This project sequenced all levels of equine brain and spinal cord transcriptome to analyze global gene expression in the CNS of natural, outbred equine hosts during grave WNV encephalitis. The data generated from this project provides invaluable insight into WN encephalitis in horses and possibly humans, and is a useful platform for future studies for both pathological and non-pathological applications. In the sequencing and analysis of the transcriptome, 41,040 sequences were identified by BLAST analysis in 5 sequence databases. There was overall consensus amongst the NCBI databases as to the hits on species, with the vast majority of sequences matching to the horse. Further analysis of the sequenced transcriptome revealed that 9,504 of the identified sequences were missed by equine predicted databases, and 1,280 of the identified sequences have yet to be discovered in the equine genome project. This is most likely due to the incomplete annotation of the equine genome and differential expression of the equine brain in disease allowing for detection of rare transcripts. Since other species' genomes have undergone more comprehensive annotation and analysis, transcriptomic sequences that should be recognized by the equine databases may be recognized in other organisms. Another possibility for the discrepancy could be differences between the equine genome and transcriptome (i.e. splice variants). These issues are likely to improve with time and more tissue specific transcriptome analyses. This portion of the project also demonstrated high sequence homology between the equine and human EST database, showing that the horse may be useful in the study of the human organism.
The main hypothesis investigated was that there are families of genes that are changed in a consistent manner in horses undergoing WN encephalitis. In the analysis of the microarray data, three subhypotheses were investigated to explore whether there was a difference in gene expression based on the state of exposure, immunity/survival, and location in the CNS. Because there was high amount of overlap in our findings from these analysis, either these findings support a generalized model of WNV encephalitis based on exposure status, recovery, and CNS pathology, or the state of WN infection without regard to immunity and recovery has been primarily modeled. Alternatively, it is possible that immunity from WN encephalitis after exposure is similar to a completely naïve, nonexposed state. Additionally, it is likely that the time of sample collection (21 days for vaccinated/exposed horses, 7–9 days for nonvaccinated/exposed horses) influenced gene expression in immune horses. However, overall, it appears that horses that are exposed to WNV demonstrate similar changes in gene expression, which are highlighted by the changes in the thalamus. Finally since the results appear to be location dependent with the majority of differentially expressed genes in the gray matter coinciding with disease/survivorship state, study of WNV in the brain is topical and likely best elucidated in a tissue specific manner.
A total of 17 neurological canonical pathways were identified across the three analyses, the majority of which involved cell signaling within the nervous system. Neurotransmitter pathways were one of the top dysregulated pathways for all groups, including glutamate and dopamine pathways. Glutamate is the primary excitatory neurotransmitter in the neurological system. Previous work has demonstrated that an excess of glutamate at the synaptic cleft can lead to apoptosis of neurons through glutamate excitotoxicity as a cause of pathology in many neurological conditions 
. In this study, the nonvaccinated group of horses exposed to WNV demonstrated gene expression changes consistent with glutamate excitotoxicity when compared to both the vaccinated and normal (non-exposed) control horses. This was also true when comparing the thalamus of the non-vaccinated horses exposed to WNV to the cerebrum of these same horses. Changes consistent with glutamate toxicity included a decrease in the expression levels of NMDA glutamate receptors, metabotropic glutamate receptors, kainate glutamate receptors, ionotropic glutamate receptors, and glutamate clearance receptors. Infection with WNV may lead to a downregulation of glutamate receptors on the post-synaptic neuron as well as glutamate uptake receptors on glial cells, leading to an increase in glutamate levels in the synaptic cleft and pathology associated with glutamate excitotoxicity.
Dopamine was another neurotransmitter pathway that was significantly changed in all three groups. Dopamine is a stimulatory neurotransmitter that functions, among other things, in the control of voluntary movement 
. In the nonvaccinated group of horses exposed to WNV, a decrease was seen in the expression levels of dopamine receptor D5 as well as the downstream affector transcripts AC, PC, and PP. In addition, tyrosine hydroxylase, which catalyzes the conversion of tyrosine to dopamine, was downregulated. The expression of monoamine oxidase (MAO), which functions to breakdown dopamine, was increased in the nonvaccinated exposed group. Thus exposure to WNV may lead to a decrease in dopaminergic receptors and subsequent downstream signaling, a decrease in enzymes to create dopamine, as well as an increase in MAO. This results in a total decrease in available dopamine, which may explain many of the clinical signs seen in WNV infection that seem to mimic human disorders such as Parkinson's disease. Further study involving the detection and quantification of the transcripts from neuronal cells infected with WNV associated with the glutamic and dopaminergic pathways is necessary before any firm conclusions can be drawn.
Clinical neurological disease in horses caused by WNV is characterized by a combination of spinal cord, midbrain/hindbrain, and mentation abnormalities, with long-term residual neurological deficits 
. The clinical signs seen in horses during WN infection mimic many of the clinical signs seen in some human neurological disorders, such as Parkinson's disease, progressive motor neuropathy, Huntington's disease, neurodegeneration, amyotrophic lateral sclerosis, and multiple sclerosis. For this study, it was found that many of the pathways and transcripts previously shown to be dysregulated during these diseases are also abnormally expressed during WNV infection in horses. Thus neurological infection with WNV in horses appears to mimic many of the seemingly non-infectious neurological disorders seen in man on both the clinical disease scale and the transcriptomic level. It is possible that seemingly non-infectious neurological disease may have an infectious origin, or that the brain can only behave and react in a certain manner no matter the stimulus or insult. This study demonstrated that infection with WNV leads to dysregulation in known neurological disease gene pathways, including those involved with neurotransmission and downstream signaling. This corresponds with clinical signs of disease in affected hosts, and also suggests a correlate between the neuropathology induced by viral infection of the CNS and the neuropathology seen in non-infectious neurological disease.
The similarities between the three analyses can also be seen when examining the immunological pathways and functions. This study demonstrated that changes in both the innate (inflammatory response, antigen presentation, immune cell trafficking) and adaptive (humoral immune response, cell-mediated immune response, cytotoxicity, immune cell trafficking) immune pathways are present during WNV infection. In general, the majority of immune transcripts and pathways were decreased in expression in the nonvaccinated horses exposed to WNV, providing evidence of downregulation of a balanced immune response during WNV infection at the peak of clinical disease.
In contrast, some immune pathways, such as the interleukin-15 signaling pathway, were upregulated during WNV infection in nonvaccinated horses exposed to WNV. IL-15 has been shown to be particularly important in providing a protective immune response to viral infection 
. For all three analyses, IL-15 was upregulated over 2-fold, as well as the transcription factor STAT1, which was upregulated over 2–3 fold. Interestingly, the downstream elements of IL-15 were downregulated in the unvaccinated horses exposed to WNV. The virus, either directly or indirectly, may be blocking the downstream effector elements of the IL-15 pathway to prevent the host immune response to the virus. There could also be other elements in the IL-15 pathway that are not yet elucidated. It is also possible that this finding is only a reflection of the timing when the naïve horses exposed to WNV were euthanized (at the onset of clinical signs) and a beneficial response from IL-15 to viral infection could not be realized in these horses. Thus it appears that IL-15 is upregulated in response to WNV infection, and while it may play a key role in recovery from viral infection, its dysregulation may be a key component of the immunopathology of this disease. Continued work targeting the quantification of IL-15 levels during viral infection at different time points is necessary for further clarification of this data.
Other pathways that were upregulated in non-vaccinated horses exposed to WNV were the IL-22, the IL-9, and the interferon signaling pathways with IL-22 and IL-9 activating similar transcripts. Both of these pathways activate JAK and TYR transcripts, which in turn phosphorylate and activate STAT (Signal Transducers and Activators of Transcription)- specifically STAT1, STAT3, and STAT5. These STAT transcripts induce the expression of ISGs (interferon stimulated genes) through a variety of mechanisms, and lead to the induction of an innate antiviral response 
. As expected, expression of these JAK/STAT transcripts is upregulated during WNV infection in the unvaccinated horses exposed to WNV. Of interest as well is the finding that the SOCS3 (suppressor of cytokine signaling 3) is also upregulated in the exposure and survival analyses. SOCS3 functions as a negative feedback inhibitor on the JAK/STAT pathway, thereby inhibiting the innate immune response 
. Transcripts of SOCS1 and SOCS3 have been shown to increase during WNV infection of the murine brain 
. Upregulation of SOCS3 allows the virus to escape the innate immune response and has also been shown to lead to chronic infection and inflammation. This is further supported by decreased expression of the transcriptional regulators ASB1 and ASB5, which function to suppress SOCS expression. Thus it is possible that while the JAK/STAT pathway is upregulated in response to WNV infection for the activation of innate immunity, WNV may induce the expression of the SOCS3 molecule to suppress this pathway and evade the innate immune response.
The transcript increased the most in expression for all analyses was pentraxin 3 (PTX3). This was the case for all analyses when comparing the unvaccinated exposed horses to the vaccinated exposed and normal horses, as well as the thalamus to the cerebrum (thus the highest levels of expression were in the unvaccinated controls). This molecule has many functions, including an integral role in the pathway of pattern recognition receptors in recognition of viruses and bacteria 
. This gene is induced by IL-1b, and functions in the phagocytosis and opsonization of antigens, as well as in the inflammatory response. Thus infection with WNV and recovery from disease may be associated with an increase in this molecule that plays an integral role in innate immunity. Another transcript that was highly increased in expression for all analyses (greatest level of expression in the non-vaccinated controls compared to other groups and in the thalamus compared to the cerebrum) was the brain specific molecule CTNND2, which functions to connect cell junctions and cytoskeletal architecture with signaling pathways 
. This may provide evidence that dysregulation of neurological tissue, such as that induced during WNV infection, leads to re-arrangement of neuronal architecture and the induction of signaling. This may also be important in viral entry into the cell. Apoptotic transcripts that were upregulated in all analyses included PARP and CASP4. Some apoptotic transcripts were upregulated in only the exposure analysis, including RXR and its receptor RAR. Understanding which transcripts are upregulated or downregulated during viral infection provides a glimpse into the affect of the virus on individual transcripts and, with further studies, could lead to the elucidation of many unanswered questions. These could include an understanding of how the virus invades the cell, as well as which cell molecules the viruses uses for replication, transcription, and translation.
The changes in gene expression data were also compared to clinical signs and histopathological data. Horses that were infected and not vaccinated demonstrated abnormal neurological and clinical signs as well as viremia, warranting humane euthanasia by day 9 of the study. On histopathological analysis, these horses also demonstrated moderate to severe signs of encephalitis, worse in the thalamus than in the cerebrum. This was in comparison to the vaccinated, exposed horses and normal horses that did not display any abnormal clinical or neurological signs and did not have a detectible viremia. Only a mild encephalitis was seen in 1 of the 6 vaccinated and exposed horses, with no abnormal histology in the normal controls. This correlates well to the gene expression data, as the non-vaccinated exposed horses demonstrated the greatest changes in gene expression when compared to the other groups of horses. The severity of the clinical signs and histopathology in the non-vaccinated horses exposed to WNV, compared to the exposed vaccinates and normal controls, is consistent with the changes in gene expression seen between the analyses of exposure and survival. This is likewise true for the location analysis when examining the histopathological differences between the thalamus and cerebrum of the exposed, non-vaccinated horses.
In summary, the microarray proved to be a useful tool to understand changes in gene expression patterns during WNV infection utilizing a custom fabricated microarray enriched for neurological and immunological sequences for study in the clinically affected host. Significant changes were identified in neurological, immunological, and apoptotic pathways with associations made between viral encephalitis and non-infectious neurological disease based on a systems biology approach. This information will eventually be integrated with other components of a systems biology approach, combining interdisciplinary scientific fields to validate these findings. This information could eventually be used to combat not only outbreaks of WNV, but also as a model to understand and reduce the impact of viral encephalitis in general.