Neuron,

Vol.

6, 957-969,

June,

1991,

Copyright

0

1991

by Cell

Press

Two a-Herpesvirus Strains Are Transported Differentially in the Rodent Visual System J. P. Card,* M. E. Whealy,* A. K. Robbins,* R. Y. Moore,+ and 1. W. Enquist* *Viral Diseases Group Du Pont Merck Pharmaceutical Company Wilmington, Delaware 19880-0228 +Departments of Psychiatry, Neurology, and Behavioral Neurosciences and Center for Neuroscience University of Pittsburgh Pittsburgh, Pennsylvania 15261

Summary Uptake and transneuronal passage of wild-type and attenuated strains of a swine a-herpesvirus (pseudorabies [PRV]) were examined in rat visual projections. Both strains of virus infected subpopulations of retinal ganglion cells and passed transneuronally to infect retinorecipient neurons in the forebrain. However, the location of infected forebrain neurons varied with the strain of virus. lntravitreal injection of wild-type virus produced two temporally separated waves of infection that eventually reached all known retino-recipient regions of the central neuraxis. By contrast, the attenuated strain of PRV selectively infected a functionally distinct subset of retinal ganglion cells with restricted central projections. The data indicate that projection-specific groups of ganglion cells are differentially susceptible to the two strains of virus and suggest that this sensitivity may be receptor mediated. Introduction a-Herpesviruses (members of the Herpesviridae family) exhibit a predilection for infection of nervous tissue, particularly sensory neurons (Roizman, 1990). Although infection of sensory ganglia generally results in establishment of latency, infection of neurons in the CNS is often lytic (Roizman and Sears, 1990). Nevertheless, the lytic nature of CNS infections is not manifested by widespread necrosis. Rather, analysis of the neuro-invasive propertiesof a number of differentstrainsofhuman(herpessimplex[HSV])andswine (pseudorabies [PRV]) a-herpesviruses has demonstrated remarkable selectivity in viral transport and replication in the brain (see Kuypers and Ugolini, 1990, for review). In every instance reported to date, first orderneuronal infection isquicklyfollowed byspread of virus to other central neurons in a pattern largely consistent with transport of the virus through functional neural circuits (Kristensson et al., 1974; Field and Hill, 1975; Bak et al., 1977; Dolivo et al., 1978; Martin and Dolivo, 1983; Kuceraet al., 1985; Ugolini et al., 1987, 1989; Margolis et al., 1987, 1989; Norgren and Lehman, 1989; Rouiller et al., 1986, 1989; Strack et al., 1989a, 1989b; McLean et al., 1989; Strack and Loewy,

1990; Cardetal., 1990).Thesedemonstrationsof reproducible, projection-specific patterns of viral transport support the conclusion that transneuronal passage of virions occurs at sites of synaptic contact, but very little is known regarding the fundamental mechanisms that would impart such specificity. The circuit-specific pattern of viral transport in the CNS is certainly the product of a complex series of cellular events initiated by viral recognition, attachment, and invasion of susceptible neurons. Although the mechanisms that account for the neuro-invasive properties of a-herpesviruses have not been fully characterized, it is likely that the initial attachment of virus to neurons is a function of both virus-encoded glycoproteins present in the viral envelope and cellular receptors located on the surface of target neurons. The envelopes of a-herpesviruses contain a variety of integral membrane glycoproteins that are conserved to varying degrees among different strains of virus and are thought to serve common functions (see Wittmann and Rziha, 1989, for a review of PRV and HSV homologs). Seven of these proteins have been characterized in the envelope of HSV-1 and PRV, and at least three have been strongly implicated in virus-cell fusion (Sarmiento et al., 1979; Little et al., 1981; Fuller and Spear, 1987; Manservigi et al., 1990; Roizman and Sears, 1990). Of particular interest are the recent demonstrations that HSV-I mutants lacking gD, an essential envelope glycoprotein, are unable to enter cells (Johnson and Ligas, 1988) and that soluble forms of this glycoprotein inhibit virus entry by binding to a cellular receptor (Johnson et al., 1990). Nevertheless, little is known of the specific receptors involved in viral infection. Specific binding sites for HSV-1 and HSV-2 have been demonstrated on both neurons and glia (Vahlne et al., 1979, 1980), and lectins have been shown to inhibit HSV and PRV binding to sensory neurons in vitro (Ziegler and Pozos, 1981; Marchand and Schwab, 1986). Furthermore, recent reports indicate that one or more of the envelope glycoproteins of HSV (WuDunn and Spear, 1989) and PRV (Mettenleiter et al., 1990) bind heparin sulfate proteoglycan in the initial stages of virion attachment, and Kanar and collaborators (1990) suggest that the basic fibroblast growth factor receptor may provide a”portal of entry” for HSV infection. Interestingly, both heparin sulfate proteoglycan and the fibroblast growth factor receptor (Wanaka et al., 1990) are widely distributed throughout the neuraxis. Collectively, theseobservations support the conclusion that a-herpesvirus infections of the CNS are the product of a specific interaction between viral glycoproteins and neuronal receptors and raise the possibility that projection-specific transport of virus through neural circuits may be explained by a differential distribution of these receptors in the CNS. To gain further insight into the specific viral gene products involved in the neuro-invasiveness of a-her-

NellVXl 958

pesviruses, we examined uptake and transport of two independent isolates of PRV (Becker and Bartha) in rat visual circuitry. PRV, like HSV, is an a-herpesvirus with a broad host range, but PRV’s primary host is pigs (Wittmann and Rziha, 1989). Our wild-type strain of PRV (Becker, PRV-Be; Becker, 1967) is a virulent field isolate, whereas Bartha (PRV-Ba; Bartha, 1961) is an attenuated vaccine strain characterized by several well-mapped mutations that affect neuro-virulence (Lomniczi et al., 1984a, 1984b,l987; Hampl et al., 1984; Petrovskis et al., 1986; Robbins et al., 1989). Of particular interest for this report is the fact that Bartha harbors a deletion affecting two prominent envelope glycoproteins (gp63 and gl) and a signal sequence mutation of a third major envelope glycoprotein (gll I), which dramatically reduces its concentration in the viral envelope. Additionally, Bartha contains an uncharacterized neuro-virulence defect in the BamHI4 fragment encoding several genes involved in capsid biosynthesis. Nevertheless, both strains of virus are known to infect efficiently functional circuits of neurons in the rodent nervous system following retrograde transport from peripheral targets (Dolivo et al., 1978; Martin and Dolivo, 1983; Rouiller et al., 1986, 1989; Strack et al., 1989a, 1984b; Strack and Loewy, 1990; Card et al., 1990). The present comparison indicates that both strains of PRV can infect the brain through anterograde, transneuronal transport and demonstrates strain-specific differences in the pattern of transport that provide a basis for examining the specificity of a-herpesvirus neuro-invasiveness. Results Both strains of virus infected ganglion cells in the retina and passed transneuronally to infect retino-recipient neurons in forebrain. With increasing survival, other infected neurons became evident in both the retina and forebrain. The location and temporal appearanceof infected neurons in each of these regions wereconsistent with passageof virus through synaptically linked populations of cells. For example, viral infection of the retina began in ganglion cells and, with increasing survival, spread through the deeper layers of retina in a manner that reflected previously documented retinal microcircuitry (see Ehinger and Dowling, 1987, for review). Similarly, the first sites of neuronal infection in the forebrain were entirelycoextensive with the known distribution of retinal afferents. The circuit-specific nature of this transport was further emphasized by the unique and reproducible patterns of transport produced by PRV-Be and PRV-Ba. Both strains produced patterns of neuronal infection that could be reliably distinguished on the basis of the temporal course of infection and the location of neurons infected by each strain. In no instance did we observe infection of neurons that would not be predicted on the basis of either the termination of primary visual projections or the subsequent transport of virus through functional neuronal circuits. In

this report we concentrate upon the differential infection of primary retino-recipient neurons that we observed following injection of the two strains of PRV. Details of the organization of neurons identified by subsequent transneuronal passage of virus through brain circuitry are beyond the scope of the present investigation and will be the subject of another communication. Viral Uptake and Replication in the Retina Three aspects of viral uptake and replication in retinal ganglion cells were common to both PRV-Be and PRVBa. First, there was aclear temporal aspect to the viral invasion of the retina. At the earliest time point examined (24 hr postinjection) viral immunoreactivity was confined to ganglion cells on the vitreal surface of the retina (Figure IA). However, with advancing survival, viruspassedtransneuronallyto infectcolumnsofcells in the retina that spanned its full cross-sectional extent (Figure IB). Second, each strain of virus selectively infected subgroups of the total population of ganglion cells at all survival times. Examination of retinal sections at postinjection intervals extending to 81 hr following PRV-Be injection and 122 hr after injections of PRV-Ba (the longest survival times included in either experimental group) clearly showed that the number of infected cells in any given section was a small percentage of the total population of ganglion cells. Although the spatial distribution of infected ganglion cells could not be determined with confidence in this material, two cases (PRV-Be at 48 hr survival and PRV-Ba at 96 hr survival) in which immunohistochemical localizations were conducted on retinal whole mounts confirmed the above observation and also showed that infected cells were distributed across the full extent of the retina (Figure IC). Finally, infection of ganglion cells by either strain of virus led to pronounced accumulation of viral immunoreactivity in optic axons on the vitreal surface of the retina and in the proximal portion of the optic nerve (Figure ID). The appearance of PRV immunoreactivity in optic axons always occurred after virus had been detected in ganglion cells and before infected neurons were apparent in the forebrain. Forebrain Targets of PRV-Be Unilateral injection of the virulent, wild-type virus led to two waves of central neuronal infection that were separated by approximately 24 hr (Figures 2-5). Each wave of infection targeted functionally distinct components of the central visual projection fields, and ultimately all subcortical visual nuclei exhibited prominent neuronal infection. This included retino-recipient neurons in the hypothalamus (suprachiasmatic nuclei [SCN] and other cell groups in retinohypothalamic projection fields), the thalamus (dorsal geniculate nucleus, intergeniculate leaflet [IGL], and ventral geniculate nucleus [VGN]) and distinct cell groups in the midbrain (tectum and accessory optic nuclei). The visual cortex was not included in this analysis. In each

Ditierential

Transport

a-Herpeswrus

in CNS

959

Figure

1. Uptake

and Transneuronal

Passage of PRV

Representative patterns of viral immunoreactivity in the retina (A-D) and in the geniculate complex of the thalamus resulting from intravitreal injection of PRV-Be are illustrated. lmmunohistochemically detectable virus is first apparent in retinal ganglion cells (A) and at longer survival times passes transneuronally into the deeper layers of the retina (B). Localization of virus in retinal whole mounts (C)demonstrates that infected neurons are a subset of the total population of ganglion cells. Uptake and replication of virus by ganglion cells is followed by passage of virus into optic axons and by subsequent anterograde transport of virus to forebrain (D). Anterograde transport of PRV to forebrain leads to transneuronal infection of neurons in a pattern that is entirely coextensive with the distribution of retinal afferents and also reflects the heavier crossed retinal projections (E and F). (E) and (F) illustrate the distribution of infected neurons in the dorsal geniculate nucleus and IGL (small arrows) contralateral (E) and ipsilateral (F) to the injected eye. Note that the ipsilateral projection to the dorsal geniculate nucleus is confined to a focal area in the dorsomedial aspect of the nucleus (large arrow in [F]) and that the number of infected neurons is larger in the nucleus contralateral to the injected eye.

of the rons

above overlapped

regions, the

the

distribution

terminal

of infected

arborization

of

neuretinal

afferents, which have previously been characterized with anterograde tracers (see Parnavelas et al., 1989, for review). Furthermore, the number of infected ceils ipsilateral and contralateral to the injected eye reflected the heavier crossed visual projections that typifythe rodent visual system. This was readily apparent in the distribution of infected neurons in the dorsal geniculate nuclei (DGN) following intravitreal injection of PRV-Be (Figures IE and IF). In the DGN contra-

lateral to the injected eye, infected neurons were prevalent in the ventral and lateral aspect of the nucleus (Figure IE). In contrast, only a moderate number of infected cells were present in the ipsilateral DGN, and they were confined to a circumscribed area in the medial aspect of the nucleus (Figure IF). In both nuclei, the location and relative number of infected cellscorresponded tothe reported distribution of retinal afferents labeled by injection of classic anterograde tracers into one eye (Parnavelas et al., 1989). The first indication of viral immunoreactivity in-

NellrOll 960

Bartha

Becker

OC

74hrs

1 '1) \

VIII *

c '?

.

,.

-

c

l

c

S

. ‘i OC

,

“OC

81hrs

VIII

0 VIII

Figure

2. Strain-Specific

Viral Infection

of the SCN

lmmunohistochemical detection of virus in the SCN illustrates the patterns of viral infection resulting from intraocular injectron oi the wild-type (A, C, and E) and attenuated (B, D, and F) strains of virus. The first significant concentration of neurons infected by either strain occurs at approximately 74 hr, and the number of infected cells increases progressively at longer survival intervals. VIII, third ventricle; OC, optic chiasm.

duced by PRV-Be injection occurred approximately 48 hr following intravitreal injection of the virus with the appearance of infected neurons in the DGN, the medial terminal nucleus of the accessory optic system, the pretectal area, and the superior colliculus (Figure 3A; Figure 5). Only scattered infected neurons were present in theVGN atthis time point, and the IGL (Figure 3A) and SCN (Figure 2A) of the hypothalamus exhibited virtually no infected ceils. The second wave of infection began approximately 72 hr postinjection and was marked by the appearance of a moderate

number of infected neurons in the SCN (Figure 2C), the IGL (Figure 3C), and the lateral terminal nucleus of the accessory optic system. In addition, the number of infected neurons in areas targeted by the first wave of infection (DGN, VGN, medial terminal nucleus, and superior colliculus) increased (Figure 3C; Figure 4A). At longer survival times, the number of infected neurons in all retino-recipient regions increased progressively, but remained confined to regions defined by the terminal arborization of retinal afferents (Figure 2E; Figure 3E; Figure 4C; Figure 5). The longest survival

C ifferentlal 9 11

a-Herpesvirus

Transport

in CNS

Becker

Bartha

51 hrs

74 hrs 9

90hrs

Figure

3. Strain-Specific

Viral

Infection

of the Geniculate

, 9

.

122hrs

1.

Complex

lmmunohistochemical localization of virus in the geniculatecomplexdemonstrates remarkably different patternsof viral infection after intravitreal injection of the two strains of PRV. The wild-type virus (Becker) initially infects neurons in the DGN, but ultimately afflicts neurons in all three subdivisions (A, C, and E). In contrast, the attenuated strain of PRV (Bartha) selectively infects neurons in the IGL (defined by arrows) and the VGN; the DGN is distinguished by an absence of infected cells (B, D, and E).

times were also characterized by the spread of PRV-Be infection to glia. Nevertheless, neuronal infection in these cases always preceded the glial infection and was confined to areas coextensive with distribution of retinal afferents.

Hypothalamus PRV-Be infection of the hypothalamuswas largelyconfined to the SCN at all survival times. During the first wave of infection of forebrain (onset at approximately 48 hr), only scattered cells were present in the SCN

(Figure 2A). However, at the onset of the second wave of infection (approximately72 hr), moderate numbers of cells became apparent (Figure 2C), and the number of infected neurons increased progressively through 81 hr (Figure 2E; Figure 5). As noted above, the majority of these cells were confined to the ventrolateral aspect of the nucleus, which receives a dense retinal innervation. Additionally, many more infected neurons were present in the nucleus contralateral to the injected eye in a manner consistent with the denser

Neuron 962

Becker

Bartha 96 hrs

Figure 4. Strain-Specific

Viral Infection

of the Colliculus

lntravitreal injectionof PRV-Beand PRV-Baproducesdramaticallydifferent patternsoftransneuronal infection of thesuperior colliculus. While the PRV-Be infects large numbers of neurons in the superficial layers of the colliculus (A and C), the same areas are essentially devoid of infected neurons at survival intervals extending to 122 hr after injection of the PRV-Ba.

contralateral retinohypothalamic projection (Moore and Lenn, 1972). At the longest survival times (72-81 hr), scattered infected cells were evident in the lateral hypothalamic area and preoptic hypothalamus in a pattern that overlapped the termination of retinohypothalamic afferents in these areas (Pickard, 1982; Johnson et al., 1988). Thalamus Transneuronal infection of DGN neuronswith PRV-Be occurred in a pattern that mirrored the distribution and density of retinal afferents in this area (Figures IE and IF). Infected neurons were first apparent at 48 hr in the ventrolateral aspect of the DGN contralateral to the injected eye (Figure 3A); the ipsilateral DGN was unaffected. By 74 hr the number of infected neurons in the ventral and lateral aspect of the contralateral DGN increased substantially (Figure 3C; Figure 5), and infected neurons also became apparent in the medial subdivision of the ipsilateral nucleus (Figure IE). By 81 hr postinjection, large numbers of infected neurons were present throughout the contralateral DGN (Figure IE; Figure 3E), but viral immunoreactivity in the ipsilateral DGN remained confined to the medial subfield (Figure IF).

Viral replication in the other two lateral geniculate subdivisions (IGL and VGN) also correlated with the distribution and density of retinal afferents, but the temporal appearance of virus in the IGL occurred later. Scattered infected neurons were first apparent in the ventral and lateral aspect of the contralateral VGN early during the first wave of infection and increased progressively in these regions with advancing survival time (Figures 3C and 3E; Figure 5). Small numbers of infected neurons were also present in comparable areas of the ipsilateral VGN by 51 hr and increased in number with advancing survival. In contrast to the VGN and DGN, infected neurons were not apparent in the IGL prior to 74 hr (Figures 3A and 3C). Thereafter, the number of infected neurons increased progressively, and substantial numbers of cells were present by 81 hr survival (Figure 3E; Figure 5). Once again, the distribution and density of infected neurons reflected the heavier crossed retinal projection (compare Figures IE and IF). Midbrain The most prominent infection of midbrain neurons by PRV-Be occurred in the superior colliculus. Small numbers of infected neurons became apparent in the

Differential 963

a-Herpesvirus

Transport

in CNS

contralateral colliculus during the first wave of infection. The initial infection occurred in the superficial layers of the medial half of the colliculus and rapidly spread laterally (compare Figures 4A and 40. By 81 hr postinjection, the infection of neurons and glia in these regions was so dense as to preclude identification of individual cells. Nevertheless, only scattered infected cells occurred in the deeper layers of the contralateral colliculus,and the ipsilateral tectum was virtually unafflicted. This distribution was consistent with previous reports that have shown the retinotectal projection to be entirely crossed in the rat with a restricted terminal arborization in superficial layers of the nucleus. Other retino-recipient areas of midbrain were also infected with PRV-Be in a manner consistentwith passage of virus through retinal afferents. The optic pretectal nuclei exhibited bilateral infectionof largenumbersof neuronswithin hr of eye injection. Similarly, thecontralateral medial terminal nucleusof the accessory system exhibited prominent viral immunoreactivity by48 hr postinjection, and neurons in the lateral terminal nucleus became infected in the same temporal sequence exhibited by the SCN and IGL (72 hr and later).

TemDoral z

so

5

40

AsDects

of

Viral

TransDort

i $30 2 ,s

20 10 50

Figure 5. Quantitation

70

80

90

100

120

of Viral Infectivity

Forebrain Targets of PRV-Ba

Quantitative analysis of the number of infected cells in the SCN, ICL, DCN, and tectum at various survival times after injection of each strain of virus is illustrated. The horizontal axis indicates the postinjection survival time, and the vertical axis indicates the average number of infected neurons that occurred in individual sections through the largest cross-sectional diameter of each area. Asterisks indicate that the average number of cells per tissue section exceeded 50. Injection of PRV-Be produces two, temporally separated waves of infection that ultimately afflict neurons in all known retino-recipient areas of forebrain. By contrast, PRV-Ba infects only those neurons targeted by the second wave of PRV-Be infection.

The pattern of viral infection resulting from intravitreal injection of PRV-Ba was markedly different from that observed after injection of PRV-Be. Regions that characteristically exhibited large concentrations of infected neurons during the first wave (48-72 hr) of PRV-Be infection (DGN, tectum, and medial terminal nucleus) remained essentially devoid of infected neuronsat postinjection intervalsextendingto122 hrafter injection of PRV-Ba (Figures 38,3D, and 3F; Figures 48 and 4D). However, neurons targeted by the second wave of PRV-Be infection (72 hr and longer) were susceptibleto infection by PRV-Ba,and thetimecourseof infection in these areas (SCN and IGL) was essentially identical with both viral strains. Hypothalamus The first signs of viral infection by PRV-Ba in any area of the hypothalamus occurred 72 hr following intravitreal injection of the virus (Figure 2B; Figure 5). At this time point, scattered neurons were present in the ventrolateral aspect of the SCN, and in some instances, infected cells were also present in the adjacent anterior and lateral hypothalamic areas. This distribution is consistent with the dense retinal innervation of the ventrolateral SCN (Moore and Lenn, 1972) and the recent studies using sensitive anterograde tracers that have demonstrated expanded retinohypothalamic fields extending into both the anterior and lateral hypothalamic regions (Pickard, 1982; Johnson et al., 1988). With increasing survival the number of infected neurons in the ventrolateral SCN increased progressively (Figure 2D), and at periods exceeding 100 hr, virtually every neuron in both the ventrolateral and dorsomedial subdivisions of the nucleus exhibited vi-

ral immunoreactivity (Figure 2F). The fact that neurons in the dorsomedial subdivision were ultimately infected with virus at extended survival times is probably due to transneuronal passage of the virus through the extensive local circuit projections that are known to connect both SCN subfields (Card et al:, 1981; van den Pol and Tsujimoto, 1985). The relative number of neurons infected in the SCN at early time points was also consistent with the larger crossed retinal projection; larger numbers of infected neurons were observed in the SCN contralateral to the injected eye. Thalamus and Midbrain The most dramatic demonstration of differential transport of the two strains of PRV was evident in the geniculate complex and the superior colliculus. In the geniculate, viral immunoreactivity was first apparent in IGL neurons at 72 hr postinjection, and no infected neurons were present in the DGN (Figure 3B). With increasing time, the number of infected IGL neurons increased progressively, but the DGN remained free of immunoreactive neurons at survival times extending to 122 hr (Figures 3D and 3F). Similarly, the pattern of viral infection in the VGN resulting from PRV-Ba injection differed from that which occurred after intravitreal injection of the wild-type (Becker) strain of PRV. Rather than infecting neurons in the ventral and lateral aspectsof the nucleus, PRV-Ba preferentially infected neurons in the internal lamina of the VGN in a pattern essentially opposite to that resulting from injection of PRV-Be (Figures 3D and 3F).

Neuron 964

Although infected neurons became apparent in the pretectal nuclei at 72 hr and increased substantially thereafter, no neuronal infection was observed in retino-recipient regions of the superior colliculus of the same animals after intravitreal injection of PRV-Ba (Figures 48 and 4D; Figure 5). Small numbers of infected neurons were observed in the deeper, nonretino-recipient layers of the colliculus at the longest survival times (116-122 hr; Figure 4D), but these cells appeared to be a product of transneuronal passage of PRV-Ba through neurons of the oculomotor and Edinger-Westphal nuclei, which were infected by leakage of the virus from the vitreous into the orbit. Discussion These experiments demonstrate striking differences in the transneuronal transport of a virulent and an attenuated a-herpesvirus. The two patterns are remarkable in that they represent differential uptake and transport of virus by functionally distinct components of the visual system rather than variations of transportwithin the same projection pathway. Following intravitreal injection of either strain of PRV, viral replication was firstnoted in retinal ganglion cells and subsequently spread to deeper layers of the retina and to the forebrain, in a pattern that was entirely consistentwith transneuronal transport.Thedistribution of infected cells in forebrain was coextensive with the terminal arborization of central retinal projections, and the relative number of infected neurons paralleled the density of retinal innervation in each of the subcortical visual centers (see Parnavelas et al., 1989, for review of central visual projections). Replication of virus in glia did occur at longer survival times, but always followed the neuronal infection in temporal appearance and did not compromisethe reproducible, projection-specific transport of virus. Furthermore, the reactive gliosis induced by viral infection of neurons isolated these cells and thus appeared to contribute to the projection-specific pattern of transport by limiting spread of virus (see Card et al., 1990, for a more detailed discussion of glial infection). Collectively, these observations indicate that both strains of virus initially infect forebrain neurons by transsynaptic passage of virus through retinal afferents. The distinctive patterns of infection that occur in both the retina and forebrain further suggest that the two strains of PRV are selectively infecting projectionspecific populations of ganglion cells. The first site of viral replication that we are able to detect with either strain of virus occurred in the ganglion cell layer of the retina. With advancing survival, both strains of virus passed transneuronally into deeper layers of retina, apparently by transneuronal passage of virions through synapses in the inner plexiform layer. The latter observation is consistent with the demonstration that injection of HSV into central visual centers resulted in retrograde transport of virus to retinal ganglion cells and subsequent transneuronal passage of

virus through columns of cells traversing the full extent of the retina (Norgren and Lehman, 1989). Our findings also indicate that selective infection of subpopulations of ganglion cells persisted through even the longest survival intervals. This was verified in an extensive cross-sectional analysis of the localization of viral immunoreactivity at a variety of survival times and in a limited sample of retinae examined in wholemount preparations at advanced postinjection intervals. These findings strongly support the conclusion that each viral strain selectively infects subpopulations of ganglion ceils that are distributed across the full extent of the retina. Further studies are clearly necessary to establish definitively that each strain of PRV infects different, projection-specific groups of ganglion cells. Nevertheless, the present demonstration that each viral strain only infects subsets of ganglion cells does provide a basis for explaining the two distinct patterns of central neuron infection that result from intravitreal injection of these two closely related strains of virus. Although the mechanism(s) underlying the different patterns of transneuronal infection remains to be identified, available evidence suggests they are not simply due to variable rates of transport of the two strains. As noted previously, the Becker strain of virus produced two waves of neuronal infection that ultimately targeted all known retino-recipient regions of the forebrain, whereas the Bartha strain infected only a subset of these circuits. In the first wave of PRV-Be infection (beginning at approximately 48 hr), substantial numbers of infected neurons became prominent in the dorsal geniculate and tectum, and virtually no infected neurons were present in the SCN or IGL. In contrast, we could not detect PRV-Ba-infected neurons in any region of the forebrain prior to 72 hr postinjection, and both the DGN and superior colliculus never exhibited PRV-Ba-infected cells at survival times extending to 122 hr (over 3 days after PRV-Be-infected neurons were first detected in these same regions). At 72 hr (onset of the second wave of PRV-Be infection), moderate numbers of PRV-Be- and PRV-Ba-infected neurons were apparent in the SCN and IGL, and the number of infected cells rapidly increased in these regions at subsequent survival times. In fact, the appearance and progression of infection induced by both strains of virus in these regions were virtually indistinguishable from their onset at 72 hr through the longest periods of PRV-Be survival (81 hr). When the distance of these retino-recipient nuclei from the eye is also considered, it becomes clear that varying rates of transport of the two strains of virus are an unlikely explanation for the two distinct patterns of viral infection. The SCN are situated at the rostra1 aspect of the forebrain, whereas the geniculate complex and tectum are further caudal in the central aspects of the neuraxis. All three subdivisions of the geniculate lie within the same plane of the diencephalon while the colliculus is farther caudal. Nevertheless, PRV-Ba never infected neurons in the DGN or supe-

Clfferential

a-Herpesvirus

Transport

in CNS

91 t.5

Figure 6. Differential \ iral Glycoproteins

Infection

of Ganglion

Cells Is Mediated

by

CVe suggest that the two strain-specific patterns of viral infection i 1 the forebrain result from differential infection of projectionspecific populations of ganglion cells in the retina. The wild-type \‘irus (Becker) contains a full complement of envelope glycoproteins and ultimately infects all ganglion cells. The attenuated strain of virus (Bartha) possesses glycoprotein mutations and tleletions that prevent it from recognizing ganglion cells that project to the dorsal geniculate and tectum.

~ior colliculus at periods extending to 122 hr, even though substantial numbersof infected neuronswere present in the IGL and pretectal nuclei of the same ‘mimals by80 hr postinjection. Similarly, PRV-Be injection produced substantial infection of DGN neurons by 50 hr postinjection, but did not cause significant neuronal infection in the SCN prior to 72 hr. Thus, it bieems unlikely that our findings could be explained !~y different modes of transport for the two strains of ,lirus in the same neuron or selective uptake of the ,/iral strains by two populations of ganglion cells with different transport mechanisms. Several aspects of our findings lead us to conclude hat the unique patterns of central infectivity achieved %with the two viral strains involve differential infection If projection-specific populations of retinal ganglion :ells. Numerous studies have demonstrated that retilal ganglion cells can be separated on the basis of ‘-heir central retinal projections (Rodick, 1979; ParnaJelas et al., 1989), and our findings are consistent with rhese studies. In particular, Pickard (1985) has demon;trated a separate population of retinal ganglion cells :hat project to the SCN and IGL. If this population 3f ganglion cells was selectively infected by virus, it Nould be expected to produce a pattern of transneu-onal infection nearly identical to that observed with PRV-Ba in this study. It seems logical that the basis for the selective uptake of the different strains of PRV lies in the interaction of envelope glycoproteins with receptors on the surface of the ganglion cells. In this model (Figure 6), PRV envelope glycoproteins interact with two receptors differentially distributed on pro jection-specific classes of ganglion cells, and the mutations affecting glycoprotein expression in PRV-Ba compromise the ability of this strain of virus to recognizeoneof these receptors. Consequently, the Becker

strain, which contains a full complement of envelope glycoproteins, would ultimately infect neurons in all subcortical projection fields, whereas PRV-Ba would maintain the ability to infect ganglion cells projecting to the SCN and IGL, but would not infect ganglion cells projecting to the DGN, medial terminal nucleus, and superior colliculus. Implicit in this model is the assumption that the first wave of PRV-Be infection is mediated by a high affinity receptor that leads to rapid internalization, replication, and transport of virus to CNS targets (within 50 hr), while the second wave of infection results from virus interaction with a less efficient (low affinity?) receptor that delays internalization and/or transport of virus to the SCN and IGL by approximately 24 hr. Neither the receptors nor the vtral gene products that mediatetheir recognition in this model have been elucidated, but there is a basic understanding of the pathway of viral adsorption that ultimately leads to cellular infection. Studies of both HSV (WuDunn and Spear, 1989) and PRV (Zuckermann et al., 1989; Mettenleiter et al., 1990) indicate that the primary route of cellular infection is through an interaction of thevirus with a cellular heparin-like receptor. Analysis of PRV (Zuckermann et al., 1989; Mettenleiier et al., 1990) has shown that this adsorption is mediated, at least in part, by the virally encoded glll envelope glycoprotein. However, recent work by Zuckermann and collaborators (1989) has also demonstrated a second, glll-independent, mode of viral adsorption that leads to slower virion penetration. In comparing the uptake of wild-type PRV with mutants lacking the gill glycoprotein (glll-) these investigators demonstrated that adsorption and penetration of the gIlI- virus was slower and less efficient than that exhibited by the wild-type virus. Furthermore, polyvalent PRV antisera and monospecific sera generated against glll and gp50 blocked adsorption of the wild-type virus, but did not inhibit adsorption of the glll- mutants. This and other observations led these investigators to postulate two modes of viral adsorption, one mediated by glll, which leads to rapid penetration of the virus, and another route that occurs in the absence of gill and results in slower and less efficient penetration of virions. These observations have obvious implications for the two patterns of infectivity that we observed in the present analysis. PRV-Ba is characterized by a large deletion that eliminates all of gl (HSV glycoprotein E homolog) and a large portion of gp63 (HSV glycoprotein I homolog). In addition, this strain contains a signal sequence mutation that substantially reduces the concentration of glll (HSV glycoprotein C homolog). Consequently, it seems reasonable that the first wave of infection that we observed following injection of PRV-Be is the result of the efficient, glycoproteinmediated mode of adsorption, whereas the transneuronal infection of neurons in the SCN and IGL reflects the less efficient, glycoprotein-independent infection of another group of retinal ganglion cells. Because

Neuron 966

many of the mutations affecting glycoprotein expression by PRV-Ba have been mapped on the PRV genome (Lomniczi et al., 1984a, 1984b, 1987; Hampl et al., 1984; Robbins et al., 1989), it should be possible to construct well-defined mutant strains of virus that would test these hypotheses. It is also becoming clear that other receptors may be involved in a-herpesvirus cellular recognition and entry. Kanar and collaborators (1990) have reported that transfection of the basic fibroblast growth factor receptor into CHOcells endowed this previously nonpermissive cell line with the ability to recognize and take up HSV-1. Both heparin sulfate proteoglycan and basic fibroblast growth factor receptor (Wanaka et al., 1990) are widely distributed in the CNS and could be involved in the circuit-specific infection of the neuraxis. Significantly, Ferguson and co-workers (1990) have shown that intravitreal injection of 1251-labeled basic fibroblast growth factor leads to specific binding and internalization of this peptide. In addition, their work has shown that the iodinated ligand is preferentially transported to the same subcortical visual centers targeted by the first wave of PRV-Be infection in the present analysis. Consequently, the circuitspecific transport of a-herpesviruses demonstrated in this and other investigations may be the product of viral recognition of receptors that are differentially distributed in the CNS. The targeted, projection-specific transport of a-herpesviruses through neural circuits evident in this and other investigations should also have considerable impact upon recent studies that have shown that these viruses are useful vectors for delivery of genes to the nervous system (see Breakefield and Geller, 1987 for a recent review). Several investigations have demonstrated stable expression of 8-galactosidase both in vitro (Geller and Breakefield, 1988; Geller and Freeze, 1990; Geller et al., 1990) and in vivo (Ho and Mocarski, 1988; Dobson et al., 1990; Chiocca et al., 1990) following delivery of the /acZ gene to neurons with herpesvirus vectors. Pathogenesis of the herpesvirus infection was reduced in these cases by substitution of early (thymidine kinase) or immediate early (ICPO and ICP4) genes with the /acZ reporter gene. In the limited number of in vivo studies that have been conducted, the viral vector was injected either into neurally innervated peripheral tissues (Ho and Mocarski, 1988; Dobson et al., 1990), into a peripheral nerve (Dobson et al., 1990), or directly into the CNS (Chiocca et al., 1990). Injection of peripheral tissues or nerves resulted in expression of 8-galactosidase in the neurons with axons either traveling in the injected nerve or innervating the injected tissue. Injection into the CNS led to expression of the foreign gene in neurons infected by these replication-deficient mutants both at the site of injection and in areas known to project to the injected region. Further information on the mechanisms that account for differential uptake and transneuronal passage of virus through functional cir-

cuits of neurons in a manner analogous to that observed in the present study should have important implications for determining the utility of viral vectors for targeted delivery of genes to functionally defined populations of neurons. In summary, we have demonstrated differential uptake and transport of two closely related strains of a swine a-herpesvirus in a manner that implies selective sensitivity of different classes of retinal ganglion cells to the invasive characteristics of the two strains. Thus, it is apparent that the specificity that characterizes viral transport in the nervous system will prove useful for defining the fundamental basis of viral neurotropismaswellasforexaminingtheorganizationoffunctionally related neural circuits. Experimental

Procedures

Animals Adult, male Sprague-Dawley rats weighing 200-350 g at the time of sacrificewere used in the analysis. Food and water were freely available throughout the course of each experiment, and the photoperiod was standardized to 14 hr light, 10 hr dark (light on at 0600). Experimental protocols were approved by the local Animal Welfare Committee and were consistent with the regulations stipulated by the American Association for Accreditation of Laboratory Animal Care and those in the Animal Welfare Act (public law 99-198). All animals were confined to a biosafety level 2 laboratory dedicated exclusively to these studies. Specific details regarding the regulations for operation of this laboratory have been published previously (Card et al., 1990). Virus The Becker and Bartha strains of virus were grown in porcine kidney fibroblasts (PK15) in a biosafety level 2 containment facility. Virus-infected cells were scraped into the media, freeze thawed, sonicated, cleared of cellular debris by centrifugation, and stored at -80°C in 200 PI aliquots. For these experiments, titers of plaque forming units revealed 5 x 108 pfu/ml for the Becker stock and 1 x IO9 pfu/ml forthe Bartha stock. Fresh stocks of virus were thawed for each injection, and unused virus was deactivated with Chlorox and discarded. Separate Hamilton microliter syringes were dedicated to each viral strain. Antisera Three rabbit polyclonal antisera were used to localize both strains of virus. Each of these antisera were generated by injecting rabbits with acetone-inactivated wild-type (Becker) virus. Comparative localization of virus with each of the antisera in adjacent sections from the same animal revealed identical patterns of immunoreactivity in animals injected with PRV-Be and PRV-Ba. In addition, immunoprecipitation and SDS-PAGE of [3H]glucosamine-labeled, Becker-infected PK15 cells demonstrated that all of the major envelope glycoproteins were recognized by these antisera. All of the antisera produced identical patterns of immunoreactivity. However, in the majority of experiments, we used rabbitantiserum#134, which we previouslycharacterized in an analysis of the neural transport of PRV-Be (Card et al., 1990). Virus Injections and Tissue Processing Our previous analysis of the virulent strain of PRV (PRV-Be) demonstrated that thevirus is highly neurotropic and can infect central neurons by either anterograde transport through the retina or retrograde transport from the viscera (Card et al., 1990). In addition to lytic neuronal infections, these routes of administration also induce peripheral infections of the lungs that appear tocontributeto thedebilitatingeffectsofviral inoculation.These effects occur within 65 hr of intravitreal injection and are severe

Clifferential 957

a-Herpesvirus

Transport

in CNS

by80 hr. Strackand Loewy(1990) reportthat theattenuated strain of virus (PRV-Ba) does not compromise the health of rats up to 1. days after injection into either the anterior chamber of the eye or the pinna of the ear. We obtained the same result after intravitreal injection of PRV-Ba, but noted that the animals developed signs of infection similar to those induced by PRV-Be on the fifth day postinjection and generally died on day 6. For these reasons, the majority of experiments involving PRV-Be were terminated at 72 hr or sooner, and the analysis of PRV-Ba transport was limited to 122 hr. Injections were timed so that animals were inoculated during the light phase of the photoperiod. Each animal wasdeeplyanesrhetized by intramuscular injection of ketamine and xylazine Jrrior to unilateral injection of 2 ul of virus into the vitreous body of the right eye. Seventeen animals received injections of “RV-Be, and 20 animals were injected with PRV-Ba. Postinjection urvival times for animals in the analysis of PRV-Be transport ,anged from 24-81 hr; animals injected with PRV-Ba were anayzed 24-122 hr following injection. Each animal was carefully nonitored throughout the course of the study, and experiments Nereterminated by transcardiac perfusion fixation with buffered .rldehyde solutions as detailed previously (Card et al., 1990). After ranscardiac perfusion fixation, the brain, spinal cord, and inected eye were removed, cryoprotected, and sectioned serially it 30 Urn per section with a freezing microtome. The brain was ;ectioned in either the coronal or sagittal plane, the spinal cord n the horizontal plane, and the eyewas sectioned perpendicular o the plane of the retina (two retinal samples were processed as wholemount preparations). Every other section (intervals of :jO pm) was incubated in the primary antiserum at a primary dilution of I:1000 to I:2000 for 24-48 hr at 4OC and processed with the avidin-biotin modification of the immunoperoxidase procedure (Hsu et al., 1981). Further details regarding these reagents and the application of this procedure in our laboratory have been published (Card et al., 1990). All intravitreal injections of PRV resulted in uptakeand replication of virus by retinal ganglion cells, and, with sufficient postinjection survival, each strain passed transneuronallyto infect neurons in the CNS. This stands in contrast to the relatively low infection rate (approximately 20% of injected animals) reported by Strack and collaborators (Strack et al., 1989a, 1989b; Strack and Loewy, 1990) in their analysis of uptake and transport of PRV-Ba through CNS circuitry modulating sympathetic outflow. This discrepancy may be related to the higher titer of inoculum used in our analysis. On the average, we injected 1 x IO6 pfu of virus per animal, while the studies conducted by Strack and collaborators used approximately 1 x IO3 pfu per injection (Strack et al., 1989a, 1989b; Strack and Loewy, 1990). Quantitative Analysis The temporal aspects of transport of both strains of virus to the SCN, the ICL, the dorsal geniculate nucleus, and the tectum were subjected toquantitativeanalysis. In each case, weselected coronal sections that sampled the largest cross-sectional diameter of each region and counted the number of cells that exhibited viral immunoreactivity. The analysis was standardized so that we sampled the same rostrocaudal level in each of the four areas. Animals were grouped according to postinjection survival (4554,65-74,75-84,85-94,95-104, and 105-125 hr), and the average number of cells per area in each of the four cell groups was graphed. Acknowledgments

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We gratefully acknowledge the expert technical assistance of Henry Pautler and Michelle Harner in conducting these studies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenf” in accordance with 18 USC Section 1734 solely to indicate this fact.

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