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Uptake, replication, and transneuronal passage of a swine neurotropic herpesvirus (pseudorabies virus, PRV) was evaluated in the rat CNS. PRV was localized in neural circuits innervating the tongue, stomach, esophagus and eye with light microscopic immunohistochemistry. In each instance, the distribution of PRV-immunoreactive neurons was entirely consistent with that observed following injection of cholera toxin- horseradish peroxidase conjugate (CT-HRP). Injections of the tongue resulted in retrograde transport of PRV and CT-HRP to hypoglossal motor neurons, while preganglionic neurons in the dorsal motor vagal nucleus or somatic motor neurons in the nucleus ambiguus were labeled following injections of the stomach or esophagus, respectively. At longer times after infection, viral antigens were found in astrocytes adjacent to infected neurons and their efferent axons and second-order neuron labeling became apparent. The distribution of second-order neurons was also entirely dependent upon the site of PRV injection. Following tongue injection, second-order neurons were observed in the trigeminal complex, the brain-stem tegmentum and in monoaminergic cell groups. Injection of the stomach or esophagus led to second-order neuron labeling confined to distinct subdivisions of the neucleus of the solitary tract and monoaminergic cell groups. Comparative quantitative analysis of the number of PRV immunoreactive neurons present in the diencephalon and brain stem following injection of virus into both the eye and stomach musculature of the same animal demonstrated that retrograde transport of PRV from the viscera was more efficient and occurred at a much faster rate than anterograde transport of virus. These data demonstrate projection-specific transport of PRV in the nervous system and provide further insight into the means through which this neurotropic virus infects the nervous system.

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.

Pseudorabies virus (PRV) has been used extensively to map synaptic circuits in the CNS and PNS. A fundamental assumption of these studies is that the virus replicates within synaptically linked populations of neurons and does not spread through the extracellular space or by cell- to-cell fusion. In the present analysis we have used electron microscopy to characterize pathways of viral replication and egress that lead to transneuronal infection of neurons, and to document the non-neuronal response to neuronal infection. Three strains of PRV that differ in virulence were used to infect preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV). The data demonstrate that viral replication and transneuronal passage occur in a stepwise fashion that utilizes existing cellular processes, and that the non-neuronal response to infection serves to restrict nonspecific spread of virus by isolating severely infected neurons. Specifically, capsids containing viral DNA replicate in the cell nucleus, traverse the endoplasmic reticulum to gain access to the cytoplasm, and acquire a bilaminar membrane envelope from the trans cisternae of the Golgi. The outer leaf of this envelope fuses with the neuron membrane to release virus adjacent to axon terminals that synapse upon the infected cell. A second fusion event involving the viral envelope and the afferent terminal releases the naked capsid into the bouton. Systematic analysis of serial sections demonstrated that release of virus from infected neurons occurs preferentially at sites of afferent contact. Nonspecific diffusion of virus from even the most severely infected cells is restricted by astrocytes and other non-neuronal elements that are mobilized to the site of viral infectivity. The ability of glia and macrophages to restrict spread of virus from necrotic neurons is the product of (1) temporal differences in the mobilization of these cells to the site of infection, (2) differential susceptibility of these cells to PRV infection, and (3) abortive viral replication in cells that are permissive for infection. The findings provide further insight into the intracellular routes of viral assembly and egress and support the contention that transneuronal spread of virus in the brain results from specific passage of virions through synaptically linked neurons rather than through cell fusion or release of virus into the extracellular space.