West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in horses and humans. WNV is endemic in parts of Africa, Europe, the Middle East, and Asia, and since 1999 has spread to North America, Mexico, South America, and the Caribbean. WNV infects the central nervous system (CNS) and can cause severe disease in a small minority of infected humans, mostly immunocompromised or the elderly. This review discusses some of the mechanisms by which the immune system can limit dissemination of WNV infection and elaborates on the mechanisms involved in pathogenesis. Reasons for susceptibility to WNV-associated neuroinvasive disease in less than 1% of cases remain unexplained, but one favored hypothesis is that the involvement of the CNS is associated with a weak immune response allowing robust WNV replication in the periphery and spread of the virus to the CNS.

Recent outbreaks of Chikungunya virus (CHIKV) infection have been characterized by an increasing number of severe cases with atypical manifestations including neurological complications. In parallel, the risk map of CHIKV outbreaks has expanded because of improved vector competence. These features make CHIKV infection a major public health concern that requires a better understanding of the underlying physiopathological processes for the development of antiviral strategies to protect individuals from severe disease. To decipher the mechanisms of CHIKV infection in the nervous system, a kinetic analysis on the host proteome modifications in the brain of CHIKV-infected mice sampled before and after the onset of clinical symptoms was performed. The combination of 2D-DIGE and iTRAQ proteomic approaches, followed by mass spectrometry protein identification revealed 177 significantly differentially expressed proteins. This kinetic analysis revealed a dramatic down-regulation of proteins before the appearance of the clinical symptoms followed by the increased expression of most of these proteins in the acute symptomatic phase. Bioinformatic analyses of the protein datasets enabled the identification of the major biological processes that were altered during the time course of CHIKV infection, such as integrin signaling and cytoskeleton dynamics, endosome machinery and receptor recycling related to virus transport and synapse function, regulation of gene expression, and the ubiquitin-proteasome pathway. These results reveal the putative mechanisms associated with severe CHIKV infection-mediated neurological disease and highlight the potential markers or targets that can be used to develop diagnostic and/or antiviral tools.

Mass bird mortality has been observed in North America after the introduction of West Nile virus (WNV), most notably massive die-offs of American crows (Corvus brachyrhynchos). In contrast, WNV epidemic activity in Europe has been characterized by very low incidences of bird mortality. As the general susceptibility of European corvids to strains of WNV remains in question, European jackdaws (Corvus monedula) were inoculated with WNV strains circulating currently in Greece (Greece-10), Italy (FIN and Ita09) and Hungary (578/10), as well as a North American (NY99) genotype with a demonstrated corvid virulence phenotype. Infection with all strains except WNV-FIN resulted in mortality. Viraemia was observed for birds inoculated with all strains and virus was detected in a series of organs upon necropsy. These results suggested that jackdaws could potentially function as a sentinel for following WNV transmission in Europe; however, elicited viraemia levels might be too low to allow for efficient transmission of virus to mosquitoes.

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These birds are highly susceptible to strains circulating in Europe and, thus, may serve as surveillance sentinels.

West Nile virus (WNV) and chikungunya virus (CHIKV) are arboviruses that are constantly (re-)emerging and expanding their territory. Both viruses often cause a mild form of disease, but severe forms of the disease can consist of neurological symptoms, most often observed in the elderly and young children, respectively, for which the mechanisms are poorly understood. To further elucidate the mechanisms responsible for end-stage WNV and CHIKV neuroinvasive disease, we used transcriptomics to compare the induction of effector pathways in the brain during the early and late stage of disease in young mice. In addition to the more commonly described cell death pathways such as apoptosis and autophagy, we also found evidence for the differential expression of pyroptosis and necroptosis cell death markers during both WNV and CHIKV neuroinvasive disease. In contrast, no evidence of cell dysfunction was observed, indicating that cell death may be the most important mechanism of disease. Interestingly, there was overlap when comparing immune markers involved in neuroinvasive disease to those seen in neurodegenerative diseases. Nonetheless, further validation studies are needed to determine the activation and involvement of these effector pathways at the end stage of disease. Furthermore, evidence for a strong inflammatory response was found in mice infected with WNV and CHIKV. The transcriptomics profile measured in mice with WNV and CHIKV neuroinvasive disease in our study showed strong overlap with the mRNA profile described in the literature for other viral neuroinvasive diseases. More studies are warranted to decipher the role of cell inflammation and cell death in viral neuroinvasive disease and whether common mechanisms are active in both neurodegenerative and brain infectious diseases.

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.

SummaryWest Nile virus (WNV) and Usutu virus (USUV) are arboviruses that are maintained in enzootic transmission cycles between mosquitoes and birds and are occasionally transmitted to mammals. As arboviruses are currently expanding their geographic range and emerging in often unpredictable locations, surveillance is considered an important element of preparedness. To determine whether sera collected from resident and migratory birds in the Netherlands as part of avian influenza surveillance would also represent an effective source for proactive arbovirus surveillance, a random selection of such sera was screened for WNV antibodies using a commercial ELISA. In addition, sera of jackdaws and carrion crows captured for previous experimental infection studies were added to the selection. Of the 265 screened serum samples, 27 were found to be WNV-antibody-positive, and subsequent cross-neutralization experiments using WNV and USUV confirmed that five serum samples were positive for only WNVneutralizing antibodies and seven for only USUV. The positive birds consisted of four Eurasian coots (Fulica atra) and one carrion crow (Corvus corone) for WNV, of which the latter may suggest local presence of the virus, and only Eurasian coots for USUV. As a result, the screening of a small selection of serum samples originally collected for avian influenza surveillance demonstrated a seroprevalence of 1.6% for WNV and 2.8% for USUV, suggesting that this sustained infrastructure could serve as a useful source for future surveillance of arboviruses such as WNV and USUV in the Netherlands. K E Y W O R D Sreservoir hosts, surveillance, Usutu virus, vector-borne diseases, West Nile virus, wild birds