Richard J. Ziegler

Uptake and Transport of Herpes Simplex Virus in Neurites of Rat Dorsal Root Ganglia Cells in Culture

Attachment and neuritic transport of herpes simplex virus (HSV) type 1 (McIntyre) were studied in a cell culture system with dissociated cells of rat dorsal root ganglia. The two-chamber cell culture system containing a diffusion barrier penetrated by neurites of cultured sensory neurons permitted infection of neurites extending outside the diffusion barrier without exposure of the neuronal cell soma. HSV adsorbed to neuritic extensions and reached the neuronal soma within 1.5 h post-inoculation. Neuritic uptake and transport of HSV were inhibited in the presence of cytochalasin B. Internalization of virus in neurites was preceded by attachment of virus to the neurite plasma membrane. Neurites transported viral nucleocapsids (NC) through the diffusion barrier of the cultures. Destruction of the neuritic extensions before or shortly after peripheral virus inoculation blocked spread of infection to the cell soma. No infection was established when neuritic extensions were exposed to viral NC and NC were then not observed inside the neurite plasma membrane. Virus produced in neurons, when HSV was inoculated into the inner culture chamber containing the neuronal cell bodies, was transported as enveloped virus in cytoplasmic vesicles from the neuronal cell body towards the periphery. Schwann cells were infected by viropexis. Shortly after infection virions were observed in vacuoles of the cytoplasm.

Electrophysiological Changes of HSV-1-Infected Dorsal Root Ganglia Neurons in Culture

Dissociated rat dorsal root ganglion cultures were infected with herpes simplex virus type 1 and studied electrophysiologically throughout the course of infection. Alterations in the electrical parameters of the action potentials of these neurons occurred as early as 4 hours post infection. The maximum rate of rise, spike amplitude, and overshoot were decreased, and the full width at half maximum and resting membrane potential were increased. At 6 hours post infection there is a movement back to normal values for these parameters. This reversal is transient, however, and the types of alterations seen at 4 hours post infection are more pronounced at 12 hours post infection. The first morphological alterations occur at 16 hours post infection, at which time less than 50% of the neurons impaled produced action potentials. Application of cyclohexamide to inhibit protein synthesis 1 hour post infection prevented electrophysiological changes normally seen at 4 hours and 6 hours post infection. It is concluded that the sodium conductance is specially reduced by acute HSV infection, and that this reduction is attributed to viral specific or modified host cell protein synthesis.

Herpes simplex virus infection of the rat sensory neuron Effects of interferon on cultured cells

SummaryEmbryonic rat dorsal root ganglion neurons were cultured in a two-chamber system allowing infection of neuritic extensions without exposure of neuronal cell bodies or vice versa. Herpes simplex virus type 1 was used to infect interferon-α and -β treated or untreated neurons and the production of virus and interferon was assayed.Treatment of nerve cell bodies with interferon inhibited virus replication in a dose-dependent manner, whether virus was inoculated directly onto the nerve bodies or peripherally on the neuritic extensions. In contrast no antiviral effect was noted when neurites were treated with interferon suggesting possible lack of interferon receptors on neurites. On infection with herpes simplex virus the rat sensory neuron cultures did not produce interferon in amounts above the detection limit (0.5 units per ml) of the interferon assay used.

Mumps Virus-induced Alterations in Cellular Excitability During Persistent Infections

Persistent mumps virus infections were established in rat pheochromocytoma (PC-12) and human medulloblastoma (TE-671) continuous cell lines. Significant amounts of infectious virus were produced by the PC-12 cells; infectious virus production by the TE-671 cells was limited. This restricted replication may be due to decreased production of viral envelope glycoproteins by TE-671 cells. The presence of virus changed the distribution of stimulus-evoked electrical responsiveness of both cell lines from responsiveness composed primarily of normal, rapidly rising, all-or-nothing action potentials to one dominated by abnormal, slowly rising, graded responses or by no response at all. Such changes have the potential to disrupt neural integration within the nervous system, and suggest a new mechanism by which persistent virus infections might play a role in chronic neurological and/or mental disease.

Loss of Functional Voltage-gated Sodium Channels in Persistent Mumps Virus-infected PC12 Cells

Rat pheochromocytoma (PC12) cells, persistently infected with mumps virus (MV), failed to generate full-sized stimulus-evoked action potentials (SEAPs) when examined by intracellular electrophysiological recording techniques. Application of tetrodotoxin (TTX) had little or no effect on MV-reduced SEAPs, indicating that the number of functional voltage-gated Na+ channels was decreased or their operation was blocked by the virus. In contrast, MV-infected cells generated normal Ca2+ spikes when bathed in a solution containing TTX, tetraethylammonium ions and a high concentration (20 mM) of Ca2+. In addition, when infected cells bathed in TTX were superfused with Co2+ the SEAP profile reverted to that typical of PC12 cells with functional voltagegated K+ channels only. These observations indicate that MV affects voltage-gated Na+ channels, but spares voltage-gated Ca2+ and K+ channels of persistently infected cells.

Ultrastructural Effects of Herpes Simplex Virus Type 2 Infection of Rat Dorsal Root Ganglia in Culture

The ultrastructural consequences of herpes simplex virus type 2 (strain R-2) infection of organotypic cultures of embryonic rat dorsal root ganglia were studied. The initial consequences (dilation of endoplasmic reticulum and/or Golgi apparatus and distortion of mitochondrial structure) occurred in the cytoplasm. These effects were followed by several nuclear changes including loss of nucleoplasm, and margination of chromatin. Extensive nuclear membrane proliferation was accompanied by the viral maturation process. Two previously unreported observations were made. First, productive virus replications occurred in glial cells, as well as in neurons. Mature, enveloped virus was produced by nuclear budding and envelopment in the cytoplasm in both cell types. Second, neurons were observed to participate in polykaryocyte formation with other neurons and with glial cells. These polykaryocytes were usually composed of only three or four cells. Neuronal-glial polykaryocytes were more prevalent than neuronal-neuronal polykaryocytes. In general, however, the ultrastructural changes in neurons and glial cells in culture were consistent with previously reported changes occurring in nervous tissue of experimental animals suffering from acute herpes simplex virus infections. Therefore, the utilization of this in vitro system to further study the pathogenesis of acute herpetic infections of sensory ganglia appears to be reasonable.

Neuritic uptake and transport of antiviral drugs modifying herpes simplex virus infection of rat sensory neurons

SummaryThe neuritic uptake and transport of three antiviral drugs were studied in a cell culture system with dissociated cells of rat dorsal root ganglia. Cultured sensory neurons extended neuritic projections which penetrated a vacuum grease sealed diffusion barrier in the culture. The peripheral infection with herpes simplex virus (HSV) type 1 (McIntyre) resulted in uptake and transport of HSV by neuritic extensions causing a neuronal infection inside the diffusion barrier. By varying the route of administration and concentration of drug and by manipulating the nerve cell culture system, neuritic uptake and transport also of the antiviral drugs (acyclovir, adenine-arabinoside and foscarnet) were demonstrable. The findings are discussed in relation to axonal transport and antiviral treatment of HSV infections of the nervous system.

Protective effects of rat splenic lymphocytes and peritoneal exudate cells on herpes simplex virus infection of rat dorsal root ganglia in culture.

An in vitro model system was developed to study the effects that immune cells might have on herpes simplex virus (HSV) infection of rat dorsal root ganglia in culture. Our results demonstrate that rat splenic lymphocytes and peritoneal exudate cells are incapable of replicating HSV even if these cells come from sensitized animals and are stimulated in vitro. Both cell preparations can offer some protection to rat dorsal root ganglia from the effects of HSV infection. The data suggest that an immunologically non-specific (not mediated by sensitized cells) type of protection is important to neurons, while an immunologically specific (mediated by sensitized cells) protection is most beneficial to fibroblasts. This system can be utilized to study the mechanism of latency since the neurons of sensory ganglia are the natural site of latent herpes virus.

The Herpes Simplex Virus Infection of the Rat Sensory Neuron

Conditions with confusion, anxiety, excitation, or aggression may be the earliest and the most prominent symptoms of an acute viral CNS infection. Various kinds of personality changes including a more or less severe mental deterioration may remain as sequelae after a virus infection of the brain. Undoubtedly, observations like these have kept alive the interest in virus infections as potential triggers of psychotic disorders. Of all the possible viral candidates, the herpes viruses are perhaps those that have attracted most of the attention because of their neurotropic properties, the capacity to induce reactivatable latent infections, and a ubiquitous occurrence.