Glutamate and lipid metabolic perturbation in the hippocampi of asymptomatic borna disease virus-infected horses

Abstract

Borna disease virus (BDV) is a neurotropic, enveloped, non-segmented, negative-stranded RNA virus that infects a wide variety of vertebrate species from birds to humans across a broad global geographic distribution. Animal symptomatology range from asymptomatic infection to behavioral abnormalities to acute meningoencephalitis. Asymptomatic BDV infection has been shown to be more frequent than conventionally estimated. However, the molecular mechanism(s) underyling asymptomatic BDV infection remain largely unknown. Here, based on real-time quantitative PCR and Western blotting, a total of 18 horse hippocampi were divided into BDV-infected (n = 8) and non-infected control (n = 10) groups. A gas chromatography coupled with mass spectrometry (GC-MS) metabolomic approach, in conjunction with multivariate statistical analysis, was used to characterize the hippocampal metabolic changes associated with asymptomatic BDV infection. Multivariate statistical analysis showed a significant discrimination between the BDV-infected and control groups. BDV-infected hippocampi were characterized by lower levels of D-myo-inositol-1-phosphate, glutamate, phosphoethanolamine, heptadecanoic acid, and linoleic acid in combination with a higher level of ammonia. These differential metabolites are primarily involved in glutamate and lipid metabolism. These finding provide an improved understanding of hippocampal changes associated with asymptomatic BDV infection.

Figure 1. BDV Detection in Horse Hippocampal Tissues by RT-qPCR and Western Blotting.

Amplification plots of RT-qPCR for BDV p24 RNA from all samples are showed (A). The cycle number on the horizontal axis is plotted against the normalized fluorescence on the vertical axis. Signals were regarded as positive if and only if the fluorescence intensity exceeded 10 times the standard deviation of the baseline fluorescence (threshold). Cq values greater than 40 were regarded as negative. BDV p24 proteins in all hippocampal samples were detected by Western blotting (B). Lanes 1–8, p24 protein positive tissue. PC: positive control sample, hippocampal tissue from persistently BDV-infected rat. NC: negative control sample, hippocampal tissue from healthy rat.

Figure 2. Representative GC-MS Total Ion Current Chromatograms from BDV-Infected and Control Horses.

Figure 3. 2D-PCA Scores Map (PC1 versus PC4) (A) and 2D Cross-Validated OPLS-DA Score Map (B) of GC-MS Data.

Each dot denotes an individual sample (BDV-infected, n = 8; control, n = 10). The ellipse represents the Hotelling's T2 with 95% confidence in score plots.