Pseudorabies virus envelope glycoprotein gI influences both neurotropism and virulence during infection of the rat visual system.

We previously demonstrated that intraocular injections of virulent and attenuated strains of pseudorabies virus (PRV) produce transneuronal infection of functionally distinct central visual circuits in the rat. The virulent Becker strain of PRV induces two temporally separated waves of infection that ultimately target all known retinorecipient neurons; the attenuated Bartha strain only infects a functionally distinct subset of these neurons. In this study, we demonstrate that deletion of a single viral gene encoding glycoprotein gI is sufficient to reproduce both the novel pattern of infectivity and the reduced neurovirulence of the Bartha strain of PRV. Glycoprotein gIII, a major viral membrane protein required for efficient adsorption of virus in cell culture, has no obvious role in determining the pattern of neuronal infectivity, but appears to function with gI to influence neurovirulence. These data suggest that neuroinvasiveness and virulence are the products of an interaction of viral envelope glycoproteins with as yet unidentified cellular receptors.     

The gE and gI homologs from two alphaherpesviruses have conserved and divergent neuroinvasive properties.

The membrane glycoproteins gE and gI are encoded by pseudorabies virus (PRV), a neurotropic, broad-host-range alphaherpesvirus of swine. PRV gE and gI are required for anterograde spread to a restricted set of retinorecipient neurons in the brain after infection of the rat retina. A related alphaherpesvirus, encoding gE and gI homologs, is called bovine herpesvirus 1.1 (BHV-1.1). BHV-1.1 is a respiratory pathogen of highly restricted host range and, in contrast to PRV, is unable to propagate in or cause disease in rodents. We have shown previously that the BHV-1.1 gE and gI proteins are capable of complementing the virulence functions of PRV gE and gI in a rodent model (A. C. Knapp and L. W. Enquist, J. Virol. 71:2731-2739, 1997). We examined the ability of the BHV-1.1 gE and gI homologs to direct circuit-specific invasion of the rat central nervous system by PRV. Both complete open reading frames were cloned into a PRV mutant lacking the PRV gE and gI genes. Recombinant viruses were analyzed for the ability to invade the rat brain after infection of the retina. Surprisingly, in a portion of the animals tested, the BHV-1.1 gE and gI proteins functioned autonomously to promote spread of PRV to a subset of retinorecipient regions of the brain. First, the presence of BHV-1.1 gI alone, but not PRV gI alone, promoted viral invasion of the optic tectum. Second, expression of BHV-1.1 gE alone facilitated PRV infection of a subset of neurons in the hippocampus not normally infected by PRV. When both BHV-1.1 proteins were expressed in a coinfection, all retinorecipient regions of the rat brain were infected. Therefore, depending on the viral source, homologs of gE and gI differentially affect spread between synaptically connected neurons in the rat.     

Retrograde, transneuronal spread of pseudorabies virus in defined neuronal circuitry of the rat brain is facilitated by gE mutations that reduce virulence

The pseudorabies virus (PRV) gE gene encodes a multifunctional membrane protein found in infected cell membranes and in the virion envelope. Deletion of the gE gene results in marked attenuation of the virus in almost every animal species tested that is permissive for PRV. A common inference is that gE mutants are less virulent because they have reduced ability to spread from cell to cell; e.g., gE mutants infect fewer cells and, accordingly, animals live longer. In this report, we demonstrate that this inference does not hold in a rat experimental model for virus invasion of the brain. We find that animals infected with gE mutants live longer despite extensive retrograde, transneuronal spread of virus in the rat brain. In this model of brain infection, virus is injected into the stomach musculature and virions spread to the brain in long axons of brain stem neurons that give rise to the tenth cranial nerve (the vagus). The infection then spreads from neuron to neuron in well-defined, and physically separated, areas of the brain involved in autonomic regulation of the viscera. We examined the progression of infection of five PRV strains in this circuitry: the wild-type PRV-Becker strain, the attenuated PRV-Bartha vaccine strain, and three gE mutants isogenic with the PRV-Becker strain. By 60 to 67 h after infection, all PRV-Becker-infected animals were dead. Analysis of Becker-infected rats killed prior to virus-induced death demonstrated that the virus had established an infection only in the primary vagal neurons connected directly to the stomach and synaptically linked neurons in the immediate vicinity of the caudal brain stem. There was little spread to other neurons in the vagus circuitry. In contrast, rats infected with PRV-Bartha or PRV-Becker gE mutants survived to at least 96 h and exhibited few overt signs of disease. Despite this long survival and the lack of symptoms, brains of animals sacrificed at this time revealed extensive transsynaptic infection not only of the brain stem but also of areas of the forebrain synaptically linked to neurons in the brain stem. This finding provides evidence that the gE protein plays a role in promoting symptoms of infection and death in animals that is independent of neuron-to-neuron spread during brain infection. When this early virulence function is not active, animals live longer, resulting in more extensive spread of virus in the brain.     

Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications

The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.     

Pseudorabies virus recombinants expressing functional virulence determinants gE and gI from bovine herpesvirus 1.1.

In the Alphaherpesvirinae subfamily, the gE and gI genes are conserved and encode membrane glycoproteins required for efficient pathogenesis (virulence). The molecular mechanism(s) responsible is not well understood, but the existence of similar phenotypes of gE and gI mutations in diverse Alphaherpesvirinae implies conservation of function(s). In this report, we describe construction of pseudorabies virus (PRV) recombinants that efficiently express the bovine herpesvirus 1 (BHV-1) membrane proteins gI and gE at the PRV gG locus. Each BHV-1 gene was cloned in a PRV mutant lacking both the PRV gI and gE coding sequences. All recombinant viruses expressed the BHV-1 proteins at levels similar to or greater than that observed after infection with parental BHV-1, and there were no observable differences in processing or ability to form gE-gI oligomers. The important observation resulting from this report is that the BHV-1 gE and gI proteins functioned together to complement the virulence defect of PRV lacking its own gE and gI genes in a rodent model, despite being derived from a highly restricted host range virus with a different pathogenic profile.     

Two modes of pseudorabies virus neuroinvasion and lethality in mice

We describe two distinct modes of neuroinvasion and lethality after murine flank inoculation with virulent and attenuated strains of pseudorabies virus (PRV). Mice infected with virulent (e.g., PRV-Becker, PRV-Kaplan, or PRV-NIA3) strains self-mutilate their flank skin in response to virally induced pruritus, die rapidly with no identifiable symptoms of central nervous system (CNS) infection such as behavioral abnormalities, and have little infectious virus or viral antigen in the brain. In distinct contrast, animals infected with an attenuated PRV vaccine strain (PRV-Bartha) survive approximately three times longer than wild-type PRV-infected animals, exhibit severe CNS abnormalities, and have an abundance of infectious virus in the brain at the time of death. Interestingly, these animals have no skin lesions and do not appear pruritic at any time during infection. The severe pruritus and relatively earlier time until death induced by wild-type PRV infection may reflect the peripheral nervous system (PNS) and immune responses to infection rather than a fatal, virally induced CNS pathology. Based on previously characterized afferent (sensory) and efferent (motor) neuronal pathways that innervate the skin, we deduced that wild-type virulent strains transit through the PNS via both afferent and efferent routes, whereas PRV-Bartha travels by only efferent routes in the PNS en route to the brain.     

Glycoprotein D-negative pseudorabies virus can spread transneuronally via direct neuron-to-neuron transmission in its natural host, the pig, but not after additional inactivation of gE or gI.

Envelope glycoprotein D (gD) is essential for entry of pseudorabies virus (PRV) into cells but is not required for the subsequent steps in virus replication. Phenotypically complemented gD mutants can infect cells and can spread, both in vitro and in mice, by direct cell-to-cell transmission. Progeny virions released by infected cells are noninfectious because they lack gD. The aim of this study was to determine the role of gD in the neuropathogenicity of PRV in its natural host, the pig. We investigated whether gD-negative PRV can spread transneuronally via synaptically linked neurons of the olfactory and trigeminal routes. High doses of a phenotypically complemented gD mutant and gD mutants that are unable to express either gI or gI plus gE were inoculated intranasally in 3- to 5-week-old pigs. Compared with the wild-type virus, the virulence of the gD mutant was reduced. However, pigs inoculated with the gD mutant still developed fever and respiratory signs. Additional inactivation of either gI or gI plus gE further decreased virulence for pigs. Immunohistochemical examination of infected pigs showed that a PRV gD mutant could replicate and spread transneuronally into the central nervous system (CNS). Compared with the wild-type virus, the gD mutant had infected fewer neurons of the CNS on day 2. Nevertheless, on day 3, the gD-negative PRV had infected more neurons and viral antigens were present in second- and third-order neurons in the olfactory bulb, brain stem, and medulla oblongata. In contrast, gD mutants which are unable to express either gI or gI plus gE infected a limited number of first-order neurons in the olfactory epithelium and in the trigeminal ganglion and did not spread transneuronally or infect the CNS. Thus, transsynaptic spread of PRV in pigs can occur independently of gD. Possible mechanisms of transsynaptic transport of PRV are discussed.     

The alpha-herpesviruses: molecular pathfinders in nervous system circuits

Several neuroinvasive viruses can be used to study the mammalian nervous system. In particular, infection by pseudorabies virus (PRV), an alpha-herpesvirus with broad host range, reveals chains of functionally connected neurons in the nervous systems of a variety of mammals. The specificity of PRV trans-neuronal spread has been established in several systems. One attenuated strain, PRV-Bartha, causes a reduced inflammatory response and also spreads only from infected post- to pre-synaptic neurons. We review the basics of PRV tracing and then discuss new developments and novel approaches that have enabled a more detailed understanding of the architecture of the nervous system. As questions and techniques evolve in the field of neuroscience, advances in PRV tracing will certainly follow.     

Different Patterns of Neuronal Infection after Intracerebral Injection of Two Strains of Pseudorabies Virus

Pseudorabies virus (PRV), a swine neurotropic alphaherpesvirus, is known to invade the central nervous system (CNS) of a variety of animal species through peripherally projecting axons, replicate in the parent neurons, and then pass transsynaptically to infect other neurons of a circuit. Studies of the human pathogen herpes simplex virus type 1 have reported differences in the direction of transport of two strains of this virus after direct injection into the primate motor cortex. In the present study we examined the direction of transport of virulent and attenuated strains of PRV, utilizing injections into the rat prefrontal cortex to evaluate specific movement of virus through CNS circuitry. The data demonstrate strain-dependent patterns of infection consistent with bidirectional (anterograde and retrograde) transport of virulent virus and unidirectional (retrograde) transport of attenuated PRV from the site of injection. The distribution of infected neurons and the extent of transsynaptic passage also suggest that a release defect in the attenuated strain reduces the apparent rate of viral transport through neuronal circuitry. Finally, injection of different concentrations of virus influenced the onset of replication within a neural circuit. Taken together, these data suggest that viral envelope glycoproteins and virus concentration at the site of injection are important determinants of the rate and direction of viral transport through a multisynaptic circuit in the CNS.     

Pseudorabies virus membrane proteins gI and gE facilitate anterograde spread of infection in projection-specific neurons in the rat

The membrane proteins gI and gE of Pseudorabies virus (PRV) are required for viral invasion and spread through some neural pathways of the rodent central nervous system. Following infection of the rat retina with wild-type PRV, virus replicates in retinal ganglion neurons and anterogradely spreads to infect all visual centers in the brain. By contrast, gI and gE null mutants do not infect a specific subset of the visual centers, e.g., the superior colliculus and the dorsal lateral geniculate nucleus. In previous experiments, we suggested that the defect was not due to inability to infect projection-specific retinal ganglion cells, because mixed infection of a gE deletion mutant and a gI deletion mutant restored the wild-type phenotype (i.e., genetic complementation occurred). In the present study, we provide direct evidence that gE and gI function to promote the spread of infection after entry into primary neurons. We used stereotaxic central nervous system injection of a fluorescent retrograde tracer into the superior colliculus and subsequent inoculation of a PRV gI-gE double null mutant into the eye of the same animal to demonstrate that viral antigen and fluorescent tracer colocalize in retinal ganglion cells. Furthermore, we demonstrate that direct injection of a PRV gI-gE double null mutant into the superior colliculus resulted in robust infection followed by retrograde transport to the eye and replication in retinal ganglion neuron cell bodies. These experiments provide additional proof that the retinal ganglion cells projecting to the superior colliculus are susceptible and permissive to gE and gI mutant viruses. Our studies confirm that gI and gE specifically facilitate anterograde spread of infection by affecting intracellular processes in the primary infected neuron such as anterograde transport in axons or egress from axon terminals.     

Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.     

Role of pseudorabies virus Us9, a type II membrane protein, in infection of tissue culture cells and the rat nervous system

The protein product of the pseudorabies virus (PRV) Us9 gene is a phosphorylated, type II membrane protein that is inserted into virion envelopes and accumulates in the trans-Golgi network. It is among a linked group of three envelope protein genes in the unique short region of the PRV genome which are absent from the attenuated Bartha strain. We found that two different Us9 null mutants exhibited no obvious phenotype after infection of PK15 cells in culture. Unlike those of gE and gI null mutants, the plaque size of Us9 null mutants on Madin-Darby bovine kidney cells was indistinguishable from that of wild-type virus. However, both of the Us9 null mutants exhibited a defect in anterograde spread in the visual and cortical circuitry of the rat. The visual system defect was characterized by restricted infection of a functionally distinct subset of visual projections involved in the temporal organization of behavior, whereas decreased anterograde spread of virus to the cortical projection targets was characteristic of animals receiving direct injections of virus into the cortex. Spread of virus through retrograde pathways in the brain was not compromised by a Us9 deletion. The virulence of the Us9 null mutants, as measured by time to death and appearance of symptoms of infection, also was reduced after their injection into the eye, but not after cortical injection. Through sequence analysis, construction of revertants, measurement of gE and gI protein synthesis in the Us9 null mutants, and mixed-infection studies of rats, we conclude that the restricted-spread phenotype after infection of the rat nervous system reflects the loss of Us9 and is not an indirect effect of the Us9 mutations on expression of glycoproteins gE and gI. Therefore, at least three viral envelope proteins, Us9, gE, and gI, function together to promote efficient anterograde transneuronal infection by PRV in the rat central nervous system.     

A Chicken Embryo Eye Model for the Analysis of Alphaherpesvirus Neuronal Spread and Virulence

We describe use of developing chicken embryos as a model to study neuronal spread and virulence of pseudorabies virus (PRV). At embryonic day 12, β-galactosidase-expressing PRV strains were injected into the vitreous humor of one eye, and virus replication and spread from the eye to the brain were measured by β-galactosidase activity and the recovery of infectious virus from tissues. The wild-type PRV strain, Becker, replicated in the eye and then spread to the brain, causing extensive pathology characterized by edema, hemorrhage, and necrosis that localized to virally infected tissue. The attenuated vaccine strain, Bartha, replicated in the eye and spread throughout specific regions of the brain, producing little to no overt pathology. Becker mutants lacking membrane proteins gE or gI replicated in the eye and were able to spread to the brain efficiently. The pathology associated with replication of these mutants in the brain was intermediate to that induced by Becker or Bartha. Mixed infection of a gE deletion mutant and a gI deletion mutant restored the pathogenic phenotype to wild-type levels. These data indicate that the replication of virus in embryonic brain tissue is not sufficient to induce the characteristic pathological response and that the gE and gI gene products actively affect pathological responses in the developing chicken brain.     

Quantitative proteomics study of host response to virulent and attenuated pseudorabies virus infection in mouse brain

Bartha, the pseudorabies virus (PRV) vaccine strain, is widely used in studies of neuronal circuit-tracing, due to its attenuated virulence and retrograde spreading. However, we know little regarding the molecular mechanisms of PRV infection and spreading between structurally connected neurons. In this study, we systematically analyzed the host brain proteomes after acute infection with PRV, attempting to identified the proteins involved in the processes. Mice were injected with PRV-Bartha and PRV-Becker (PRV-Bartha's wild-type parent strain) in the olfactory system, the proteomes of the brain and synaptosome were analyzed and compared at various infection intervals using mass spectrometry-based proteomics techniques. In all, we identified > 100 PRV-infection regulated proteins at the whole-tissue level and the synaptosome level. While at whole-tissue level, bioinformatics analyses mapped most of the regulations to the inflammation pathways, at the synaptosome level, most of those to synaptic transmission, cargo transport and cytoskeleton organization. We established regulated protein networks demonstrating distinct cellular regulation pattern between the global and the synaptosome levels. Moreover, we identified a series of potentially PRV-strain-specific regulated proteins with diverse biological functions. This study may provide new clues for molecular mechanisms for PRV infection and spread.     

Role of a structural glycoprotein of pseudorabies in virus virulence.

The virulence of deletion mutants of pseudorabies virus defective in the expression of glycoprotein gI, gp63, or both was tested in 1-day-old chickens and young pigs. In the absence of expression of gI, the virulence of a fully virulent laboratory strain, PrV(Ka), for 1-day-old chickens was reduced approximately fourfold. Inactivation of glycoprotein gp63 appeared also to affect the virulence of PrV(Ka) only slightly, as did inactivation of both gI and gp63. The level of reduction in virulence, however, was considerably more marked in Bartha 43/25aB4, a less virulent virus strain. Inactivation of the expression of gI in Bartha 43/25aB4 reduced virulence for chickens at least 100-fold. The results obtained when the virulence of the mutants for pigs was determined were compatible with those obtained for chickens. These results indicate that gI plays a role in virulence, but that it does so in conjunction with at least one other viral function (a function that is defective in Bartha 43/25aB4).     

Role of glycoprotein gIII of pseudorabies virus in virulence.

Deletion mutants of pseudorabies virus unable to express glycoprotein gIII, gI, or gp63 or double and triple mutants defective in these glycoproteins were constructed, and their virulence for day-old chickens inoculated intracerebrally was determined. Mutants of wild-type pseudorabies virus defective in glycoprotein gIII, gI, or gp63 were only slightly less virulent (at most, fivefold) for chickens than was the wild-type virus. However, mutants defective in both gIII and gI or gIII and gp63 were avirulent for chickens, despite their ability to grow in cell culture in vitro to about the same extent as mutants defective in gIII alone (which were virulent). These results show that gIII plays a role in virulence and does so in conjunction with gI or gp63. The effect of gIII on virulence was also shown when the resident gIII gene of variants of the Bartha vaccine strain (which codes for gIIIB) was replaced with a gIII gene derived from a virulent wild-type strain (which codes for gIIIKa); gIIIKa significantly enhanced the virulence of a variant of the Bartha strain to which partial virulence had been previously restored by marker rescue. Our results show that viral functions that play a role in the virulence of the virus (as measured by intracerebral inoculation of chickens) may act synergistically to affect the expression of virulence and that the ability of the virus to grow in cell culture is not necessarily correlated with virulence.     

Transneuronal tracing of neural pathways that regulate hindlimb muscle blood flow

Despite considerable interest in the neural mechanisms that regulate muscle blood flow, the descending pathways that control sympathetic outflow to skeletal muscles are not adequately understood. The present study mapped these pathways through the transneuronal transport of two recombinant strains of pseudorabies virus (PRV) injected into the gastrocnemius muscles in the left and right hindlimbs of rats: PRV-152 and PRV-BaBlu. To prevent PRV from being transmitted to the brain stem via motor circuitry, a spinal transection was performed just below the L2 level. Infected neurons were observed bilaterally in all of the areas of the brain that have previously been shown to contribute to regulating sympathetic outflow: the medullary raphe nuclei, rostral ventrolateral medulla (RVLM), rostral ventromedial medulla, A5 adrenergic cell group region, locus coeruleus, nucleus subcoeruleus, and the paraventricular nucleus of the hypothalamus. The RVLM, the brain stem region typically considered to play the largest role in regulating muscle blood flow, contained neurons infected following the shortest postinoculation survival times. Approximately half of the infected RVLM neurons were immunopositive for tyrosine hydroxylase, indicating that they were catecholaminergic. Many (47%) of the RVLM neurons were dually infected by the recombinants of PRV injected into the left and right hindlimb, suggesting that the central nervous system has a limited capacity to independently regulate blood flow to left and right hindlimb muscles.     

Role of the pseudorabies virus gI cytoplasmic domain in neuroinvasion, virulence, and posttranslational N-linked glycosylation

The glycoproteins I and E of pseudorabies virus are important mediators of cell-to-cell spread and virulence in all animal models tested. Although these two proteins form a complex with one another, ascribing any function to the individual proteins has been difficult. We have shown previously, using nonsense mutations, that the N-terminal ectodomain of the gE protein is sufficient for gE-mediated transsynaptic spread whereas the cytoplasmic domain of the protein is required for full expression of virulence. These same studies demonstrated that the cytoplasmic domain of gE is also required for endocytosis of the protein. In this report, we describe the construction of viruses with nonsense mutations in gI that allowed us to determine the contributions of the gI cytoplasmic domain to protein expression as well as virus neuroinvasion and virulence after infection of the rat eye. We also constructed double mutants with nonsense mutations in both gE and gI so that the contributions of both the gE and gI cytoplasmic domains could be determined. We observed that the gI cytoplasmic domain is required for efficient posttranslational modification of the gI protein. The gE cytoplasmic domain has no effect on gE posttranslational glycosylation. In addition, we found that infection of all gE-gI-dependent anterograde circuits projecting from the rat retina requires both ectodomains and at least one of the cytoplasmic domains of the proteins. The gI cytoplasmic domain promotes transsynaptic spread of virus better than the gE cytoplasmic domain. Interestingly, both gE and gI cytoplasmic tails are required for virulence; lack of either one or both results in an attenuated infection. These data suggest that gE and gI play differential roles in mediating directional neuroinvasion of the rat; however, the gE and gI cytoplasmic domains most likely function together to promote virulence.