Equine herpesvirus type 1 (EHV-1) replication in primary murine neurons culture

Abstract

Equine herpesvirus-1 (EHV-1) infections cause significant economic losses for equine industries worldwide as a result of abortion, respiratory illness, and neurologic disease in all breeds of horses. The occurrence of abortions caused by EHV-1 has repeatedly been confirmed in Poland, but neurological manifestations of the infection have not been described yet. Also it is unknown how the infection of neurons with non-neuropathogenic strains is regulated. To further understand the virus-neuron interaction we studied two strains of EHV-1 in murine primary neuron cell cultures. Both strains were isolated from aborted fetuses: Rac-H, a reference strain isolated by Woyciechowska in 1959 (Woyciechowska 1960) and Jan-E isolated by Bańbura et al. (Bańbura et al. 2000). Upon infection of primary murine neuronal cell cultures with Jan-E or Rac-H strains, a cytopathic effect was observed, manifested by a changed morphology and disintegration of the cell monolayer. Positive results of immunofluorescence, nPCR and real-time PCR tests indicated high virus concentration in neurons, meaning that both EHV-1 strains were likely to replicate in mouse neurons in vitro without the need for adaptation. Moreover, we demonstrated that some neurons may survive (limited) virus replication during primary infection, and these neurons (eight weeks p.i.) harbour EHV-1 and were still able to transmit infection to other cells.

Replication kinetics of neuropathogenic and non-neuropathogenic equine herpesvirus type 1 (EHV-1) strains in primary murine neurons and ED cell line

“…The concept of EHV-1 neuropathogenicity indicates the ability to cause pathological changes in the nervous tissue which is associated with the occurrence of clinical symptoms (Cymerys et al 2010). The neuropathogenic mechanism of the virus has been partially explained by other researchers.…”

“…To address the question of EHV-1 neuropathogenicity we have established an in vitro culture system of embryonal murine neuronal cells, as described before (Cymerys et al 2010). Neuronal cells were suspended in B-27 Neuron Plating Medium consisting of neurobasal medium, B27 supplement, glutamine (200 mM), glutamate (10 mM), antibiotics (penicillin and streptomycin) with 10% supplement of fetal and equine serum (Gibco) and plated onto coverslips coated with poly-L-lysine, or poly-D-lysine with laminin (10 4 -10 5 neurons per well) and incubated at 37 o C with 5% CO 2 .…”

Acyclovir and trichostatin A modulate EHV-1 replication in murine neurons in vitro

“…Balb/c (H-2 d ) mice, genetically susceptible to EHV-1 infection, were used to establish primary cultures of murine neurons as described previously (6,9). For this purpose, cells isolated from fetal brains were cultured in B-27 Neuron Plating Medium containing B-27 supplement, 200 mM of glutamine, 10 mM of glutamate, penicillin/ streptomycin antibiotics, 5% of fetal and 5% equine serum.…”

Function of myosin during entry and egress of equid herpesvirus type 1 in primary murine neurons

“…Balb/c (H-2 d ) mice genetically susceptible to EHV-1 infection were used to establish primary culture of murine neurons, as described before (Cymerys et al, 2010). Primary murine neurons were cultured in B-27 Neuron plating medium, consisting of neurobasal medium, B-27 supplement, 200 mmol/l of glutamine, 10 mmol/l of glutamate and penicillin/streptomycin antibiotics with 10% supplement of fetal bovine (5%) and equine serum (5%) (Gibco Life Technologies).…”