Abstract
BackgroundThe recent West Nile virus (WNV) outbreaks in developed countries, including Europe and the United States, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. The changing epidemiology, the constant risk of (re-)emergence of more virulent WNV strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. Thus, to gain insight into the pathophysiological processes in severe WNV infection, a kinetic analysis of protein expression profiles in the brain of WNV-infected mice was conducted using samples prior to and after the onset of clinical symptoms.Methodology/Principal FindingsTo this end, 2D-DIGE and gel-free iTRAQ labeling approaches were combined, followed by protein identification by mass spectrometry. Using these quantitative proteomic approaches, a set of 148 proteins with modified abundance was identified. The bioinformatics analysis (Ingenuity Pathway Analysis) of each protein dataset originating from the different time-point comparisons revealed that four major functions were altered during the course of WNV-infection in mouse brain tissue: i) modification of cytoskeleton maintenance associated with virus circulation; ii) deregulation of the protein ubiquitination pathway; iii) modulation of the inflammatory response; and iv) alteration of neurological development and neuronal cell death. The differential regulation of selected host protein candidates as being representative of these biological processes were validated by western blotting using an original fluorescence-based method.Conclusion/SignificanceThis study provides novel insights into the in vivo kinetic host reactions against WNV infection and the pathophysiologic processes involved, according to clinical symptoms. This work offers useful clues for anti-viral research and further evaluation of early biomarkers for the diagnosis and prevention of severe neurological disease caused by WNV.
Highlights
West Nile virus (WNV) is a small, enveloped, positive-stranded RNA virus belonging to the Flaviviridae family
On day 3, approximately 6.5 log10 of positive strand RNA copies were present in the brain, which steadily increased until day 6 to approximately 9 log10 RNA copies
The bioinformatics analysis (Ingenuity Pathway Analysis) of each protein dataset of the different time-point comparisons revealed that four major functions were altered during the course of WNVinfection in the mouse brains: i) modifications of cytoskeleton maintenance associated with virus circulation; ii) deregulation of the protein ubiquitination pathway; iii) modulation of the inflammatory response; and iv) alteration of neurological development and neuronal cell death
Summary
West Nile virus (WNV) is a small, enveloped, positive-stranded RNA virus belonging to the Flaviviridae family (genus Flavivirus). This arbovirus is widespread, occurring on all continents with the exception of Antarctica [1]. Since its isolation from a febrile woman in Uganda in 1937 [2], WNV has been responsible of high morbidity and mortality in infected birds, horses and humans [3]. Horses or humans are considered incidental or ‘‘dead-end’’ hosts due to the insufficient blood viremia to infect a naıve feeding mosquito. Human WNV infections are asymptomatic in more than 80% of cases, 1% of persons with clinical illness could develop neurologic symptoms such as meningitis, encephalitis and acute flaccid paralysis, with low mortality rates [5]. To gain insight into the pathophysiological processes in severe WNV infection, a kinetic analysis of protein expression profiles in the brain of WNV-infected mice was conducted using samples prior to and after the onset of clinical symptoms
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