Vol. 23, No. 2INFECTION AND IMMUNITY, Feb. 1979, p. 373-3830019-9567/79/02-0373/11$02.00/0

Route of Infection, Systemic Host Resistance, and Integrity ofGanglionic Axons Influence Acute and Latent Herpes Simplex

Virus Infection of the Superior Cervical GanglionRICHARD W. PRICE,2 * AND JONATHAN SCHMITZ2

George C. Cotzias Laboratory of Neuro-Oncology and Department of Neurology, Memorial Sloan-KetteringCancer Center, and Department of Neurology, Cornell University Medical College,2 New York, New York

10021

Received for publication 20 November 1978

The character of acute and latent herpes simplex virus (HSV) infection of thesuperior cervical ganglion (SCG) in mice depended on the route by which thevirus reached the ganglion, the level of systemic host resistance, and the integrityof postganglionic nerves. Prevention of ganglionic infection by postganglionicneurectomy carried out before intraocular (i.o.) virus challenge established theimportance of the neural route in the development of SCG infection. However,hematogenous virus dissemination also led to SCG infection although withreduced frequency compared to that with i.o. inoculation. Enhanced host systemicantiviral resistance had two divergent effects on ganglionic infection dependingon the dose and timing of virus inoculation. Thus, both acute and latent ganglionicinfections were concomitantly reduced when resistant C57B1/6 mice were chal-lenged with low doses of virus or when less resistant BALB/c mice were activelyimmunized 1 week before virus challenge. On the other hand, when resistant micewere challenged with high doses of virus or when either active or passive(antibody) immunization was delayed long enough to assure viral access to theganglion, intraganglionic viral replication during the acute phase of infection wasreduced, but the prevalence of subsequent latent infection was either unaffectedor actually enhanced. Postganglionic neurectomy, performed after virus hadreached the ganglion, altered the course of SCG infection in a direction oppositethat of immunization, augmenting the acute phase of viral replication whilereducing latency. In athymic nude mice and mice immunosuppressed with cyclo-phosphamide, intraganglionic viral replication was prolonged. These results em-

phasize that host factors both extrinsic and intrinsic to the SCG modify thecourse of ganglionic infection.

Herpes simplex viruses (HSV) types 1 and 2,are exquisitely adapted for survival in the hu-man community. Once these viruses infect thehuman host, they are capable of sustaining aprolonged, perhaps lifelong, latent infection. La-tent virus may then periodically reactivate andbe shed at epithelial surfaces or into externalsecretions where it is available to infect suscep-tible contacts. These three stages of HSV infec-tion, i.e. primary infection, latency, and recur-rence, are evidence of a variability in the virus-host relationship. Initial invasion is accompa-nied by a productive infection resulting in asufficient quantity and distribution of viral prog-eny to allow the virus to reach the site(s) ofeventual latency. Productive infection then givesway to the latent stage, during which virus eitherno longer replicates or replicates in insignificantamounts. Finally, recurrent infection indicates aswitch back to an active productive phase with

release of HSV in amounts sufficient to infectcontacts. Host responses are important in deter-mining the course of each phase of infection andpresumably fall within a limited range for bothhost and virus to survive. If the host is toorestrictive, initial infection will not occur or re-currence will be aborted. On the other hand, ifthe host fails to restrict virus replication, it willbe overwhelmed, and the period during whichinfection is passed on will be markedly abbrevi-ated. A number of recent studies have providedsubstantial support for the hypothesis that sen-sory ganglia of the nervous system play a centralrole in latent and reactivated HSV infections (2,13). Sensory ganglia have been shown to harborlatent HSV types 1 and 2 in both experimentallyinfected animals (14, 17) and in humans (1, 3).To explore those factors which modify or reg-

ulate the course of HSV infection, we have em-ployed an experimental model of infection of the

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superior cervical ganglion (SCG). When the an-terior chamber of the mouse eye is inoculatedwith HSV, infection of the ipsilateral SCG en-sues. This ganglion, located in the neck adjacentto the bifurcation of the common carotid artery,is part of the sympathetic division of the auto-nomic nervous system and supplies postgan-glionic nerve fibers to target organs in the head,including the iris within the anterior chamber ofthe eye. Infection of this sympathetic ganglionresembles in all respects thus far tested experi-mental infection ofsensory ganglia (10, 12). Aftereye inoculation, an acute infection of the SCG isproduced, during which active viral replicationtakes place and HSV can be detected in homog-enates of the ganglion. This acute phase subsideswithin 7 to 10 days, and, thereafter, the conven-tional homogenization assay no longer allowsrecovery of virus. However, the continued pres-ence of latent HSV can be demonstrated whenexplantation and co-cultivation methods areused for virus detection. Latent virus can bereactivated in vivo when postganglionic nervefibers emanating from the SCG are surgicallysevered (12). Recently, HSV has been recoveredfrom the SCG of humans (18) supporting thesuggestion that the autonomic nervous systemmay participate in the pathogenesis of humanherpetic infection (11).We now describe a series of experiments which

demonstrate that the character of ganglionicinfection depends upon the route by which virusreaches the ganglion, systemic host resistance toHSV, and the integrity of postganglionic nerves.

MATERIALS AND METHODSTissue culture and media. Rabbit kidney (RK)

cells were used either as primary cell monolayers orafter one or two passages, whereas human embryoniclung (HEL) cells were obtained commercially (Micro-biological Associates, Rockville, Md.) and used be-tween passages 17 and 26. Cultures were maintainedas previously described (12).

Virus propagation and inoculation. The F strainof HSV type 1 was prepared on RK monolayers (6)with the stock virus pool for inoculation containing 2x 107 plaque-forming units (PFU) of HSV/ml. Intra-ocular (i.o.) inoculation was carried out as previouslydescribed (12); unless otherwise specified, mice re-ceived 4 pl of undiluted virus stock pool i.o. on theright side.

Animals. With the exception of two experiments,4- to 6-week-old BALB/c female mice, purchased fromCharles River Breeding Laboratories, Inc., Wilming-ton, Mass., were used for these studies. C57B1/6 miceand the BALB/c mice used in the experiment com-paring these two mouse strains were simultaneouslypurchased from Jackson Laboratories, Bar Harbor,Maine; both strains were received at 6 weeks of ageand used when 10 weeks old. Nude mice (nu/nu) andheterozygous littermates (nu/+), obtained from theSloan-Kettering Institute Animal Care Facility, were

used at 3 to 5 weeks of age and possessed an ICRgenetic background.

Preparation of antisera and determination ofserum antibody titers. Anti-HSV antiserum wasprepared in both BALB/c mice and rabbits. Mouseantiserum was induced by two intraperitoneal (i.p.)injections of 2 x 105 PFU of live HSV suspended in0.2 ml of Dulbecco phosphate-buffered saline (PBS)spaced 2 weeks apart. Mice were bled 10 to 14 daysafter the second injection. Rabbit antiserum was ob-tained by using a similar schedule with intradermalinjections of undiluted stock virus pool for induction.Serum neutralizing antibody titers, determined usinga plaque-reduction assay (16), were expressed as thereciprocal of the highest serum dilution producing a50% reduction in viral plaques.Assay of ganglia and eyes for virus. SCG were

analyzed for HSV by either homogenization or explan-tation-co-cultivation techniques as previously de-scribed (12). Viral titers of ganglion homogenates weredetermined using RK cell monolayers and results wereexpressed as logo mean viral titers (sum of logo viraltiters of individual ganglia per number of ganglia as-sayed); in cases where ganglia were negative for virus,the convention that 1 PFU of HSV logoo value of 0)was detected in these ganglia was employed in calcu-lating mean values. Significance of differences in viraltiters was assessed by the Student t test. For theexplantation-co-cultivation assay, ganglia were placedon HEL monolayers and monitored for 3 weeks for theappearance of HSV-induced cytopathology. Results ofthis assay were presented as a ratio (the number ofganglion explants positive for virus per number ofganglia assayed), and the significance of results wereassessed by the chi-square contingency method or,when the number of ganglia was less than 20, by theFisher exact test. Viral titration of eye homogenateswas carried out in a manner similar to that used forganglia. In brief, eyes were washed three times in PBScontaining antibiotics, homogenized with a TenBroeck tissue grinder, frozen and thawed three times,and, after centrifugation at 5,000 rpm for 10 min, HSVtiters of the supernatants were determined by usingRK monolayers.

Surgery. All neurectomies and sham operationswere performed aseptically under pentobarbital anes-thesia with the aid of a dissecting microscope.

RESULTSEffect of postganglionic nerve section on

the acquisition of SCG infection. To deter-mine whether HSV reaches the SCG over neuralpathways after inoculation of the anterior cham-ber of the eye, mice were subjected to postgan-glionic nerve section 1 day before i.o. HSV injec-tion. Under ether anesthesia, the three majorpostganglionic nerve fibers emanating from theright SCG were severed; the efficacy of operationwas judged by the appearance of eyelid ptosison the operated side. On the day after surgery,the neurectomized mice, along with unoperatedcontrol mice, were infected by i.o. HSV inocula-tion of the right eye. Mice were then sacrificed

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for ganglion assay both at the height of the acuteinfection (day 5) and during the latent phase ofinfection (day 21). The ipsilateral (right) SCG ofmice sacrificed during the acute phase of infec-tion were assayed for HSV by homogenization,whereas ganglia of mice sacrificed during thelatent phase were assayed by explantation-co-cultivation. The data in Table 1 show that post-ganglionic neurectomy almost completely pre-vented ganglionic infection. While in the controlgroup during the acute phase of the infectionganglion homogenates of all 13 animals con-

tained HSV with a mean viral titer approaching2.9 x 103 PFU per ganglion, in none of theneurectomized mice was HSV detected in gan-glion homogenates. Similarly, during the latentphase in only 1 of 11 SCG explants in the neu-

rectomized group was HSV detected, comparedto 10 of 15 ganglia in the control group.The most likely explanation for these results

is that the postganglionic nerves serve as themajor route of virus passage from the eye to theSCG. An alternative explanation is that viruscirculating in the bloodstream preferentially in-fects the non-operated ganglion, i.e., that post-ganglionic neurectomy inhibits virus uptake andsubsequent replication or latency. To test thispossibility and also to assess the efficiency withwhich hematogenously disseminated virus in-fects the SCG, mice were subjected to unilateral(right) postganglionic neurectomy and inocu-lated intravenously (i.v.) (tail vein) with 1.0 x

106 PFU of virus diluted in 0.2 ml of PBS; boththe right (ipsilateral to neurectomy) and the left(nonoperated) SCG were then assayed for HSV.Table 2 summaizes the results of three experi-ments in which mice were neurectomized atvarious intervals from 21 days to 1 day beforei.v. virus inoculation; the animals were sacrificedeither 5 days (preliminary results indicated thatthis was the optimal time to detect virus in the

TABLE 1. Effect ofpostganglionic neurectomybefore inoculation of the ipsilateral eye on the

acquisition of acute and latent HSV infection of theSCG

Acute phase (ho-mogenates)

Ratio of la-Group Mean tent phase

Ration titer explants(PFU,

logO)bUnoperated con- 13/13 3.5 10/15

trolsPostganglionic 0/12 0 1/11neurectomya p < 0.001.bP < 0.001.P< 0.01.

HSV INFECTION OF THE SCG 375

TABLE 2. Acute and latent SCG infection after i.v.HSV inoculationa

Acute phase Latent

Ganglion ayed Homo- phase

genatesb Explants (explants)'Left (unoperated) 2/49 13/51 2/31Right (postgan- 2/49 15/52 2/31

glionic neurec-tomy)

a Results all express the ratio of the number ofganglia positive for HSV per number of ganglia as-sayed. The table gives the combined results of threeexperiments (see text).

b Quantitative assay of viral titers in positive gan-glion homogenates revealed 1 PFU and 30 PFU in theleft-sided ganglia and 2 PFU and 33 PFU in the right-sided ganglia. These ganglia were from four differentmice.

'Positive ganglion explants were obtained fromthree mice; in one of these both ganglia were positive,whereas in single animals only the right or only theleft ganglia was positive for HSV.

SCG after i.v. injection either by homogeniza-tion or explantation) or 21 days after virus injec-tion. As the data in the table show, there was nodifference in the acquisition of infection betweenthe neurectomized and non-neurectomized gan-glia. During the acute phase, SCG homogenateswere negative in all but approximately 4% of theganglia, and in those ganglia in which HSV wasdetected, the viral titers were low, ranging from1 to 33 PFU per ganglion. Although the moresensitive explant method detected HSV in be-tween 25 and 29% of explants during the acutephase, the prevalence of detectable latent infec-tion fell to less than 7% of ganglia in both groupsby 21 days, indicating that the efficiency ofestablishing latent infection by the i.v. route wasfar lower than that following ipsilateral i.o. virusinjection.Latent SCG infection in C57B1/6 mice

compared with that in BALB/c mice. Themorbidity and mortality of mice subjected tosystemic HSV infection varies among inbredmouse strains (9). To determine whether geneticdifferences in systemic resistance to virus influ-ence the development of latent ganglionic infec-tion, SCG infection of C57B1/6 mice, a resistantstrain, was compared to infection in BALB/cmice, the strain used in previous studies that ismoderately susceptible to systemic HSV infec-tion (9). Because resistance to infection changesrapidly with age in young mice, 10-week-oldanimals were used for this study. Female miceof the two strains were received, housed, in-fected, and sacrificed, and ganglia were assayedat the same time to minimize variables thatmight arise if the groups had been tested sepa-

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376 PRICE AND SCHMITZ

rately. To compare the development of latentganglionic infection, mice of both strains were

inoculated i.o. with from 8 to 8 x 105 PFU ofHSV suspended in 4 pl of PBS, and 3 weekslater ganglia ipsilateral to the inoculated eye

were assayed for latent HSV by explantation-co-cultivation. The data in Fig. 1 demonstrate thatalthough both mouse strains were capable ofsupporting a latent SCG infection, the dose-re-sponse curves of the two strains were quite dif-ferent. In the case of the C57B1/6 mice, theanimals were resistant to the development oflatency at lower doses of virus inoculum, butthere was a steady increase in the percentage ofmice harboring latent infection as the dose in-creased, and maximum prevalence of latencyoccurred in the group receiving the highest virusdose. In contrast, latent infection of BALB/cmice developed at a lower dose (e.g., the 40%infection rate in the BALB/c mice occurred ata virus dose of less than one one hundredth ofthat inducing a similar rate in the C57B1/6mice). In addition, the highest frequency of la-tent infection was detected in BALB/c miceinjected with a relatively low concentration ofvirus (8 x 102 PFU), whereas higher concentra-tions of virus inoculum led to progressively lowerrates of latency.

loor o0-wo C57 BL/6*--- BALB/c

80F

qua

-7Q

(A

c

60H

40F

20h

8xl60 8xlOl 8xlO2 8x1o0 8xl1 8x10Virus inoculum (PFU)

FIG. 1. Comparison of latent SCG infection inC57B1/6 and BALB/c mice. Mice of both strainswere inoculated i.o. with the dilutions ofHSVshownsuspended in 4 ul ofPBS and were sacrificed 3 weekslater. Each point represents the results of explanta-tion of ipsilateral ganglia from nine or more mice.

To determine if the extent of HSV replicationeither in the SCG or at the site of virus inocula-tion during the acute phase of infection corre-lated with the subsequent development of latentganglionic infection, viral titers of homogenatesof the SCG as well as of the injected eyes ofBALB/c and C57B1/6 mice inoculated with thehighest dose of virus (8 x 105 PFU) were com-

pared during the acute phase of infection. Asshown in Table 3, the level of virus replicationin the two mouse strains during the acute phaseof infection differed, and, in fact, correlated in-versely with the subsequent development of la-tency. Viral titers of ganglia and eyes fromBALB/c mice were more than 10 times higherthan titers of the same organs of C57B1/6 mice,indicating that interference with viral replicationdid not account for the lower prevalence of la-tency at high doses in the BALB/c mice.

Effect of active immunization before i.o.virus challenge on the development of la-tent SCG infection. To explore further theeffect of systemic viral resistance on both thedevelopment of latent infection and on thecourse of the acute lytic phase of the infection,a series of manipulations of anti-HSV immunityin BALB/c mice was carried out. In the first ofthese studies, the effect of active immunizationwith live HSV, given i.p. at various intervalsbefore virus challenge, was assessed. Initial stud-ies showed that when mice were actively im-munized by i.p. injection of 4 x 105 PFU of HSV7 days before virus challenge, latent SCG infec-tion was completely prevented with none of 13explants positive for HSV; this agrees with theresults of previous observations (10) and alsoindicates that i.p. virus inoculation is unlikely toitself lead to latent SCG infection. Immunization1 or 3 days before i.o. challenge, on the otherhand, failed to reduce the prevalence of latencycompared to that in unimmunized controls with55 to 60% of explants positive for virus in eachgroup. Further studies were then carried out toassess whether similar active immunization

TABLE 3. Comparison of viral titers of inoculatedeyes and ipsilateral SCG ofBALBIc and C57B1/6

mice during the acute phase of infection'Mean tissue titers (acute phase)

Mouse strain (PFU, logo)Eyes' SCGe

BALB/c 5.4 3.0C57BI/6 4.2 1.1

Mice were sacrificed 5 days after i.o. inoculation.Results express the mean values of eye and SCGhomogenates from five animals of each mouse strain.bp< 0.02.cP< 0.02.

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shortly before i.o. HSV challenge would influ-ence the course of the acute phase of ganglionicinfection. The data in Table 4 indicate that whileactive immunization before i.o. virus challengeagain did not reduce the subsequent prevalenceof latency, immunization effectively reduced theextent ofvirus replication during the acute phaseof infection by over 100-fold compared to theunimmunized controls.Effect ofpassive immunization with anti-

HSV antibody on the development oflatentSCG infection. Similar studies assessing theinfluence of passively administered antiviral an-tibody on acute and latent infection were nextundertaken. BALB/c mice were immunized byi.p. injection of0.2 ml ofimmune BALB/c mouseserum possessing an anti-HSV neutralizing an-tibody titer of >1,024. Mice receiving serum 1day before, 1 day after, and 3 days after right-sided i.o. HSV challenge were compared to un-immunized control animals. As shown in Fig. 2,during the acute phase of infection, mean viraltiters in ipsilateral ganglion homogenates of con-trol animals (group D) were high, approaching1.0 x 104 PFU per ganglion. In each of theantibody-treated groups viral titers of ganglionhomogenates were reduced. Greatest reductionoccurred in mice receiving immune serum 1 dayafter virus challenge (group B) with lesser re-duction in groups A and C. In contrast to theresults of ganglion assay during the acute phaseof infection, when latent infection in thesegroups was assessed by explantation, the highestprevalence of latency was found in group B, withprogressively lower rates of latent infection ingroups A, C, and D. Thus, an inverse relationwas observed between viral titers of ganglion

TABLE 4. Effect of active immunization by i.p.injection ofHSV I day before i.o. virus challenge on

acute and latent SCG infection'Acute phase (homog-

enates) Ratio of la-

Group Mean ti- tent phaseRatioh ter (PFU, explants'

logO)cUnimmunized 16/17 3.6 17/20Immunized 3/18 1.3 20/20

'Mice were sacrificed on day 5 after i.o. HSV in-oculation during the acute phase of infection and onday 28 after Lo. inoculation during the latent phase ofinfection. Immunized mice received 4 x i05 PFU ofHSV i.p. 1 day before io. virus challenge while unim-munized controls were injected i.p. with PBS on thesame schedule.

11 P < 0.001.CP<0.001.d Not significant.

HSV INFECTION OF THE SCG 377

I ACUTE PHASE

4-

i2,3bH LATENT PHASE

at

c

CL

co0o

1O°r

50s

A B C DI doy before Idoyafter 3 doys after control:challenge challenge challenge no antibodyTiming of Passive Antibody Administration

FIG. 2. Effect on the development of acute andlatent SCG infection of passive immunization withmouse anti-HSV antibody administered at varioustime intervals. Mice were injected i.p. with anti-HSVantibody I day before (group A), 1 day after (groupB), and 3 days after (group C) i.o. virus challenge;group D did not receive antibody. (I) Acute phase:Mice were sacrificed on day 5, and viral titers inganglion homogenates were determined. The col-umns depict the mean viral titers and standard errorofthe mean, whereas the numbers within the columnsgive the number ofganglion homogenates positive forHSVper number ofganglia assayed. Significance: Avs. D, P < 0.01; B vs. C, P < 0.05; B vs. D, P <0.001;C vs. D, P < 0.02; other comparisons not significant.(II) Latent phase: Mice were sacrificed 21 days afterviral challenge, and ganglia were assayed by ex-plantation-co-cultivation. Columns depict the per-centage of ganglion explants positive for HSV,whereas the numbers within the columns give theactual number ofganglion explants positive for HSVper number ofganglia assayed. Significance: A vs. C,P < 0.05; A vs. D, P < 0.01; B vs. C, P < 0.01; B vs D,P < 0.01; other comparisons not significant.

homogenates acutely and the subsequent prev-alence of latency.The suppression of viral replication and en-

hancement of latent infection by passively ad-ministered antibody were reproducible whenlarger numbers of animals were tested and whenhigher titer antiserum produced in rabbits wasused. The data in Table 5 show that rabbit anti-HSV serum administration (0.2 ml of serum i.p.with a neutralizing titer of >4,000 resulting in aserum titer of 128 in recipient mice) 1 day afterio. virus challenge markedly decreased both thepercentage of positive homogenates and the

10/10

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378 PRICE AND SCHMITZ

mean viral titers in ganglia during the acutephase while again enhancing the development oflatent infection.

Effect ofcyclophosphamide on the courseof acute SCG infection. The effect of immu-nosuppression with cyclophosphamide on thecourse of ganglionic infection was then assessed.BALB/c mice were divided into four groups:untreated controls; mice treated with cyclophos-phamide (250 mg/kg i.p.) 1 day after i.o. viruschallenge; mice receiving rabbit anti-HSV serum(0.2 ml i.p. of serum with a neutralizing titer of>4,000) 1 day after i.o. virus challenge; and micesimilarly treated with both cyclophosphamideand immune serum. Figure 3 depicts the results

TABLE 5. Effect ofpassive immunization withrabbit anti-HSV antiserum on acute and latent SCG

infection'Acute phase (homog-

enates) Ratio of la-Group Mean ti- tent phase

Ratioh ter (PFU, explantsdlogO))

Unimmunized 22/23 3.5 24/32Immunized 3/23 1.2 31/32"Mice were immunized with rabbit anti-HSV serum

injected i.p. 1 day after i.o. HSV challenge (see text).P < 0.001.

dP < 0.001.dp< 0.02.

5rHSV alone

-.- HSV. cyclophosphamide4+- HSV. cyclophosphomide. antibody--_- HSV. antibody

N'-~~~~~~~~~~~~~~U- L4

12 3 4 5 6 7 8 9 10 11Time (days) a

FIG. 3. Effect of cyclophosphamide treatment onthe course of acute SCG infection. All mice werechallenged i.o. with HSV on day 0, and viral titers ofganglion homogenates from mice sacrificed between2 and 11 days after HSV challenge were measured.Both cyclophosphamide (250 mg/kg) and rabbit anti-HSV antiserum were administered 1 day after i.o.virus challenge. The four groups included mice re-ceiving no additional treatment, mice treated withcyclophosphamide alone or in combination with an-tibody, and those passively immunized with antibodyonly. All remaining mice in the cyclophosphamidealone group died between the days 7 and 11. Eachpoint represents the results of determinations on fiveor more ganglia.

a 1

2

of SCG homogenates of these four groups ofanimals when sacrificed on days 2 through 11after virus inoculation. In the untreated animals,maximum viral titers were noted on day 4, de-creasing thereafter to undetectable levels by day7. In the cyclophosphamide-treated mice, viraltiters continued to rise after day 4, peaked atday 5 and then declined to a mean of approxi-mately 2.0 x 102 PFU per ganglion on day 7; allmice in this group died before day 11. Immuneserum treatment, as in previous studies, mark-edly reduced viral titers in SCG homogenates ofimmunocompetent mice on each day tested. Inthe mice receiving both cyclophosphamide andimmune serum, viral titers of ganglion homoge-nates were somewhat reduced early in the courseof the infection, but further virus eliminationwas delayed, with HSV being detected in all ofthe five ganglia tested at day 7, and at day 11one of four ganglia contained a small amount ofinfectious HSV. Antibody levels were measuredat day 5 using pooled serum specimens fromeach group. In the two groups of mice passivelytransferred with rabbit anti-HSV serum, anti-body titers were 128; in the untreated mice aserum titer of 4 was measured, whereas in ani-mals treated only with cyclophosphamide, se-rum antibody was undetectable.Acute SCG infection in nude mice. Mice

homozygous for the nude trait (nu/nu) wereinjected i.o. with HSV bilaterally, and both theright and left SCG were assayed for HSV byhomogenization at 5, 8, and 10 days after inoc-ulation. Results in nude mice passively immu-nized with 0.2 ml of immune rabbit serum (anti-

.--. No antibodyS--40 Antibody

5 6 7 8 9 10

Time (days)

FIG. 4. Acute SCG infection in nude mice. Nudemice were injected with either immune rabbit serum(anti-HSVneutralizing antibody titer >4,000) or withnormal rabbit serum 1 day after bilateral i.o. viruschallenge (see text). Animals were sacrificed for assayof SCG homogenates 5, 8, and 10 days after i.o.challenge. Each point represents the results of assayoffour or more ganglia.

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HSV antibody titer of >4,000) given i.p. 1 dayafter i.o. HSV challenge were compared to un-immunized nude mice similarity injected i.p. with0.2 ml of normal rabbit serum devoid of neutral-izing antibody. Figure 4 shows that in the un-immunized nude mice highest viral titers wereobserved on day 8 after infection and that infec-tious virus remained detectable in SCG homog-enates at day 10. Antibody treatment of thenu/nu mice reduced SCG infection to undetect-able levels on the days assayed. Infection inunimmunized and passively immunized hetero-zygous (nu/+) littermates was also assayed andresembled that observed in BALB/c mice (datanot shown).

Effect of postganglionic neurectomyafter i.o. HSV injection on the course ofacute SCG infection in passively immu-nized mice. We have previously shown thatpostganglionic neurectomy reproducibly reacti-vates HSV in the SCG of latently infected mice(12). We next sought to determine whether thissame operative procedure would alter the acutephase of ganglionic infection if neurectomy weredelayed until after virus reached the ganglion.BALB/c mice were injected with 0.2 ml of rabbitanti-HSV serum (titer >4,000) i.p. 1 day afteri.o. virus challenge and subjected to postgan-glionic neurectomy at various time intervals be-fore and after HSV inoculation. All animals weresacrificed for ganglion assay by homogenization5 days after i.o. virus challenge. As shown inTable 6, when neurectomy was carried out be-

TABLE 6. Effect ofpostganglionic neurectomy onthe acute phase ofSCG infection in passively

immunized miceAcute phase'

Time (homogenates)of

Operative procedure opera- Meantion' ai titer(h) Ratio (PFU,

log,0)Postganglionic neurectomy -24 0/4 0Postganglionic neurectomy -1.5 0/4 0Postganglionic neurectomy +6 0/5 0Postganglionic neurectomy +20 2/4 3.8Postganglionic neurectomy +29 2/5 3.8Postganglionic neurectomy +48 4/5 4.6Postganglionic neurectomy +72 4/4 2.9Sham +20 1/5 0'

a Time of operative procedure in relation to time ofi.o. challenge. Negative sign indicates operation beforevirus injection, whereas positive sign indicates opera-tion following virus injection.

'Mice were sacrificed 5 days after i.o. HSV chal-lenge.

' The single positive ganglion contained 1 PFU ofvirus.

HSV INFECTION OF THE SCG 379

tween 24 h before and 6 h after virus challenge,HSV was undetectable in SCG homogenates.However, in animals neurectomized between 20and 72 h after i.o. virus challenge, ganglionicHSV replication was detected, with highest viraltiters being noted in mice subjected to neurec-tomy at 48 h. The results of ganglion assay inthese neurectomized, passively immunized micecontrasted sharply with those in sham-operated(20 h), immunized animals in which mean viraltiters of SCG homogenates remained below 1PFU per ganglion.A further experiment was undertaken to eval-

uate the time course of neurectomy-induced en-hancement of virus replication during the acuteinfection. Mice were divided into animals sub-jected to ipsilateral postganglionic neurectomy48 h after i.o. HSV inoculation and those simul-taneously subjected to sham operation in whichthe SCG was surgically exposed but left un-touched. The two groups were in turn subdi-vided into those passively immunized with rab-bit anti-HSV serum (titer >4,000) 24 h aftervirus challenge and those receiving normal rab-bit serum at the same time. As indicated by thedata in Fig. 5, neurectomy profoundly influenced

5

0

U-03N

0 3 4 5 6 7Time (days)

FIG. 5. Time course ofpostganglionic neurectomy-induced augmentation ofHSV replication during theacute phase of SCG infection. All mice received i.o.HSV challenge on day 0. The immunized groups wereinjected i~p. with rabbit anti-HSV antiserum and theunimmunized groups were injected with normal rab-bit serum I day after i.o. virus challenge. Postgan-glionic neurectomies and sham operations were done2 days after i.o. injection. Mice were sacrificed ondays 3 through 7, and viral titers ofganglion homog-enates were determined. Each point represents theresults of assay of between four and six ganglia.Symbols: O. unimmunized, sham operation; *, im-munized, sham operation; O. unimmunized, neurec-tomy; *, immunized, neurectomy.

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the course of viral replication. In the unimmu-nized mice subjected to sham operation, viraltiters peaked at day 4 and declined to nearlyundetectable levels at day 7. In contrast, in theneurectomized, unimmunized group viral titerscontinued to rise after day 4 reaching a meantiter above 4.5 x 104 PFU per ganglion on day 5;this compares with a mean titer of 5.9 x 102 onthe same day in the sham-operated, unimmu-nized group. Even more striking, however, wasthe course of viral replication in the immunized,neurectomized group. In these animals, HSVwas detected at day 3 and rose to a peak level ofnearly 1.0 x 104 PFU per ganglion on day 5,again demonstrating that neurectomy inducedhigh viral titers in the SCG despite passive trans-fer of immune serum which, in the sham-oper-ated group, resulted in minimal viral titers inthe SCG at days 3 and 4 and undetectable levelsthereafter. Mice from the two immunized groupswere also sacrificed at 21 days to assess the effectof neurectomy on the development of latentinfection. In the neurectomized group 5 of 15(33%) ganglion explants were positive for HSV,whereas 11 of 14 (79%) ganglia from sham-op-erated control mice were positive for virus (P <0.05), indicating that neurectomy not only aug-mented viral replication acutely but also led toa reduction in the subsequent prevalence of la-tency.

DISCUSSIONThe present studies show that the course of

experimental SCG infection with HSV is influ-enced by a number offactors, including the routeby which the virus reaches the ganglion, thelevel of systemic host resistance, and the integ-rity of postganglionic axons. These factors im-portantly determine not only whether or not theganglion becomes infected but also the characterof both the acute and latent phases of ganglionicinfection.

Viral access to the SCG may occur via eitherneural or hematogenous pathways. Disruptionof the nerves connecting the eye and the SCGby postganglionic neurectomy before Lo. viruschallenge prevented both acute and latent gan-glionic infection, thereby demonstrating the im-portance of the neural route in establishing au-tonomic infection. Whether neural passage en-tails primarily intra-axoplasmic (retrograde)transport (8) or perineural virus passage (7) can-not be directly inferred from these studies, al-though both the detection of virus in ganglia asearly as 24 h after i.o. injection (5) and thefailure of neurectomy performed later than 24 hafter inoculation to prevent ganglionic infectionsuggest involvement of the more rapid intra-axoplasmic route. Similarly, the failure of pas-

sively administered antiviral antibody, whichmight be expected to interfere more effectivelywith perineural than with axoplasmic passage ofvirus (5), to prevent latent SCG infection alsofavors the importance of axoplasmic viral trans-port. However, in unimmunized animals HSVmay well reach the ganglion by both intra-axonaland perineural passage. Moreover, the presentstudies also demonstrate that virus can reachthe ganglion hematogenously, although withpoor efficiency. Thus, i.v. injection of HSV ledto low levels of virus replication in the ganglionand to a lower prevalence of subsequent latencycompared to anterior chamber inoculation.Both immunologically specific and nonspecific

defenses influenced whether or not HSV gainedaccess to the SCG. The protective effect of spe-cific immunity was demonstrated by the preven-tion of ganglionic infection in BALB/c miceactively immunized i.p. with HSV 7 days beforeLo. virus challenge. Although anti-HSV anti-body may have contributed to this protection,the failure of antibody alone, when administered1 day before Lo. virus challenge, to exert a similarprotective effect suggests that other, presumablyvirus-specific, cell-mediated, immune mecha-nisms were also involved in preventing virusfrom reaching the ganglion in actively immu-nized mice. On the other hand, the resistance tothe development of ganglionic infection seen inthe C57B1/6 mice compared with that ofBALB/c mice when low doses ofHSV were usedfor inoculation suggests that nonspecific hostdefenses can also participate in preventing gan-glionic infection. Virus gains access to the SCGwithin 24 to 48 h of i.o. injection, and, therefore,early events at the site of inoculation and per-haps along the nerve are paramount in deter-mining whether the ganglion becomes infected.Because these early events take place rapidly,before specific immune defenses can be mobi-lized by the virgin host, it is likely that othergenetically determined but immunologicallynon-specific antiviral defenses account for thedifferences in ganglionic infection seen in thetwo mouse strains at low virus doses.The ganglionic infection which ensues once

HSV reaches the SCG has been analyzed in twophases, an acute and a latent phase. The acutephase was quantitatively assessed by the viraltiters of ganglion homogenates which reflect theextent of intraganglionic viral spread and repli-cation. The latent phase was assessed by theexplantation-co-cultivation assay which detectsthe continued presence ofthe HSV viral genome.The present studies demonstrate that the out-come of the acute and latent phases of infectionmay be dissociated. Thus, appropriately timedimmunization or genetically determined sys-

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temic resistance decreased the magnitude ofviral replication acutely while failing to reduce,or actually enhancing, the subsequent preva-lence of latent infection. Conversely, postgan-glionic neurectomy 48 h after i.o. virus challengeaugmented the acute phase of ganglionic infec-tion while reducing subsequent latency.How does systemic host resistance favor the

development of latency over acute productiveinfection? Stevens and Cook have hypothesizedthat one particular aspect of host defenses, an-tiviral immunoglobulin G antibody, exerts a di-rect effect on the infected ganglion cell andmodulates the permissiveness ofthe cell for virusreplication (15). Aspects of the present studiescan be interpreted as consistent with this hy-pothesis. Passively administered antiviral anti-body, particularly when given 1 day after viruschallenge, not only reduced productive infectionbut potentiated latency. Likewise, active immu-nization induced by i.p. virus injection betweendays 1 and 3 before i.o. virus challenge reducedacute virus replication without reducing latency;this effect could also be interpreted as secondaryto circulating antibody which becomes detecta-ble between days 4 and 6 after immunization. Inboth instances it can be speculated that antiviralantibody directly signaled ganglionic cells to"commit themselves" to the latent state ratherthan allowing productive infection.The results of other experiments, however,

suggest that antiviral immunoglobulin G aloneis inadequate to explain the influence that sys-temic host resistance exerts in inhibiting acuteproductive infection and enhancing latency. Inthe studies of timed passive immunization, itmight have been expected that transfer of anti-serum 1 day before i.o. virus challenge (Fig. 2,group A) would have produced the greatest re-duction in the acute infection if antibody alonewas responsible for inhibiting virus replication.In fact, maximal reduction occurred in micegiven antibody 1 day after i.o. virus challenge(Fig. 2, group B). One possible explanation forthis result is that the 24-h interval between i.o.virus challenge and later antibody administra-tion in group B allowed clearer exposure of thehost's own immune system to the virus, therebystimulating a stronger endogenous cell-mediatedimmune response. Furthermore, in mice immu-nized with anti-HSV antibody and also treatedwith cyclophosphamide, productive infectioncontinued beyond the usual peak at 4 to 6 days,indicating that antibody, by itself, was incapableof inducing the ganglion cells in these animalsto enter the latent state, but rather required theparticipation of a cyclophosphamide-sensitivecell population. The experiments employingathymic nude mice indicate that T-lymphocytes

contribute to eliminating intraganglionic HSVreplication, but the observation that anti-HSVantibody largely abrogates the acute infection inthe SCG of nude mice suggests that one reasonfor the prolonged infection in the thymus-defi-cient animals may have been an absence ofantibody production rather than a lack of T-lymphocyte effector cells; HSV acts as a thymus-dependent antigen in these animals, and thusthey require T-lymphocyte helper function toproduce neutralizing antibody (4). The results ofstudies of C57B1/6 compared to BALB/c miceinoculated with higher titers ofvirus also suggestthat mechanisms other than antibody contributeto concomitant reduced acute infection and in-creased latency (9).Although the present investigations do not in

each instance precisely define the immune de-fenses involved, they do suggest that a constel-lation of host defenses can cooperate in reducingthe level of intraganglionic HSV replicationacutely and that the effect ofantibody is perhapscomplemented by T-lymphocytes and other cellpopulations. But how do these antiviral mecha-nisms, which eliminate infectious virus acutely,actually promote the development of latency?In part, they may act by preserving the anatomicintegrity of the ganglion. In unimmunizedBALB/c mice, infected ganglia have at timesbeen noted to be reduced in size when assayedfor latency, and light microscopic examinationhas revealed a loss of ganglionic cells in theseanimals. Immunization, by either active or pas-sive administration of antibody, lessened thedegree of atrophy and neuronal loss (unpub-lished data). Ganglionic atrophy was similarlyless evident in C57B1/6 than in BALB/c mice.Reduced latency may then result from a quan-titative reduction in surviving neurons. On theother hand, it can be argued that ganglionicatrophy provides only a partial answer and doesnot fully explain why the prevalence of latencyis higher in the surviving cells of the immunizedmice compared with that of the nonimmunizedanimals. Despite destruction of some cells, thesurviving cells of both groups are still faced witha "decision" as to whether latent or productivelytic infection will ensue following viral expo-sure. An alternative hypothesis to explain thepotentiation of latency by increased host resist-ance is that the input multiplicity plays a criticalrole in determining whether a ganglion cell is tobe productively or latently infected, and thatwhen a cell is infected with a low input, latentinfection occurs, although higher doses of infect-ing virus lead to productive infection. Such amechanism would account for the diverse cir-cumstances in which heightened resistance leadsto latency. The several means discussed earlier

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by which host defenses dampen productive in-fection might also thereby insure latency byreducing, but not preventing, initial viral access

to the ganglion, by eliminating productively in-fected cells and by quantitatively reducing intra-ganglionic virus spread with the overall effect ofreducing the input multiplicity to individual gan-glion cells. It may additionally be speculated asa corollary to this hypothesis that the route bywhich virus reaches the ganglion also influencesthe outcome of infection. The ability of hostdefenses to more effectively prevent perineuralpropagation of HSV, which entails multiple cy-

cles of virus replication and spread to contiguouscells, compared to axoplasmic viral transportprovokes the question of whether the balancebetween productive and latent infection dependson which of these neural routes serves to intro-duce virus to the ganglion. If virus reaches theneuronal soma as part of a wave of infectioninvolving Schwann cells, other non-neuronalsupporting cells, and adjacent neurons, produc-tive infection may be more likely to occur thanif virus reaches the soma via its own axonalprocess.Whatever the mechanisms by which

heightened resistance reduces acute infectionand promotes latency, the experiments employ-ing postganglionic neurectomy establish that thestate of the ganglion cells also profoundly influ-ences the balance between productive and latentinfection although in a direction opposite thatconferred by immunization. When postgan-glionic neurectomy was delayed 48 h after i.o.virus challenge so that viral access to the gan-

glion was assured, HSV replication during theacute phase was augmented. This was evident innonimmunized mice, but was even more strikingin mice passively immunized with antiviral an-

tibody in which viral titers of nearly 1.0 x 104PFU per ganglion were detected, compared tominimal viral titers in sham-operated immu-nized controls. Indeed, the effect of neurectomywas similar in magnitude, although not in dura-tion, to that seen when mice were immunosup-pressed with cyclophosphamide. Furthermore,this augmented viral replication in the acutephase was followed by a reduced prevalence ofsubsequent latency. The mechanism involved inthis neurectomy-induced alteration ofacute gan-glionic infection is uncertain, although verylikely it is the same as that responsible for reac-tivation of latent infection by postganglionicneurectomy (12). We have previously speculatedthat neurectomy might alter permissiveness ofganglion cells for HSV replication either as a

part of the axon reaction (chromatolysis) whichis accompanied by augmented and redirectedRNA and protein synthesis, or as a result of

altered trophic influence conferred by inner-vated target tissue (12). Whatever the mecha-nisms involved, it is evident that although in-creased systemic host resistance favors an out-come of latent rather than productive SCG in-fection, postganglionic neurectomy favors theopposite. Also, neurectomy can clearly overcomethe effect of antiviral antibody, allowing vigorousproductive infection to occur despite high levelsof serum neutralizing antibody.Host factors both extrinsic (e.g., systemic an-

tiviral resistance) and intrinsic (e.g., the integrityof postganglionic axons) to the ganglion deter-mine the course of acute and latent ganglionicinfection. For the most part the mechanismsunderlying these host influences remain uncer-tain. For this reason, the foregoing discussionhas been rather speculative. Support for or re-jection of the offered hypotheses requires addi-tional studies of in vivo ganglionic infection, and,perhaps more importantly, awaits the develop-ment of relevent in vitro models of ganglioniclatency.

ACKNOWLEDGMENTSThis work was supported by Public Health Service research

grant NS-12396 from the National Institute of Neurologicaland Communicative Disorders and Stroke and Basil O'ConnorStarter research grant 5-123 from the National Foundation-March of Dimes.We gratefully acknowledge the additional technical assist-

ance of Joanne Levin.

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