Pseudorabies Virus Infection Alters Neuronal Activity and Connectivity In Vitro

Abstract

Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV), infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP) firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural function seen in PRV-infected animals.

Virus Cell-to-Cell Transmission

“…To mention one more example in support of this model, while herpesviruses can spread through neurological synapses, they can also establish new cell-cell contacts outside the synapse to promote viral spread to neighboring cells (28,108). The ability to use viral glycoprotein/receptor pairs with dominant adhesion features will also allow viruses to manipulate existing synapses (78,126).…”

Function and regulation of the fusogen EFF-1 during axonal repair

“…Concentration of EFF-1 to cell borders prior to and during fusion has been observed in vivo in C. elegans as well as in heterologous systems 2,[38][39][40] . In contrast, a study of endogenous EFF-1 in the embryonic hypodermis has suggested only transient localization of EFF-1 to the membrane for fusion 37 . Some authors have postulated that this discrepancy may be related to how closely two fusing cells are apposed 40 .…”

“…Firstly, even within the hypodermis of the animal, the localization pattern appears to differ between the embryonic and larval stages of development. Localization studies in the embryo suggested only transient localization of EFF-1 to the membrane 37 , whereas in larvae, there was clear accumulation of EFF-1 at the site prior to fusion 82 . It has been postulated that this discrepancy may be due to differences in the extent of cellular apposition at these different proteins.…”

“…Later studies confirmed that PrV infection was able to induce fusion between infected sympathetic neurons in vitro, based on the diffusion of high molecular weight dyes from the soma of an injected neuron to neighboring neurons 37 . More recently, neuronal cell-cell fusion caused by PrV infection has been observed in vivo in the autonomic ganglia of infected mice 38 .…”

“…However, another study using immunostaining demonstrated that endogenous EFF-1 instead localized predominantly to intracellular compartments, and was present only transiently at the membrane. These authors suggested that accumulation might only be seen in models that involve ectopic expression of EFF-1 37 . They also noted that one of these studies 34 utilized an EFF-1::GFP reporter that was subsequently found to contain two mutations in its coding sequence 37 .…”