The Journal of Neuroscience. June 1993. 13(6): 2515-2559

Pseudorabies Virus Infection of the Rat Central Nervous System: Ultrastructural Characterization of Viral Replication, Transport, and Pathogenesis

J . P. Card,’ L. Rinaman,2-8 R. 6. Lynn,3 B.-H. Lee,4 Ft. P. Meade,’ R. R. Miselis,5 and L. W. Enquistl Viral Diseases Research, Du Pont Merck Pharmaceutical Co., Wilmington, Delaware 19880, *Medical College of Pennsylvania, Department of Anatomy and Neurobiology, Philadelphia, Pennsylvania 19129, 3Thomas Jefferson University, Philadelphia, Pennsylvania 19107, 4Gyeongsang National University, Chinju, Korea, and 5Department of Animal Biology, University of Pennsylvania Veterinary School, Philadelphia, Pennsylvania 19104

Pseudorabies virus (PRV) has been used extensively to map (3) abortive viral replication in cells that are permissive for synaptic circuits in the CNS and PNS. A fundamental as- infection. The findings provide further insight into the intra- sumption of these studies is that the virus replicates within cellular routes of viral assembly and egress and support the synaptically linked populations of neurons and does not contention that transneuronal spread of virus in the brain spread through the extracellular space or by cell-to-cell fu- results from specific passage of virions through synaptically sion. In the present analysis we have used electron micros- linked neurons rather than through cell fusion or release of copy to characterize pathways of viral replication and egress virus into the extracellular space. that lead to transneuronal infection of neurons, and to doc- [Key words: pseudorabies virus, transneuronal transport, ument the non-neuronal response to neuronal infection. Three vagal motor neurons, glia, ultrastructure, immunocytochem- strains of PRV that differ in virulence were used to infect istty] preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV). The data demonstrate that viral replication Pseudorabicsvirus (PRV) is a swinea-herpesvirus that has been and transneuronal passage occur in a stepwise fashion that used extensively to map neural circuits (Martin and Dolivo, utilizes existing cellular processes, and that the non-neu- 1983; Rouiller et al., 1986, 1989; Strack et al., 1989a,b; Card ronal response to infection serves to restrict nonspecific et al., 1990, 1991, 1992; Strack and Loewy, 1990; Jansenet al., spread of virus by isolating severely infected neurons. Spe- 1992; Nadelhaft et al., 1992; Rotto-Percelay et al., 1992). Like cifically, capsids containing viral DNA replicate in the cell other members of this family of viruses (i.e., herpes simplex nucleus, traverse the endoplasmic reticulum to gain access virus), PRV exhibits a pronounced affinity for neuronsthat leads to the cytoplasm, and acquire a bilaminar membrane en- to uptake, replication, and transneuronal passageof virions. velope from the trans cisternae of the Golgi. The outer leaf Productive infection of neuronsproduces cytopathic effectsthat of this envelope fuses with the neuron membrane to release could compromise the circuit related transport of virus. Con- virus adjacent to axon terminals that synapse upon the in- sequently, knowledge of the intracellular pathways of viral as- fected cell. A second fusion event involving the viral enve- sembly and egress,as well as the responseof non-neuronal cells lope and the afferent terminal releases the naked capsid to neuronal infection are important considerationsin evaluating into the bouton. Systematic analysis of serial sections dem- the transport of virus through any neural circuit. onstrated that release of virus from infected neurons occurs A substantial number of investigations have examined the preferentially at sites of afferent contact. Nonspecific dif- morphological events of cu-herpesvirusassembly and egressin fusion of virus from even the most severely infected cells is vitro (Morgan et al., 1959, 1968; Nii et al., 1968a,b, McCracken restricted by astrocytes and other non-neuronal elements and Clarke, 1971; Miyamoto and Morgan, 197 1; Fong et al., that are mobilized to the site of viral infectivity. The ability 1973; Smith and de Harvin, 1974; Johnson and Spear, 1982, of glia and macrophages to restrict spread of virus from 1983; Lycke et al., 1984, 1988; Marchand and Schwab, 1986; necrotic neurons is the product of (1) temporal differences Lee et al., 1987; Jonesand Grose, 1988; Pol et al., 1989, 199la,b; in the mobilization of these cells to the site of infection, (2) Whealy et al., 199 1; Peeterset al., 1992). In addition, a limited differential susceptibility of these cells to PRV infection, and number of studieshave examined theseprocesses in vivo (Field and Hill, 1974; Kristensson et al., 1974, 1978, 1982; Bak et al., 1977, 1978; Dolivo et al., 1978). Analysis of viral replication Received Aug. 18, 1992; revised Dec. 3, 1992; accepted Dec. 17, 1992. Work appearing in this article was supported by Du Pont Merck Pharmaceutical in immortalized pig kidney fibroblasts infected with PRV has Company and NIH Grant GM27739 awarded IO R.R.M. We gratefully acknowl- demonstrated that acquisition of a bilaminar membrane from edge the insight provided by Dr. Pat Levitt. Mary Whealy, Henry Pautler, Claudia the trans cistemae of the Golgi complex may be a critical step Williams, and Janice Lancaster provided expert technical assistance. Correspondence should be addressed lo J. Patrick Card, Ph.D., Department of in the replication of the virus (Whealy et al., 1991). Virions of Behavioral Neuroscience, 446 Crawford Hall, University ofPittsburgh, Pittsburgh, similar morphology have been idcntificd in cultured human PA 15260. sensoryneurons infected with herpessimplex virus (HSV) (Lycke x Present address: Department of Behavioral Neuroscience, 446 Crawford Hall, University of Pittsburgh, Pittsburgh. PA 15260. ct al., 1988).These studiessuggest that releaseofinfectious virus Copyright 0 1993 Society for Neuroscience 0270-6474/93/132515-25$0%00/O is dependent upon fusion of the outer membrane of the newly 2516 Card et al. * Transsynaptic Passage of Pseudorabies Virus replicated vu-ions with the plasma membrane of the infected the present studies, all work with PRV was confined to a Biosafety Level cell. However, this hypothesis has not been established with 2 (BSL-2) containment facility dedicated exclusively to this work. The conduct of experiments in this laboratory conformed to the BSL-2 reg- certainty and the mode of intracellular transit and the precise ulations detailed in Health and Human Services Publication No. 88- site of virion release in the CNS have not been examined in a 8395 (Biosqfety in Microbiological and Biomedical Laboratories). All systematic fashion. animals were conlined to the BSL-2 laboratory throughout the course Characterization of the route of viral passage through the of the analysis and infectious virus was inactivated in each animal at death by transcardiac infusion of buffered aldehyde solutions (see be- nervous system is also dependent upon a thorough understand- low). ing of the cytopathic events associated with viral infection. Vir- Preparation ofPR P. Three strains ofpseudorabies virus (PRV-Becker, ulent and attenuated strains of HSV and PRV have been shown PRV-4, and PRV-Bartha) were used. The Becker strain of virus (PRV- to induce neuropathological changes that ultimately lead to glial Be; Becker, 1967) is a virulent held isolate that we have previously used infection (Ugolini et al., 1987; Norgren and Lehman, 1989; Card to define a multisynaptic brainstem circuit involved in gastric function (Card et al., 1990). PRV-Bartha (PRV-Ba; Bartha, I96 1) is an attenu- et al., 1990, 199 1, 1992; Strack and Loewy, 1990; Blessing ct ated, vaccine strain that possesses two prominent alterations in the viral al., 1991; Norgren et al., 1992; Rinaman et al., 1993) and it is genome that contribute to a reduction in virulence and longer postin- becoming increasingly clear that glia participate in the immune oculation survival intervals (Card et al., 1992). PRV-4 is a virulent response to neurotropic viral infections (Massa et al., 1986, strain derived from PRV-Be containing a mutation in the viral envelope glycoprotcin glll gene. Previous analysis has shown that PRV-4 is as 1987a,b; Lieberman et al., 1989; Sarmicnto and Klcincrman, virulent as PRV-Be when injected into the vitreous body of the eye 1990; Weinstein et al., 1990; Zuckermann et al., 1990). The (Card et al., 1992). All strains were grown in pig kidney fibroblasts to extent to which these responses contribute to the isolation or titers of 6 x 10” pfu/ml (PRV-Be). 5 x IO8 pfu/ml (PRV-Ba), and 7 x spread of virus in the CNS has not been fully elucidated. How- IO’ pfu/ml (PRV-4). ever, available evidence suggests that the non-neuronal response Experimental paradigms. Rats were infected by injecting l-6 ~1 of virus into the ventral wall of the stomach as described previously (Card to infection serves to isolate afflicted neurons and thereby fa- et al., 1990). The extent of transport of each strain of PRV was assessed cilitates transsynaptic spread of virus (Field and Hill, 1974; by injecting animals with equal volumes of the same viral stocks and Kristensson et al., 1974, 1978, 1982; Bak et al., 1977; Rinaman processing the brainstems for light microscopic immunohistochemistry et al., 1993). Nevertheless, ultrastructural analysis of the in- or for electron microscopic analysis. Animals injected with PRV-Be fccted CNS is necessary to characterize fully the extent of virally were killed 48 hr following inoculation, while those inoculated with PRV-4 and PRV-Ba were killed at 53 and 65 hr, respectively. induced pathology and elucidate further the roles that glia and Animals inoculated with PRV-4 were simultaneously injected with 7 macrophages play in determining the route of viral passage ~1 of 0.25% cholera toxin conjugated to horseradish peroxidase (CT- through the neuraxis. HRP). CT-HRP is transported through both the motor and sensory In the present investigation we conducted a systematic ultra- components of the vagus nerve and enzymatic reaction of tissue with diaminobenzidine produces an electron-dense reaction product that structural analysis ofthe pathways ofPRV replication, assembly identifies labeled perikarya and processes at the ultrastructural level and egress in the dorsal motor vagal (DMV) complex of the rat (Rinaman ct al., 1989). We used the CT-HRP reaction product to iden- brainstem and characterized the non-neuronal response to viral tify DMV motor neuron perikarya and processes to determine the extent infection. The DMV was chosen because we have fully char- of intracellular transport of newly replicated virus in infected neurons. Immunoperoxidase localizations. A rabbit polyclonal antiserum raised acterized the temporal aspects of PRV transport in this circuit against PRV and the avidin-biotin modification (Hsu et al., 1981) of at the light microscopic level (Card et al., 1990; Miselis et al., Stembcrgcr’s (I 979) immunoperoxidase method were used to localize 199 1; Stemini et al., 199 1; Rinaman et al., 1993). In addition, viral epitopes. Specific details of antibody specificity and application of the peripheral projections of DMV neurons and the ability to this method for light microscopic immunohistochcmical localization of infect them by injecting virus into the wall ofthe stomach (Card PRV in the DMV have been published (Card et al., 1990). Ultrastructurulpreparation. Animals were anesthetized and perfused et al., 1990) provide an ideal system for evaluating the CNS transcardially with buffered aldehyde solutions as previously described response to infection in the absence of the confounding physical (Card et al., 1986). Thercahcr, each brain was removed from the cra- insults that would occur if the virus were injected directly into nium and postfixed in the primary lixativc overnight at 4°C and the the brain. The data provide new insights into a-herpesvirus caudal brainstem was sectioned serially in the coronal plane with a vibratome (100 pm per section). Tissue sections were washed in buffer, assembly, transport, and pathogenesis and arc consistent with postlixed in a 1% osmium tetroxide solution containing 1.5% potassium the conclusion that the non-neuronal response to infection iso- ferricyanide (Langford and Coggcshall, 1980). dehydrated, infiltrated, lates infected neurons and thereby supports specific transport and flat-embedded in Epon-Araldite plastic resin. Sections at the level of PRV through synaptically linked populations of neurons. of the area postrema were trimmed to a trapezoid including the DMV and nucleus of the solitary tract (NST) and sectioned with a Reichert Jung ultramicrotome using a diamond knife. Thick sections (0.5 pm) wcrc cm, mounted on slides, stained with toluidine blue, and photo- Materials and Methods graphed with a Reichert Jung Polyvar photomicroscope. Ultrathin sec- Animals. Adult male Sprague-Dawley rats (200-300 gm body weight tions of areas immediately adjacent to the thick sections were picked at death) were used in this investigation. The animals were maintained up on Formvar-coated slot grids, stained with lead citrate and uranyl in a standardized environment throughout the experiment (12 hr light; acetate, and analyzed and photographed with a JEOL 1200EX trans- light on at 0600) with food and water freely available. Procedures for mission electron microscope. the maintenance and use of experimental animals conformed to the A serial series of 1535 consecutive ultrathin sections was collected reaulations detailed in the NIH Guide for the Cure and UseofLaboratorv from the animal infected with PRV-Bartha. This series was used to Aiimafs. Experimental protocols involving USCof the virus were ap- examine the same profiles in adjacent sections in order to gain a thorough proved by our Animal Welfare Committee, which is subject to the rules understanding of virion assembly, intracellular transport, and rclcase. and regulations of the American Association for Accreditation of Lab- CT-HRPIPRV injections. The two animals injected with a cocktail oratory Animal Cart and those stipulated in the Animal Welfare Act of PRV-4 and CT-HRP were anesthetized and killed by transcardiac (Public Law 99- 198). All surgeries were conducted on deeply anesthc- perfusion fixation 53 hr after inoculation. One animal was perfused with tized rats using aseptic procedures. sequential tramcardiac infusions ofdilute (0. I% pamformaldehyde, 0.51 Safity procedures. Procedures for use of virus in these experimental glutaraldehyde in 0. I M sodium phosphate buffer) and concentrated (5 x paradigms have been published previously (Card et al., 1990, 1991). the concentration of the dilute) fixative. The brain was then removed, Nevertheless, it is important to reiterate that specialized facilities and postfixed for I hr, washed in phosphate buffer overnight, and sectioned procedures are necessary for experiments employing herpesviruses. In in the coronal plane with a vibratome (50 rm/section). The tetramethyl- The Journal of Neuroscience, June 1993, f3(6) 2517 benzidine method of Mesulam (I 982) was used to localize HRP in every DMV and NST. These pathological changeswere present in the third section through the DMV/NST. These sections were mounted on DMV of a!! infected animals, but they were more prevalent in gelatin-coated slides, air dried, and coverslippedfor light microscopic analysis. The remaining sections were reacted with diaminobenzidine animals infected with the virulent strains of PRV. Dense de- and H,O, to visualize the HRP and processed for electron microscopy generation of chronically infected DMV neuronswas only ob- (Rinaman et al., 1989). The other animal was perfused with 4% para- served in the animals infected with the virulent strainsof virus. formaldehyde and 0.2% picric acid in 0. I M sodium phosphate buffer and processed for light microscopic immunohistochemical localization Virus replication, assembly, and egress in neurons of virus and CT-HRP conjugate. Virus was localized with the rabbit polyclonal anti-PRV antiserum, and a goat antiserum raised against Capsidformation. The appearanceof viral capsidsin cell nuclei cholera toxin (Calbiochem) was used to localize the CT-HRP conjugate. was among the first morphological changesin infected neurons The virus antiserum was diluted to I : IO00 with phosphate buffer con- (Fig. 2). Chromatin becameclumped and marginated, the nu- taining I% normal goat serum and 0.3% Triton X- 100; the CT antiserum was diluted to I:5000 with 1% normal rabbit serum and 0.3% Triton cleolus dispersed,the nuclear envelope proliferated and devel- X-100 using the same buffer. Both antigens were visualized with fluo- opcd increasingly complex invaginations, and the progressive rescence histochemistry using either rhodamine- or FITC-conjugated increase in capsidsproduced pathological inclusions in the cell secondary antisera. nuclei. Individual nucleocapsidsconsisted ofelectron-dense cores Ultrastructural analysis. Each ultrathin section was subjected to a of viral DNA within structural protein cages (Fig. 3A). The systematic method of analysis designed to characterize fully the extent of viral infection in the DMV/NST complex. This method was previ- structural protein cage was approximately 8 nm thick and the ously used to characterize the synaptic organization of vagal gastric entire cross-sectionaldiamctcr of mature capsidswas 100 nm. motor and sensory adherents in this region of brainstem (Rinaman et al., Capsids occurred individually, in large aggregations,and in 1989). Specific details regarding sampling arc available in that publi- paracrystalline arrays adjacent to the nuclear membrane. In the cation, but it is worthwhile to reiterate the most important aspects of the method and to detail those unique to the present study. A critical larger aggregations,they were often enmeshedin a granular feature of the analysis involved correlating light and electron micro- matrix that varied from a light homogeneousmeshwork to dense scopic data. Thick sections (0.5 pm) from areas immediately adjacent concentrations of 20 nm granules (Fig. 3A). to the ultrathin sections were photographed and used to direct the ul- Analysis of neurons in scria! thin sectionsdemonstrated that trastructural analysis. Landmarks evident in those photomicrographs the nucleus of every infected cell contained distinct sites of (i.e., blood vessels, myelinated fiber tracts, motor neurons) were used to identify precisely the region examined at the ultrastructural level. capsid assembly. Complex interwoven massesof tubules were The DMV was initially examined at 5000 x in sequential passes through the principal constituent of theseareas (Fig. 3R.C). Cross-sec- the nucleusto identify infectedneurons and basicfeatures of virally tional profiles of individual tubules presented a spherical to induced neuropathogcnesis. The analysis was then repeated at 20,000 x slightly oblong conformation with a widest diameter of 10-l 5 to characterize the synaptic organization of the DMV and the mor- phologyof infectedprofiles. The samemethod of analysiswas applied nm, and the entire tubule masswas a few micrometers in size. to the NST, the tractus solitarius, and the ventrolateral region adjacent Within each of theseareas, multiple tubules formed concentric to the DMV that contains efferent axons of vagal motor neurons. rings around regions of coalescingviral DNA (solid arrows in Fig. 3&C). Fully assembledcapsids were presentat the periph- Results ery of the tubule mass,sometimes with individual tubules as- Examination of viral transport with light microscopic immu- sociated with the superficial aspectof the structural protein cage nohistochemistry demonstratedthat the extent of viral transport (open arrow, Fig. 3C). in the dorsal motor vaga! complex was equivalent for each of Capsid transport to the cytoplasm. Our data support the con- the three strains of PRV, even though the postinoculation in- clusion that nucleocapsidsgain accessto the cytoplasmic com- terval was longer in the animal infected with the attenuated partment of the cell by budding through the inner membrane strain. This finding confirms our previous demonstrations that of the nuclear envelope, traversing the endoplasmic reticulum the temporal course of infection produced by attenuated virus (ER), and decnveloping to exit the ER in the vicinity of the lagsbehind that produced by virulent strains (Card et al., 1992; Golgi complex. As noted above, large numbers of capsidswere Rinaman et a!., 1993). Following injection of the ventral stom- obscrvcd adjacent to the inner nuclear membrane, often in para- ach wall, a!! three strains of PRV infected substantial numbers crystalline arrays that filled a lobule of the nucleus created by of motor neuronsin the left DMV and scattcrcd motor neurons pathological invagination of the nuclear envelope. In addition, in the right DMV. Neurons in the media! subfield of the NST we observed individual capsidsbudding through the inner leaf were also infected, but there were no infected cells in other of the nuclear envelope to acquire a spherical membrane cn- regionsofthc neuraxisat theseearly survival times. This pattern velope (Fig. 30). These enveloped virions were 120-l 60 nm in of infection was also observed in the animals injected with both widest diameter and were routinely observed between the two PRV4 and CT-HRP, but the CT-HRP reaction product was lcavcs of the nuclear envelope, either individually or in groups. confined to motor neurons and primary sensoryafferents. Cells advanced in the courseof viral infection exhibited vast arrays of ER that extended throughout the cytoplasm. Only Pathological hallmarks of viral infection small Niss! bodies were present in uninfcctcd DMV neurons Light and electron microscopic examination of the DMV re- from control material and were rare in neurons infected with vealed a number of prominent signsof viral infection. These PRV. Thus, it is unclear whether the extensive ER networks in included pathological inclusions in the nuclei of infected neu- infected neurons were the product of membrane proliferation rons, ruffling and invaginations of the nuclear membrane, en- or simply representeddisassembly ofNiss1 bodies. In any event, largement of the perivascular space,and the presenceof small infected cells displayed highly interconnected trabeculae of ER intensely staining cellsadjacent to the vascularcndothelium and that were continuous with the nuclear membrane at multiple in the neuropi! (Fig. 1%D). Virally induced changesin neurona! sites. In many instances,enveloped capsidsbetween the leaves morphology were confined to the DMV and NST, while mor- of the nuclear envelope passedinto the ER (Fig. 3E), and linear phological alterations in the vasculature and the small densely stretchesof ER in the cytoplasm contained membrane-limited staining cells also were apparent in regions bordering upon the capsids that were similar in size and morphology. Portions of 2518 Card et al. l Transsynaptic Passage of Pseudorabies Virus

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Figure 1. A illustrates the organization of vagal circuitry that innervates the stomach. The dorsal wall of the stomach is innervated predominantly by motor neurons in the right DMV, the left DMV provides the primary innervation for the ventral wall. Sensory afferents in the stomach arise from neurons in the nodose ganglion (NG), and these cells also give rise to a process that passes through the tractus solitarius (ts) to synapse on DMV dendrites and neurons in the NST. B is a light microscopic immunohistochemical localization of PRV-Ba in the left DMV/NST 65 hr following injection of the virus into the ventral stomach musculature. The solid line defines the boundaries of the DMV. Substantial numbers of DMV neurons exhibit PRV immunoreactivity, and the virus has also passed transneuronally to infect presynaptic neurons in the overlying NST. C and D are photomicrographs of 0.5 pm sections from the DMV of an animal infected with PRV-4 53 hr prior to death. Infected motor neurons characteristically exhibit pathological inclusions in the cell nuclei (small open arrows). Increased cytoplasmic density (large open arrow in C) or shrunken electron-dense profiles (large open arrows in D) reflect more advanced pathology. The other hallmark of infection by virulent strains of virus includes increases in the density and thickness of the vascular wall (solid arrow3 in C and D) and the presence of small osmophilic cells in the neuropil.

ER continuous with the nuclear envelope were studded with a common observation. Additionally, the Golgi complex was ribosomes, but gave way to complex trabecular networks of the most frequent site of capsid accumulation in the cytoplasm. smooth ER further into the cytoplasm. Virions were present Envelopment of capsids. The final envelopment of capsids throughout the full extent of these trabeculae. occurred at the Golgi complex. This processendowed capsids The passageof enveloped virions into the ER was followed with a bilaminar membrane envelope that permitted them to by fusion events that released naked nucleocapsidsinto the leave the parent cell and gain accessto other cells in the CNS. cytoplasm near the trans cistemae of Golgi complexes(Fig. 4A). Application of this envelope occurred by wrapping of Golgi- Profiles of smooth ER containing singleenveloped virions were derived membrane around individual capsids. Following fu- prevalent in the vicinity of the Golgi, and releaseof capsids sion-induced releaseof capsidsfrom the ER (Fig. 4A), naked from the ER by fusion of the virion membranewith the ER was capsidsaccumulated adjacent to the polar aspectsof the Golgi Figure 2. Forty-eight hours following injection of 1 ~1 of PRV-Be into the ventral wall of the stomach, a parasympathetic neuron in the DMV illustrates morphological characteristics typical of virus-infected neurons. Mature virions in the cytoplasm are difficult to resolve at this low magnification. However, pathological invaginations of the normally smooth nuclear envelope and dense intranuclear inclusions (arrow) resulting from virus infection are clearly apparent. The neuron in the upper portion of the field shows no overt morphological signs of infection, and the organization of the neuropil appears relatively normal in spite of the virulence of the virus and the 48 hr postinjection survival interval. As, astrocyte; C, capillary. Scale bar, 2 pm. 2520 Card et al. l Transsynaptic Passage of Pseudorabies Virus

Figure 3. Viral replication and egress from the nucleus of an infected DMV neuron are illustrated. A demonstrates that the large inclusions in the nuclei of infected cells (see Fig. 1) are composed of viral capsids. These nucleocapsids consist of electron-dense cores of viral DNA surrounded by virally encoded structural protein capsules. Aggregations of capsids are often enmeshed within a dense homogeneous matrix (arrow). B and C, Intranuclear tubule masses provide focal sites for capsid assembly. Individual tubules exhibit an average cross-sectional diameter of 10-l 5 nm and surround sites at which viral DNA coalesces (solid arrows). The open arrows show mature virions at the periphery of tubule masses. D and E, Capsids’ egress from the cell nucleus is accomplished by budding through the inner leaf of the nuclear envelope (arrow in D) to acquire a single membrane envelope and pass into the rough ER (arrow in E). N, nucleus. Scale bars: A, 400 nm; B-E, 100 nm. The Journal of Neuroscience, June 1963. 13(6) 2621

and became intimately associated with individual cisternae at branes was reduced in size and increased in density, and the the trans face of this organelle (Fig. 4B-D). Most commonly, entire virion was more spherical in conformation. This matu- distinctive C-shaped segmentsat the pcriphcral aspect ofa Golgi ration process produced a particle of smaller cross-sectional cistern surrounded individual capsids and ultimately fused to diameter (approximately 180 nm) in which it was sometimes produce a bilaminar envelope (Fig. 4C). A particularly inter- difficult to resolve the two membranescomposing the envelope esting aspect of this process was the distinct morphology ex- (Figs. 5, 6A-E). hibitcd by the portion of the Golgi membrane involved in en- Characterization of the full extent of transport of enveloped velopment. In most cases, the segment of trans cistern destined virions through the dendritic tree of infected DMV neuronswas to wrap a capsid exhibited prominent membrane decorations determined by ultrastructural analysisof tissue from the animal (Fig. 4B-D). This coat was approximately 15 nm thick, and the that received dual injection of PRV-4 and CT-HRP. The elec- dense granular components of the coat exhibited a regular pe- tron-densechromagen resulting from histochemicallocalization riodicity at about 10 nm intervals. of CT-HRP was used to identify dendrites of gastric motor Wrappings of the capsids with trans Golgi cisterns produced neurons in the DMV and overlying NST. Systematic exami- virions with an average cross-sectional diameter of 200 nm. nation of these areas at 20,000~ revealed numerous labeled Both membranes of this envelope were easily resolved in newly primary, intermediate, and distal dendrites that also contained wrapped vii-ions, and the average distance between the inner enveloped virions (Fig. 6A-C). All of these vii-ions were ap- and outer membrane was 20-25 nm. The outer membrane char- proximately 180 nm in widest diameter, and careful examina- acteristically exhibited the membrane decorations found on the tion demonstrated that their denseenvelope was composedof Golgi cistern that had wrapped it, a feature that provided a two membranesseparated by a small electron-densespace. Thus, reliable means of distinguishing these profiles from the mem- it is apparent that newly replicated vu-ions are transported brane enclosed virions traversing the ER. However, it should through the full extent of the dendritic tree. Whether this trans- be emphasized that there were instances in which the membrane port is active or occurs by passive diffusion cannot be deter- decorations were absent from trans Golgi membrane involved mined from the present data. in capsid envelopment. Material from the CT-HRP/PRV-4 experiment was also an- Lysosomal targeting of virions. All infected neurons exhibited alyzed to determinethe morphology ofvirions in axons of motor complex lysosomal inclusions that were filled with enveloped neuronsand in sensoryaxons ofthe tractus solitarius. The axons virions in various states of degradation (Fig. 5). These inclusion of motor neurons at the ventrolateral extent of the DMV only bodies were concentrated in the peripheral aspects of the cell contained nakedcapsids (Fig. 6E), indicating that capsidsgained soma and generally consisted of multiple interconnected elec- accessto motor axon terminals in the stomach wall by fusion tron-dense lobules. Bilaminar enveloped vu-ions fused with these of the virion envelope with the axonal membrane, and that larger lysosomal inclusions and released capsids surrounded by capsidswere not incorporated in a vesicle for retrograde trans- a single membrane envelope into the electron-dense core of the port to the neuronalcell body. The absenceofenveloped virions inclusion. These. inclusions were evident in neurons at all stages in motor axons further suggestedthat newly replicated vu-ions of infection and were one of the early morphological indicators were differentially sorted to the dendrites of infected cells and of viral infection. However, in spite of the prevalence of these were not transported into the axon within this survival interval. inclusions, and the large number of virions that they contained, In contrast, the few sensoryaxons in the tractus solitarius that we never observed them fusing with the external limiting mem- contained virions at this early survival time had a bilaminar brane ofa neuron to release their contents into the extracellular cnvclope (Fig. 60). This observation suggeststhat, following space. Furthermore, the necrotic state of vii-ions within each retrograde transport of capsids from the stomach to sensory inclusion suggested that they represented a degenerative cnd- neurons in the nodoseganglia, virus replicated in theseperiph- point rather than a vehicle for bulk delivery of virions to the eral neurons and virions acquired a Golgi-derived envelope plasma membrane. before being transported antcrogradely to the NST. Intracellular transport of mature virions. Mature virions were Transneuronalpassageofvirus. Virions with bilaminar mem- observed throughout the perikarya of infected neurons and in brane cnvclopcs were observed throughout the perikarya and the primary dendrites extending from those cells (Fig. 5). How- dendritic arbors of infected DMV neurons. These profiles and ever, the morphology of virions changed substantially as they the surrounding neuropil were examined (often in serial series moved away from the Golgi complex and entered other portions of electron micrographs) to elucidate the site of virion release of the ccl1 soma and dendritic tree. In particular, the Golgi- and to determine if virions were free within the extracellular derived external coat was not apparent on the outer membrane space.This analysis demonstrated that vu-ions were frequently of the envelope, the space between the inner and outer mem- releasedat sites of afferent synaptic contact, but did not gain

Figure 4. The process by which virions are released into the cytoplasm and enveloped at the Golgi complex is shown in the schematic diagram, and selective aspects of the process are shown in the accompanying electron micrographs. A illustrates a virion in a cistern of smooth ER (solid arrows). Naked capsids released into the cytoplasm by fusion of the virion envelope with a segment of smooth ER are shown at the open urrows. B-D illustrate the application of bilaminar envelopes to capsids (Band Care cross sections through Golgi profiles; D is a horizontal section through the trans portion of the Go@). Trans cisterns of the Golgi wrap naked capsids (open arrows). These membranes are continuous with trans cistemae (open arrow in C and solid m-row in D). Note also that membranes wrapping the capsids are decorated with a spiked coat that is limited to that portion of the trans cistern surrounding the capsid (best illustrated in C and D). Scale bars: A and C, 100 nm; B and D, 200 nm. Figure 5. Two infected DMV motor neurons arc illustrated. A large primary dendrite emanates from one of the neurons in the center of the field. The boxed area is shown at higher magnification in the lower inset and illustrates the large number of mature virions that are present in this segment of the dendrite (see Results for detailed description of morphology). Virions in various stages of degeneration are also present in interconnected lysosomal inclusions (upperinset) and multilaminar glial sheaths surround the cell soma (arrows). Scale bar, I pm; lower inset scale bar, 500 nm; upperinset scale bar, 200 nm. i 2524 Card et al. * Transsynaptic Passage of Pseudorabies Virus

access to the extracellular space. This was true of all infected Astrocytic response.Astrocytes exhibited a profound response neurons, even those exhibiting advanced pathology. Further- to viral infection of DMV neurons, and this responseappeared more, the data demonstrated that vii-ions did not discriminate to play a prominent role in restricting spreadof virus from the among the morphologically distinct classes of axon terminals chronically infected neurons. Reactive astrocytes were found in that synapsed upon the soma and dendrites of infected neurons. every field of viral infection and were particularly prevalent in Viral egress was observed in relation to all classes of terminals relation to neurons suffering the pathological consequencesof throughout the full extent of the dendritic arbor. infection with virulent strains of virus (seeFig. 13). They ex- Our observations indicate that the bilaminar nature of the hibited the classicalreactive morphology that has been previ- Golgi-derived envelope is essential for release of virions from ously characterized in response to neuronal injury (de1 Rio- the infected neurons as well as for the subsequent invasion of Hortega and Penfield, 1927; Duffy, 1983). This included an afferent terminals. This process involves two fusion events in increase in cell size and elaboration of large, filament-laden which (1) the outer membrane of the vu-ion fuses with the mem- processesthat pcrmeatcd throughout the neuropil. In many in- brane of the infected cell to release infectious virus adjacent to stances,the perikarya of thesecells were directly apposedto the the afferent terminal, and (2) the remaining membrane of the neuronsexhibiting virally induced neuropathology (Fig. 10A). viral envelope fuses with the afferent to deposit the capsid into At advanced stagesof infection, the intimate relationships the terminal (see schematic inset of Fig. 7). All the elements of that astrocytes establishedwith infected neuronseventually led this process are shown in Figures 7 and 8. The bilaminar en- to infection of the astrocytes themselves. Infected astrocytes veloped virion initially comes in close proximity to the site of exhibited all of the hallmarks of infection displayed by neurons afferent contact (Fig. 74, the outer membrane of the virion with the important exception that enveloped virions were never fuses with the plasma membrane to release the virion adjacent observed in the cytoplasm of the astrocytc somaor its processes. to the terminal (Figs. 7B-D, 8C), and the remaining membrane Aggregations of nucleocapsidswere prevalent in the cell nuclei ofthe envelope fuseswith the terminal to releasethe capsid into (Fig. 10.4) and these capsidsused the nuclear membrane and the aflerent (Figs. 7E, 8C). Our data also indicate that more ER in the same manner as neurons to gain accessto the cyto- than one virion may crossinto a singleafferent (Fig. 8A-C) and plasm. However, in contrast to neurons, the ER of infected that passageof multiple virions through a single site leadsto astrocytes became extremely distended (Fig. 1On-C). These pronounced buildup of membrane that marks the site of trans- edematousER profiles contained capsidssurrounded by a single neuronal passage(Fig. 70. These areas of membrane accu- membrane (Fig. IOB), and this membrane fused with the ER mulation were present throughout the DMV/NST neuropil. to releasenaked capsidsinto the astrocytc cytoplasm (Fig. IOC). In many instances,the external membrane surrounding the vi- Neuropathogenesis and the non-neuronal response to rion in the ER wascovered by a spiked coat. This coat remained neuronal infectivity on the inner surfaceof the ER following membrane fusion and Astrocytes, microglia, and macrophagesexhibited a prominent releaseof the capsid into the cytoplasm, thereby marking the responseto viral infection of DMV neurons. Systematic ex- site of release(Fig. 100 Large.numbers of naked capsidswere amination of the neuropil revealed that all three of these cell observed in the astrocyte cytoplasm (Fig. 1OD) and individual types were prevalent in the DMV of animals infected with vir- capsids were present in the filament-laden processesof these ulent strains of PRV. In contrast, although reactive astrocytcs cells. Nevertheless,well-structured Golgi cistemaewere difficult and microglia were routinely observed in the DMV ofthe animal to find in thesecells, and there was never any evidence ofcapsid infected with attenuated virus, macrophageswere rare at this envelopment. In some instances, PRV infection of astrocytes early survival interval. This finding is consistentwith our rcccnt induced densedegenerative pathology. light microscopic characterization of the temporal responseof Microglia. Reactive, ramified microglia were also prevalent these cells to infection of DMV neurons with attenuated and in the DMV of infected animals. Like astrocytes, these cells virulent strains of PRV (Rinaman et al., 1993). wcrc found in all infected animals, but were more numerousin

Figure 6. This series of micrographs illustrates the morphology of virions in central motor and sensory vagal processes. The experimental paradigm and the circuitry are shown in the schematic diagram. A cocktail of virus (PRV-4) and CT-HRP conjugate was injected into the ventral wall of the stomach, and the animal was killed 53 hr postinoculation. The electron-dense CT-HRP reaction product (solid arrows in A-C) permitted unambiguous identification of components of the vagal circuitry. T!re thick section to the right of the schematic is from the region immediately adjacent to the ultrathin sections analyzed with the electron microscope, and labeled arrows designate the region from which the corresponding electron micrographs were taken. Distal (A), intermediate (B), and proximal (C) DMV dendritesall contain double-membraned virions (open arrows). Double-membraned virions are also present in a pathologically swollen sensory afferent (D). The arrow in D designates the virion that is shown at higher magnification in the inset. In contrast, myelinated efferent axons of DMV motor neurons (E) only contain naked capsids. The solid arrow in E designates a capsid that is shown at higher magnification in the inset. and the open arrow highlights another capsid that is pathologically swollen. AP, area postrema; DMV, dorsal motor vagal nucleus; NST, nucleus of solitary tract; fs, ttactus solitarius; XII. hypoglossal nucleus, Scale bars: A, B, and E. 200 nm; C and D. 500 nm; insets, 100 nm. Figure 7. Several examples of transneuronal passage of virions are illustrated. The entire process is summarized in the schematic diagram. A illustrates a newly wrapped virion in close proximity to an axosomatic affcrent contact (arrow). Note the clear bilaminar nature of the viral envelope. B-D illustrate virions that have been released from DMV motor neurons adjacent to sites of afferent contact (arrows). E illustrates an tutenveloped capsid (arrow) within an alferent synapsing upon an infected neuron, and F illustrates the pronounced accumulation of membrane in regions where multiple vii-ions pass transneuronally at the same site. The arrow in F points to a virion. Scale bars: D, 100 nm; all other bars, 200 nm. Figure 8. A-C are from a serial series through the same site of axodendritic contact. The animal was killed 65 hr postinoculation with PRV-Ba. An axodendritic contact in the boxed area ofA is shown at higher magnification in B and in another plane of section in C. Three areas (arrowheads in B) of transneuronal passage of virions are shown in glazing sections through the vii-ions. A free virion is also apparent in the dendrite (arrow). In C, a capsid enclosed by a single membrane is interposed between the infected dendrite and the same axon terminal (open arrow and shown at higher magnification in the insef) and a free capsid is present in the affercnt (solid arrow). Scale bars: A, 500 nm; B, 200 nm; C. 100 nm. PRV + CT-I-IRP I I - Pre-synaptic -1 Cleft +-Post-synaptic - I

2528 Card et al. * Transsynapfic Passage of Pseudorabies Virus

Figure 9. This schematicdiagram summarizes the significantevents (denoted by numbers) that leadto replicationand transneuronalpassage of PRV. Virions replicatein the cell nucleus,acquire a singlemembrane envelope at the inner leaf of the nuclearenvelope (I), traversethe ER (2), and are releasedinto the cytoplasmby a fusionevent betweenthe virion membraneand the ER (3). The final envelopeis applied to the capsidat the G~lgi apparatus(4), endowingthe virion with a bilaminarenvelope derived from transcistemae. The envelopeof the virion consolidatesand becomesmore dense as the virion movesthrough the cell toward sitesof afferentsynaptic contact (5). Transneuronalpassage of virus occursby fusionof the outer membraneof the virion envelopewith the postsynapticmembrane, releasing a singleenveloped virion adjacentto the afferent (6). The remainingmembrane of extracellularvirions fuses with the membraneof the afferentto releasethe capsidinto the afferentterminal (7) or may fusewith reactiveastrocyte processes and infect thesecells. Capsids released into afferentterminals are retrogradelytransported to the parentneuron. Capsids that enter astrocyteslead to replicationof viral DNA, but thesecells are incapableof producinginfectious virus.

the two animals infected with virulent strains of PRV. The apparent in the animal infected with the attenuated virus. Ul- morphology (Milligan et al., 1991; Rinaman et al., 1991) and trastructural examination of pathological blood vesselsin ani- ultrastructural characteristics(Streit et al., 1988; Kaur and Ling, mals infected with virulent PRV revealed that the small os- 1992) of thesecells were consistent with those describedin the mophilic cells exhibited morphological characteristics of injured CNS. The cells characteristically contained a small ob- monocytesand tissuemacrophages. In severalinstances we found long nucleus with clumped heterochromatin around the inner areasin which cellsof monocyte morphology remained adherent surfaceof the nuclear envelope. The cytoplasm exhibited a ho- to the inner surface of blood vessels,in spite of intravascular mogeneousgranularity, active Golgi profiles, long linear arrays infusion of fixative at death (Fig. 12). Analysis of serial sections of rough ER, and numerous densephagocytic inclusions (Fig. through these regions demonstrated that processesemanating 11A). Thick branching processesemanated from the perikarya from these cells contacted the luminal surface of the endothe- and surrounded afflicted neuronal profiles in the neuropil (Fig. lium. These small sphericalto slightly oblong cells were 5-8 wrn 11A). These processesisolated infected neuronal profiles and in widest diameter, contained a denseorganelle poor cytoplasm, phagocytosedcellular debris (Fig. 1l A-C). However, even though and exhibited a spherical nucleus with densemarginated chro- microglia were intimately associatedwith infected neuronsand matin and few invaginations (Fig. 12). Cells of similar, but their processes,they never contained virions or exhibited any distinct, morphology were present in the distended perivascular morphological sign of infection. space.These cells were more oblong in appearance,exhibited a Monocytedmacrophages. One of the principal hallmarks of lobulated cell nucleus, and contained a richer complement of advanced viral infection was an alteration in the morphology cellular organellesand inclusions than the cells located in the of the vasculature. Blood vesselsin and immediately surround- blood vessels(Fig. 12). ing the infected DMV exhibited a distended perivascular space Small osmophilic cells were also present in the neuropil of and a thickened appearancedue to the accumulation of small the DMV, especially in relation to neuronsexhibiting advanced osmophilic cells (Fig. 1C,D; inset Fig. 12). Cellsof similar mor- degenerative changes(Fig. 13A). However, these cells differed phology were alsopresent in the surroundingneuropil, especially from thosein the perivascular spacein three important respects. in relation to neuronsexhibiting the most pronounced pathol- First, they were more irregular in conformation and gave rise ogy. The changesin vascular morphology were characteristic of to processesthat extended into the adjacent neuropil. Second, all animals infected with virulent strains of PRV, but were not they contained a multilobulated nucleus and a large collection

Figure 10. A illustratesthe perikaryon of an infectedastrocyte that is in direct contact with the somaof an infectedDMV motor neuron. Nucleocapsidsare presentin the cellnucleus, both individually and in aggregations(arrow). The boxed areas areillustrated at highermagnification in B and D. C is from anotherastrocyte in the samesample. Capsids surrounded by a singlemembrane are found within the distendedprofiles of ER (B). The limiting membranesof thesevirions fusewith the ER to releasenaked capsids into the cytoplasm(arrows in C); the point of fusion is demarcatedby the spikedcoat that is characteristicallypresent on the surfaceof the virions (largearrow in C). Capsidsreleased from the ER accumulatewithin the cytoplasmand never acquirea final envelope(0). Scalebars: A, 1 pm; B-D, 200 nm.

The Journal of Neuroscience, June 1993, 13(6) 2531

Figure 12. The vascular pathology that develops in response to infection of neurons with virulent strains of PRV is illustrated. The inset illustrates a large vessel in the DMV from an animal infected with PRV-4 53 hr prior to death. Note the thickened appearance of the vessel wall. The electron micrograph is from an ultrathin section adjacent to the area indicated by the arrow in the inset. The small spherical cell on the luminal side of the vessel exhibits ultrastructural features classically associated with monocytes. The dense cell in the perivascular space (H’s) is similar in morphology, but contains a lobulated nucleus and a larger collection of organelles and cytoplasmic inclusions. Dense packing of these cells in the perivascular space accounts for the thickened appearance of the vascular wall in thick plastic sections (inset). Scale bar, 1 pm. of electron-denselysosomal bodies in the cytoplasm. Finally, populations of neurons and that glia and macrophagesrestrict they contained many large phagocytic inclusions and were in- the spread of virus from infected neurons that become com- timately associatedwith neurons in advanced stagesof degen- promised. This conclusion is basedupon transmissionelectron eration. Often it appeared as if they were actually embedded microscopic (TEM) analysisof DMV neuronsinfected with vir- within the degeneratingcell (Fig. 13A). However, they did not ulent or attenuated strains of PRV and our previous light micro- exhibit signsof viral infection or pathology. scopic analysesof the transport of thesestrains in the CNS (Card et al., 1990, 1991, 1992; Rinaman et al., 1993). Our earlier Discussion studiesof PRV transport over longer survival intervals demon- Our data, summarized in Figures 9 and 14, support the conclu- strated that virus moves through the CNS in a circuit-specific sion that pseudorabiesvirus replicateswithin synaptically linked fashion and does not produce the widespread necrotizing pat- c Figure 11. A illustrates a ramified microglial cell in association with two infected DMV motor neurons. The cell exhibits the dense cytoplasm, active Golgi bodies, and linear stretches of rough ER typically associated with these cells. Large phagocytic inclusions are also evident in the cell body and processes (arrows). The boxed area is illustrated at higher magnification in B and in another plane of section in C. These micrographs reveal that the microglial cell is actively phagocytosing a degenerating afferent that terminates on a dendrite arising from an infected neuron (N). Ne, neuron; M, microglial cell. Scale bars: A, 2 pm; B and C, 500 nm. 2532 Card et al. l Transsynaptic Passage of Pseudorabies Virus

Figure 13. A DMV motor neuron exhibiting advanced dense vesicular degeneration is illustrated in A. The animal was infected with PRV-4 53 hr prior to death. The boxed areas are illustrated at higher magnification in B-D. In spite of the advanced pathology, the integrity of the neuron is maintained, and an afferent is still in contact with the cell soma (C). Also, note the intimate association of a tissue macrophage and a reactive The Jcumal of Neuroscience, June 1993. M(6) 2632

terns of infection that one would expect if it were spreading help us identify both the motor and sensorycomponents of the indiscriminately through the extraccllular space or by cell fusion. vagal innervation of the stomach that were infected with virus. However, other studies have shown that more virulent strains Finally, examination of multiple infected cells in each sample, of PRV and HSV sometimes produce neuropathology that ob- as well as the use of virulent and attenuated strains of virus, viously rcflccts nonspecific spread of virus (Norgren and Leh- permitted us to analyze cells in all stages of viral infection. man, 1989; Strack and Lowey, 1990). Thus, it is clear that our Collectively, this approach provided us with an accurate means findings cannot be generalized for all cu-hcrpcsvirus strains, and of assessing both the intracellular pathways of viral replication that analysis of viral transport within a well-defined temporal and egressas well as the the non-neuronal responseto viral window is essential for appropriate interpretation of circuit re- infection of neurons. lated transport of virus. Nevertheless, our data provide unique We observed three basic stagesin the CNS in responseto insights into the processes of replication and dissemination of PRV infection. The first stagewas manifested by capsid repli- well-characterized strains of PRV that are widely used for neu- cation in the cell nucleus and the appearanceof virions in lyso- ronal tract tracing, and also provide valuable insights into the somes.Lysosomal inclusions containing virions in various stages response of the CNS to viral infection. of degradation were demonstrableearly in the courseof cellular It can be argued that electron microscopy is ill suited for infection, but there was not a dramatic increasein the number making assertions about the temporal aspects of viral replication of these inclusions in neurons exhibiting more advanced pa- and transport in the CNS. The labor intensive nature of the thology. This suggeststhat the initial responseof afflicted cells method and the small sample size that can bc effcctivcly ex- was to shunt newly replicated virions to lysosomesand that this amined present formidable barriers to this type of analysis. For response was ultimately overwhelmed as the infection pro- example, it is always possible that the failure to observe non- gressed.The second stageinvolved productive replication and specific release of virus into the extracellular space is due to the specific egressof infectious virus. The directed assemblyand restricted sampling inherent to the method. We have taken the intracellular transport of virions in this phase led to transneu- following measures to address these considerations. Firsf, WC ronal passageof virus at sites of afferent synaptic contact. In are usinga system in which the neuronal architecture, connec- the third phase, infected neurons began to succumbto the in- tions, and responseto viral infection are well characterized. The fection and a non-neuronal responsewas mounted that limited polarized dcndritic tree of DMV neurons(Shapiro and Miselis, the spread of virus from compromised neurons. 1985) and its well-described connections with vagal sensory afferents (Rinaman et al., 1989) and NST neurons(Pickel et al., Virion replication and assembly 1989)present an ideal system for examining assemblyand trans- Replication and assemblyof a-herpesvirusesis a complex mul- neuronal passageof virus. Furthermore, we have previously tistep phenomenon that begins with viral DNA replication in characterized the transport of both the Becker and Bartha strains the nucleus and ends with the application of a membrane en- of PRV through this circuitry (Card et al., 1990; Miselis et al., velope (XC Roizman and Sears,1990, for a review). The precise 1991; Stemini et al., 1991; Rinaman et al., 1993). Second, we intracellular route of assembly is controversial, and a number processedanimals for light microscopic immunohistochemical of sitesof capsid envelopment have been proposed.The results analysisin parallel with thoseprocessed for TEM usingthe same of the present investigation are consistent with those revealed experimental parametrics and reagents.Consequently, WCwere in our previous in vitro analysis of PRV replication in immor- able to gaugethe relative transport of each of the three strains talized pig kidney fibroblasts (Whcaly et al., 1991) and also of PRV. Third, we standardized our analysis to the region of contain clcmcnts of models proposed for other herpesviruscs the DMV either immediately rostra1 to or at the lcvcl of the (Stackpole, 1969; Johnson and Spear, 1982, 1983; Jones and area postrema to reduce any variability in viral transport that Grose, 1988; Komuro et al., 1989). We found that, following might occur becauseofthe viscerotopic organization ofthe DMV/ viral DNA replication and packaging in the cell nucleus, nu- NST complex (Shapiro and Miselis, 1985; Rinaman and Miselis, cleocapsidsacquired a single membrane envelope by budding 1987; Altschuler et al., 199 1). Fourth, we conducted a rigorous through the inner leaf of the nuclear membrane. These mem- light microscopic analysis of thick sectionsimmediately adja- brane limited capsidsthen traversed the ER and were released cent to areas subjected to ultrastructural examination in order into the cytoplasm by a fusion event in which the limiting mem- to document our TEM analysis thoroughly and ensure that all brane of the vu-ion fused with the ER. Final envelopment oc- areas of pathology were analyzed. Fifth, many of the profiles curred at the Golgi apparatus where a bilaminar membrane were examined in serial ultrathin sections. We routinely cut derived from trans cistemae was applied to the capsid (Fig. 9). repeating seriesof 2-10 consecutive ultrathin sectionsof tissue Cupsid assembly. The structural organization of the icosa- infected with virulent strainsof PRV, and over 1500consecutive hedral structural protein capsid enclosing viral DNA is well sectionswcrc cut through the DMV of the animal infected with characterized, but the individual events leading to its assembly the attenuated strain. Sixth, the systematicfashion in which the in the cell nucleus remain elusive. Our data suggestthat the TEM analysiswas conducted ensuredthorough samplingof the complex array of 10-15 nm tubules that formed focal aggre- entire DMV/NST complex and identification ofall infcctcd pro- gations in the nuclei of infected neurons may be involved in files. Seventh, we used an electron-dense tracer (CT-HRP) to mobilization of the protein constituents of the capsid to focal c astrocyte (As) with the degenerating neuron in A. The macrophage contains several prominent phagosomes (open arrows), but shows no morphological indications of infection or degenerative pathology. There are numerous sites at which vesiculation in the neuron appears to be related to release of a cytotoxic substance from the macrophage (arrow in D). B shows a portion of the degenerating neuron being phagocytosed by a protoplasmic astrocyte that contains a prominent phagosome (arrow in A). Capsids and enveloped virions are prominent throughout the cytoplasm of the degenerating neuron (B and nrrow in D). Scale bars: A, 1 pm; B-D, 500 nm. 2534 Card et al. l Transsynaptic Passage of Pseudorabies Virus

Figure 14. The response of glia and macrophages early (A) and late (B) in the course of neuronal infection with PRV are illustrated. A (early response), Viral replication and assembly in neurons (I) leads to reactive astrogliosis that isolates infected neurons and the afferent axon terminals that synapse upon them (2). Microglia and macrophages show very little response at this stage. B (late response), Neurons advanced in the course of infection exhibit dense degeneration (I), and the astroglial response becomes more pronounced (2). Microglial cells increase in size and density in the vicinity of these cells and phagocytose degenerating neurons and processes (3). Monocytes leave the vasculature at the site of infection, differentiate into tissue macrophages, and become intimately associated with the degenerating neurons (4).

sitesin the nucleusfor assembly.These proteins are synthesized around coalescingviral DNA in the nuclei of infected cells. This in the cytoplasm of the cell and must find their way back into morphological association,along with the observation that cap- the cell nucleus to assembleand encapsulateviral DNA. Our sids in various stagesof assemblywere routinely enmeshedin data demonstrate that thesetubules often form circular arrays thesetubular arrays, leadsus to hypothesize that the intranuclear The Journal of Neuroscienca, June 1993, 13(6) 2535 tubule masses support efficient assembly of capsids by trans- gested that the viral cnvelopc is acquired either directly at the porting structural proteins to assembly sites and/or providing a Golgi (Dubois-Dalcq et al., 1982; Komuro et al., 1989) or from “scaffolding” for capsid assembly. Howcvcr, further analysis is Golgi-derived vesicles (Stackpole, 1969; Johnson and Grose, ncccssary to determine the validity of this hypothesis. 1988). Processing and packaging of glycoproteins, differential Envelopment. Previous in vitro studies of HSV and varicella sorting of vcsiclcs among cellular compartments, and membrane zoster virus have proposed that application ofthe viral envelope recycling are all essential functions of the Golgi (for recent re- to nucleocapsids occurs at the nuclear envelope (Johnson and views, see Geuze and Morre, 1991; Mollenhauer and Morre, Spear, 1982) by migration of immature particles through the I99 I ; Morre, I99 1; Mellman and Simons, 1992) that are re- Golgi cisternae (Johnson and Spear, 1983), or by budding of quircd for appropriate virion assembly and egress. While our capsids into Golgi-derived vesicles for bulk transport to the cell findings support the conclusion that the viral envelope is ac- membrane (Jones and Grose, 1988). Studies of PRV replication quired directly from the Golgi cistcmae, we cannot exclude the have also proposed a variety of sites for virion envelopment. possibility that capsids bud into Golgi-derived vesicles adjacent McCracken and Clarke (197 1) suggested that capsids acquire an to this organelle. Nevertheless, either mechanism of envelop- envelope from the inner and outer nuclear membranes, smooth ment incorporates the normal functional properties of the Golgi and rough endoplasmic reticulum, and the Golgi complex. Pal apparatus as an efficient means of endowing the capsid with an et al. (199 I b) studied three strains of PRV (including the Bartha envelope that contains mature glycoproteins, and also provides strain used in the present analysis) and concluded that there a mechanism of supplying the virus with targeting signals that were differences in the sites that virulent and attenuated strains would allow differential sorting of newly wrapped virions to were enveloped. They reported that only virulent strains ac- specific arcas of the cell (i.e., dendrites vs. axon). quircd an envelope at the nuclear membrane and further sug- gested that the failure of the Bartha strain to be enveloped at Intracellular targeting and egress of virions this site was due to the absence of the gene that encodes the gl One of the early signs of viral infection in DMV motor neurons virallenvelope glycoprotcin. In contrast, our data demonstrate included pathological invaginations of ccl1 nuclei and the ac- a common mode of envelopment for both virulent and atten- cumulation of degrading virions in lysosomes. The analysis of uated strains of PRV. All of the events associated with assembly serial ultrathin sections conducted on the PRV-Ba-infected of the two virulent strains also occurred in the assembly of the sample revealed that individual lysosomes were generally part Bartha strain, which lacks the gI gene. Thus, our observations of a larger group that were interconnected by narrow dense do not support the conclusion that the gl gene must be present tubules. These chains of variable sized varicosities are very for capsids to acquire a membrane at the nuclear envelope as similar to the continuous endosomal reticulum described by suggested by Pol et al. (1991 b). However, it remains possible Hopkins et al. (1990) and were most prevalent at the peripheral that this inconsistency is rclatcd to the different cell types (nasal aspects of the cell soma and in the proximal portions of primary mucosa vs. pig kidney fibroblasts and neurons) used in these dendrites. They appeared early in the course of infection, but studies. their numbers did not increase dramatically with advancing Our model of assembly contains many of the elements ob- viral replication. This suggests that their appearance may have served in previous studies, but we interpret thcsc events as part been the product of an initial attempt of the cell to protect itself of a larger pathway of assembly that ultimately leads to final from viral infection by targeting newly replicated virions to envelopment of capsids at the trans face of the Golgi. A critical lysosomcs for degradation, and that this response was subse- consideration in this model is that capsid envelopment at the quently overcome with advancing viral replication. We also inner membrane of the cell nucleus provides a means of trans- obscrvcd a spiked coat on the portion of the trans cistemae that porting capsids from the cell nucleus to the cytoplasm, and does wrapped capsids that was similar in morphology to the clathrin not reflect final envelopment of the virion. This hypothesis has coat applied to Golgi-derived vesicles sorted to lysosomes (see also been advanced by Lycke et al. in their in vitro analysis of review by Mellman and Simons, 1992). This coat was also ob- HSV assembly in human dorsal root ganglia sensory neurons served on the outer membrane of virions fusing with lysosomes. (Lycke et al., 1988) and is supported by a recent in vitro analysis With the available data we are unable to make an absolute of the role of viral envelope glycoproteins in PRV replication correlation of the occurrence of this coat with a particular stage and egress (Peeters et al., 1992). Lycke et al. (1988) demon- of infection. We can say that it was routinely observed on the strated capsids budding through the inner leaf of the membrane trans Golgi cistemae ofcells exhibiting early morphological signs and further postulated that fusion of this membrane with the of infection and that it was more difficult to demonstrate in cells outer leaf of the nuclear envelope was an “essential step” in with advanced pathology. However, a more systematic exam- providing capsids access to the cytoplasm. Peeters et al. (1992) ination of cells in various stages of infection is required to sup- demonstrated that capsids produced by mutant strains of PRV, port the assertion that the presence of this coat is correlated lacking the gene for envelope glycoprotein gI1, bud through the with sorting of virions to lysosomes. inner leaf of the nuclear membrane, but are incapable of fusing There was no evidence in our material that lysosomal inclu- with the outer leaf. Thus, virions accumulate within the peri- sions or fusion of infected cells contributed to dissemination of nuclear space, and infectious virus is never produced by the cell. virus in the CNS. Lysosomal inclusions filled with virions were These data support the contention that membrane acquisition confined to the soma and primary dendrites of infected cells, at the nuclear envelope simply provides nuclcocapsids with a and we never observed any of these large inclusion bodies fusing means of gaining access to the cytoplasm via the ER. with the plasma membrane to release virions into the extracel- The data from this and our previous investigation (Whealy lular space. Similarly, we observed many cases in which infected et al., 1991) indicate that the final envelope applied to newly neurons and glia were in direct apposition, but there was ncvcr replicated virions is derived from the trans face of the Golgi. any evidence that these cells had fused. Thus, the in vitro dem- Other investigations of a-herpesvirus envelopment have sug- onstration ofvirus-induced cell fusion (reviewed by Spear, 1984) 2536 Card et al. l Transsynaptic Passage of Pseudorabiis Virus

does not appear to be relevant to spread of PRV through the been elucidated. Our data strongly support the conclusion that CNS. This observation is consistent with the many studies that newly assembled virions arc differentially targeted to the den- have shown that a-herpesviruses gcncrally pass through the ncr- dritic tree of infcctcd central neurons and are specifically re- vous system in a circuit-specific fashion rather than via lateral leased at sites of afferent contact. This observation is consistent spread. with mapping studies that have shown that PRV travels through It is our impression that the morphology of virions destined CNS is dependent upon a gene designated .,34.5. Deletion of for release from infected neurons differed from those targeted et al., 1990; Strack and Loewy, 1990; Miselis et al., 199 1; Ster- to lysosomes. This conclusion is supported by our analysis of nini et al., 1991; Jansen et al., 1992; Rotto-Percelay, 1992). virion morphology in the perikarya and processes of DMV mo- However, the mechanisms involved in virion fusion with af- tor neurons as well as the early in vitro studies of cu-herpesvirus ferent terminals following release from infected neurons require assembly and egress (Morgan ct al., 1959; Nii et al., 1968a,b; further definition. It has not been established that fusion of McCracken and Clarke, 1971; Fong ct al., 1973). Fong ct al. virions with afferent terminals is a specific event or mcrcly the (1973) in particular, noted that the envelope morphology of product of reuptake processes such as those that permit HRP, guinea pig herpes-like particles varied “depending upon the lo- latex microspheres, and other retrograde tracers to gain access cation ofthe virus” in the cell. In our analysis, virions associated to axon terminals (Kristensson and Olsen, 1971; LaVail and with lysosomes characteristically exhibited membrane decora- LaVail, 1973; Katz et al., 1984; Schmued and Fallon, 1986). A tions on the outer surface of the external membrane, whereas substantial literature supports the conclusion that cY-herpesvirus virions in the dendritic tree were more spherical and slightly invasion of cells is a receptor-mediated event involving viral smaller (approximately 180 nm), and the envelope was ofgreater glycoproteins and an as yet uncharacterized receptor (Spear, density and smoother conformation. Our data also dcmonstrat- 1984; Roizman and Sears, 1990). However, these conclusions ed that axons of infected motor neurons only contained free are based primarily upon in vitro investigations of a variety of capsids, whereas vu-ions endowed with a double membrane en- cell lines, and do not consider the relatively “nonspecific” up- vclopc wcrc diffcrcntially concentrated in the somata and den- take mechanisms that permit latex microspheres to gain access drites. These data confirm the elegant in vitro findings of Lycke to axons. Consequently, it remains possible that specific trans- ct al. (I 988) and Marchand and Schwab (1986) who demon- neuronal passage of virus is dependent upon the ability of neu- stratcd that viral DNA was retrogradely transported to neuronal rons to sort virions differentially to specific areas of the cell (i.e., perikarya in free capsids, and that double-enveloped virions the postsynaptic membrane), and that viral uptake at afferent only appeared in the perikarya and processes after viral repli- terminals is a relatively nonspccilic, secondary event. Further cation had occurred. The differential intracellular distribution analysis of the uptake and transport of viral mutants lacking of enveloped virions is also consistent with the demonstration glycoproteins shown to bc essential for invasion of cells in vitro that viral glycoproteins arc sorted to the dendrites of cultured is necessary to address this question. hippocampal neurons (Dotti and Simmons, 1992). Collectively, these findings indicate that the differential intracellular distri- Neuropathogenesis bution of morphologically distinct virions recognized in earlier There is little doubt that infection of neurons with PRV ulti- in vifro studies is even more apparent in the highly polarized mately leads to cell death. This response is most evident in cellular compartments of neurons, and that the structural fea- neurons infected with virulent strains of virus, but also occurs tures of virions in different cellular compartments provide clear in neurons infected with attenuated strains after prolonged viral morphological correlates of events that occur during viral entry, replication (Card et al., 1992). The fundamental mechanisms replication, and egress. underlying this rcsponsc have not been fully characterized, but Although release sites always occurred adjacent to afferent available evidence suggests that the virally induced cytopatho- terminals, the precise site of virion egress was not always into genesis may occur in response to expression of gene products the synaptic cleft (see summary, Fig. 9). The critical cvcnt in cncodcd by the virus as well as via the ncuroimmune response rclcasc was fusion of the outer membrane of the virion envelope elicited by viral infection. Chou et al. (1990) have recently shown with the postsynaptic membrane of the infected cell. A second that the ability of HSV- 1 to induce lethal infection of the mouse fusion event between the remaining membrane of the viral en- CNS is dcpcndcnt upon a gene designated ,,34.5. Deletion of velope and the membrane of the afferent terminal then released the two copies of this gene or insertion of stop codons produces the naked capsid into the afferent. These observations are con- strains of HSV that are avirulcnt following intracerebral inoc- sistent with previous studies that have demonstrated that a ulation. It is not yet known whether PRV contains a similar membrane is necessary for both release of infectious virus as gene, but a number of recent studies have demonstrated a re- well as viral invasion of another cell (see Nii et al., 1968a, for duction in neurovirulence following deletion of genes that en- discussion). However, it should be noted that the topology of code PRV envelope glycoproteins (Ben-Porat and Kaplin, 1985; the viral glycoproteins in the outer membrane of this bilaminar Card et al., 1992; Kimman et al., 1992; Zsak et al., 1992). Thus, envelope is such that the cytoplasmic domains are on the out- at least some of the cytopathic effects observed in infected neu- side. Thus, fusion of this membrane with the host plasma mem- rons may bc due to expression of virally encoded genes. brane releases a virion in which envelope glycoproteins are in Viral infections have also been shown to elicit the expression the proper conformation necessary for performing their func- of a variety of cytokincs that may contribute, either directly or tions in recognition, attachment, and invasion of susceptible indirectly, to death of infected neurons. For example, cells. In contrast, the initial fusion event that releases the virion interleukin-6 (Frci et al., 1989; Lieberman et al., 1989) tumor from the cell involves a membrane in which these glycoproteins necrosis factor (TNF) (Lieberman et al., 1989), and interferons are in the opposite conformation. (Lieberman et al., 1989) are secreted by astrocytes in response The mechanism(s) that would confer specificity on the intra- to neurotropic viral infections. The expression of TNF under cellular targeting of virions to postsynaptic membranes has not these circumstances is particularly intriguing in light of the di- Tha Journal of Neuroscience. June 1993. U(6) 2537

verse role that this cytokine has been shown to play in neu- 1977) had commented on the absence of enveloped virions in roinflammatory responses of the CNS (Beutlcr and Cerami, satellite glia and astrocytcs, but there was no clear indication 1989). In particular, our work has demonstrated that macro- that these cells harbored a replication defect. We observed cap- phages are prevalent in areas of PRV-induced neuropathogene- sid replication in the nuclei of infected astrocytcs, and these sis (see also Rinaman et al., 1993) and that thcsc cells establish capsids budded through the nuclear envelope and were trans- very intimate associations with neurons in advanced stages of ported to the cytoplasm via the ER in the same manner that dcgencration. Our data also suggest that these macrophages are occurred in neurons. However, rather than being enveloped, derived from peripheral blood monocytcs that migrate into the naked capsids accumulated in large aggregations in the astrocyte CNS and differentiate into tissue macrophages. Both of these cytoplasm and enveloped virions were never obscrvcd in sys- observations are interesting in light of studies that have shown tcmatic examination of serial sections. The fundamental mcch- that astrocytes infected with neurotropic viruses sccretc TNF anism underlying this defect remains to be established, but the (Lieberman et al., 1989), and that TNF is involved in macro- existence ofa replication defect in astrocytes indicates that these phage differentiation (Witsell and Schook, 1992) and also in- cells act as a reservoir for vii-ions that do not gain access to duces expression of adhesion molecules by neurons (Birdsall et afferents and thereby limit indiscriminate spread of virus through al., 1992). Our ultrastructural data revealed that macrophages the extracellular space. were literally attached to the surface of necrotic neurons and Our data also support previous reports (Morahan, 1983; Mor- may have contributed to the dcgenerativc vcsiculation that was ahan et al., 1985; Inglesias et al., 1989; Rinaman et al., 1993) so prevalent in these compromised neurons. This response is demonstrating that different populations ofglia are differentially quite similar to the TNF-induced facilitation of mononuclear susceptible to viral infection. Although microglia were prevalent leukocyte adherence to human cortical neurons and ncuroblas- throughout all of our preparations and were intimately associ- toma cells in response to expression of cellular adhesion mol- ated with neurons exhibiting advanced pathology, we never ob- ecules by these cells (Birdsall et al., 1992). In addition, Thery served any evidence of viral infection in these cells. This is et al. (199 1) recently demonstrated cytotoxic effects of brain consistent with our previous light microscopic observations on macrophages on developing neurons. Collectively, these obscr- the response of immunocytochemically identified ramified mi- vations suggest a complex interaction of cytokines, adhesion croglia to PRV infection (Rinaman et al., 1993) and the dem- molecules, and cytotoxic macrophagcs in virally induced patho- onstration that microglia resist infection by HSV (Weinstein ct genesis. al., 1990). Thus, the primary function of these cells appears to bc phagocytosis of degenerating neurons. The role of glia in transneuronal passage of virus Three aspects of our findings suggest that glia play a prominent Conclusions role in ensuring circuit-specific passage of virus through the Our findings suggest that spread of PRV through the CNS fol- CNS. First, our data support the conclusion that astrocytes and lowing peripheral inoculation occurs by retrograde transneu- microglia respond to viral infection of neurons by undergoing ronal passage of virus through synaptically linked populations a reactive gliosis that effectively isolates infected neurons and of neurons. The specificity of this transport is the product of a contributes to the removal of cellular debris. Second, the data complex process of viral replication, assembly, and intracellular from this and other investigations (Field and Hill, 1974; Bak et sorting that specifically targets newly replicated vu-ions to sites al., 1977) indicate that astrocytes are susceptible to infection by ofafferent synaptic contact on the cell soma and dendritic arbor. PRV, but are incapable of releasing infectious virus. Thus, the Glial cells and infiltrating macrophages also play a prominent astrocytic cells and processes that ensconce infected neurons role in maintaining the specificity oftransneuronal passage. These provide a very effective barrier that limits spread of virus from cells act to isolate infcctcd neuronal profles and thereby prevent sites of primary infection, irrespective of the pathological state nonspecific dissemination of virus through the extracellular space. of the afflicted neuron. Third, although microglia phagocytose The potential for spread of virus through infected astrocytes is infected neurons, they are resistant to PRV infection. When obviated by the inability of these cells to produce infectious considered with the numerous reproducible demonstrations of virus. Thus, they act as a sink for virions that are released from unique, circuit-specific transport of virus in the CNS, these data necrotic neurons at advanced stages of infection. strongly support the conclusion that the glial response to neu- ronal infection effectively limits nonspecific spread of virus. Astrocytes are among the first cells to respond to PRV infec- References tion of neurons (Rinaman et al., 1993) and play a prominent Altschuler SM, Ferenci DA, Lynn RB, Miselis RR (1991) Represen- role in restricting the spread of virus. Filament-rich processes tation of the cecum in the lateral dorsal motor nucleus of the vagus nerve and commissural subnucleus of the nucleus tractus solitarii in of these cells were present on the surface of infected neurons, rat. J Comp Neurol 304:26 1-274. and their processes surrounded afferents synapsing on these cells. 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