAT relative intensity °

1° S S S S

2-

3394 3405 3442 3448 3484 Mean

L L L L L

F F F F F

0 .00 0 .21 1 .46 0 .00 0 .00

12 13 16 11 10 12 .6±2 .7N=5

4 30 3 .2 2 3

3383 3395 3443 3480 Mean

L L L L

M M M M

0 .00 0 .93 0 .00 0 .00 0 .29± .54N=9

5 4 3 3 3 .5±1 .3N=4

4 2 .5 12 2 7 .0 ± 9 .2 N = 9

5879 5926 5932 5879 Mean

1 .4 30 20 30 12 .8 ± 14 .6 N = 4

2 .27 2 .75 2 .46 2 .25 2 .43 ± 0 .24 N = 4

° Severity of acute infection, 1 ° (Days 1-14 postinfection) or spontaneous recurrent disease, 2 0 (Days 15-74 postinfection) . S, severe 1 ° disease; L, little 1 ° disease ; F, frequent recurrent disease (10-17 lesion days) ; M, moderate recurrent disease (3-4 lesion days) . Animals with severe primary disease developed severe scarring of the perineum, precluding scoring for recurrent disease . 'Average of the sum of the daily lesion scores (12) divided by the number of observation days . Daily lesion scores range from 0, corresponding to no lesions, to 4, corresponding to virtually confluent vesicles on the genital skin or death (2) . Days that a vesicular lesion was apparent on the genital skin during the observation period Days 15-74 postinfection . ° The relative intensity of the LAT signal was determined for each Northern blot as a ratio of the LAT autoradiographic signal to the total RNA load as determined by ethidium bromide staining and by the autoradiographic signal to a neurofilament-specific probe (26) .

mals, and 0/6 uninfected animals . No LAT was detected in animals that did not develop cutaneous lesions even though a low level of replicating virus was recovered from vaginal swabs collected 3 days postinfection in 3 of 4 such animals . Sacral (S1-S3) and lumbar (L4-L7) ganglia were separately analyzed for the presence of latent virus by in situ hybridization . LAT was detected only in the sacral ganglia (7/14) and not in the lumbar ganglia (0/42) . Within the ganglionic cells, expression of LATwas exclusively seen within the neuron and appeared limited to the nucleus of the cell (data not shown) . Earlier studies by Scriba (16) and Meignier at aL (17) reported the majority of latent virus in guinea pigs similarly infected, was recovered by explant cocultivation from S1-S3 but not L3-L6 . Between 0 .3 and 6% of the neuronal cells harbored latent HSV-2 . No hybridization was detected using a probe complementary to ICPO RNA . We have previously reported the complete protection of guinea pigs from HSV disease by immunization with a mixture of glycoproteins purified from viral infected cells, combined with CFA and delivered into the

footpad (18) and the same high level of protection using a single recombinant glycoprotein combined with a muramyl tripeptide as adjuvant and delivered intramuscularly, the latter being a vaccine composition and mode of administration suitable for human clinical trials (19) . An important aspect of these studies is the impact of vaccination on the establishment of a latent infection . Do vaccinated animals that are asymptomatic after viral inoculation still harbor latent virus in their sensory ganglia? In this study we detected latent virus in 4/19 immunized animals, 5/5 untreated, infected control animals, and 0/6 uninfected animals . From these results it appears that the most efficacious vaccination regimens certainly reduce the number of latently infected neurons in the sensory ganglia and in some cases may completely block the establishment of latency . The biological role of LAT is not clear . HSV-1 deletion mutants that are unable to express LAT establish latency in the mouse trigeminal ganglia and can be recovered by explant cocultivation (20-22), although the frequency of such reactivation or recovery may be



796

SHORT COMMUNICATIONS TABLE 2 EXPERIMENT 2 : CLINICAL COURSE

Group

Antigen/adjuvant route'

1

gDl/MTP-PE/FP

2

gDl/MTP-PE/IM

3

4

gD1/CFA/FP

g82 + gD2/MTP-PE/IM

5

Control (unimmunized, infected)

6

Control (unimmunized, uninfected)

OF

THE ACUTE HSV DISEASE AND DETECTION

Animal' 1015, 1022, 1023, 1082, 1084, 1085, 1046, 1050, 1053, 14252 1427 2 1429, 14302 1507 2 1510 2 1512 2 1513 2 1514 2 1516 2 1064, 1066, 1069, 1523 2 1570 2 1071, 1073, 1075, 1573 2 1574 2 1576 2

OF

LAT

LAT RNA

Mean lesion score'

Vaginal virus titer°

Lesion days'

+

0 .50 0 .07 0 .11 0 .00 0 .68 0 .07 0 .00 0 .00 0 .00 0 .00 007 0 .00 0 .00 0 .57 0 .25 1 .21 0 .00 0 .00 0 .25 1 .36 2 .00 2 .00 1 .71 2 .21 NA" NA NA NA NA NA

385 1,272 712 639 8,235 1,152 3,267 0 32 NT NT NT NT NT NT NT NT NT NT 1,782 5 .193 37,980 NT NT NA NA NA NA NA NA

NT' NT NT NT NT NT NT NT NT 0 3 0 0 2 3 4 0 0 4 NS9 NS NS NS NS NA NA NA NA NA NA

+

+ + + + + + -

a Animals were immunized with three 6-µg doses of the indicated antigen combined with MTP-PE, a muramyl tripeptide, or CFA, complete Freunds adjuvant, as adjuvant and administered by the footpad (FP) or the intramuscular (IM) route . ° In situ probed with HSV-1, subscript 1, or HSV-2, subscript 2, LAT probe . `Average of the sum of the daily lesion scores divided by the number of observation days . ° HSV-2 titer in vaginal swab samples collected 3 days postinfection . ° Days recurrent vesicles observed during the observation period, Days 15-30 postinfection . 'Not tested . s Not scored . The primary infection was very severe in these animals such that the perineum was permanently scarred, precluding scoring for recurrent disease . " Not applicable .

reduced (23) or delayed (24) . One LAT deletion mutant failed to reactivate efficiently in vivo in a rabbit eye model (25) . The present studies characterize latent HSV-2 in the guinea pig genital herpes model . This model could be very useful for additional studies on the in vivo role of LAT gene expression . ACKNOWLEDGMENTS We thank Dr . Stephen E . Straus for continued support and helpful discussions and Jeffrey Neidlema n for help with the manuscript preparation . This study was supported in part by the Biocine Company .

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