Treatment for many viral infections of the central nervous system (CNS) remains only supportive. Here we address a remaining gap in our knowledge regarding how the CNS and immune systems interact during viral infection. By examining the regulation of the immune and nervous system processes in a nonhuman primate model of West Nile virus neurological disease, we show that virus infection disrupts the homeostasis of the immune-neural-synaptic axis via induction of pleiotropic genes with distinct functions in each component of the axis. This pleiotropic gene regulation suggests an unintended off-target negative impact of virus-induced host immune responses on the neurotransmission, which may be a common feature of various viral infections of the CNS. Many viruses can invade the central nervous system (CNS), infect the neurons, propagate by exploiting cellular machinery, hijack axonal transport, and cross the synapses to disseminate within neural networks . This isPostulated mechanisms of the neuropathogenic impact of WNV infection center around the loss of neurons due to either direct cytopathic effect of virus replication or damaging effects of the inflammatory environment developing in response to infection. Yet, in order to develop potential neuroprotective therapies, we need to better understand the mechanisms of neuronal dysfunction during acute infection, before neuronal loss has occurred, as well as the characteristics of neurological deficits after recovery. Loss of synapses in the hippocampus has been suggested as one of the potential mechanisms underlying cognitive impairment in patients recovering from WNV encephalitis . The impOne unbiased approach to obtain a broad insight into complex pathophysiological changes occurring in the virus-infected CNS is to examine alterations in global gene expression. Although several studies have provided insights into changes in gene expression in the CNS after an experimental WNV infection, they mainly focused on the immune responses and apoptosis . Our undTissue samples for this study were selected from the CNS of NHPs inoculated intracerebrally with wild type WNV (NY99) . The ratWe examined gene expression changes in the cerebellar and spinal samples from the CNS of intracerebrally infected NHPs using Affymetrix microarray analysis and validated microarray results by qPCR. A broad examination of changes in global gene expression in response to WNV infection showed transcriptional changes that progressively increased during the neurological symptomatic stages in a CNS-region-specific manner (MX1 (12.2-fold change), IFIT3 (7.9-fold change), IFI44L (7.6-fold change), PARP9 (4.0-fold change), STAT1 (3.2-fold change), and TRIM22 (2.1-fold change). Annotation of these genes to gene ontology (GO) terms revealed a functional dichotomy for some of these genes in regulating both the immune and nervous system. While all six genes were annotated to GO biological process (BP) terms such as defense response to virus (GO:0051607) and immune system process (GO:0002376), two of these genes (MX1 and STAT1) were also annotated to the nervous system related terms such as the dendrite (GO:0030425), axon (GO:0030424), synapse (GO:0054202), and regulation of synapse structure or activity (GO: 0050803). These data suggest a dual immune-neural functionality of some early upregulated genes and offer a hint to a transcriptional activation of immune responses and concurrent modulation of neurotransmission, likely associated with sensing the spread of WNV within the CNS, which can occur trans-synaptically (At the presymptomatic stage of WNV-ND (3 dpi), no genes expressed in the cerebellum were significantly differentially regulated compared to mock . In the spinal cord, however, six genes were significantly upregulated  and none were significantly downregulated. The upregulated genes were: ptically . Since tAt the early-symptomatic stage of WNV-ND (7 dpi), there were many more upregulated than downregulated genes in both the cerebellum and spinal cord, with much stronger transcriptional changes in the spinal cord . At the The overrepresentation test , using the lists of genes that were upregulated or downregulated in the spinal cord at the advanced-symptomatic stage, identified the immune and nervous systems as the top biological systems significantly enriched for the upregulated  and downSince the top largest enriched GO BP term for downregulated genes was 'nervous system development', we further dissected transcriptional regulation of the developmental and repair processes in the CNS that may be altered by WNV infection using the gProfiler . AnalysiNext, we used the Reactome Knowledgebase  to identAs expected based on the identification of the immune system as the top biological system significantly enriched for the upregulated genes , ReactomAs expected based on the identification of the neuronal system as the top biological system significantly enriched for the downregulated genes , ReactomTogether, these results show that WNV infection induces progressive and CNS-structure-dependent gene expression changes that manifest in transcriptional upregulation of biological pathways ascribed to the immune system gene ontologies and downregulation of functions related to the neuronal system biological domain. Therefore, a larger overlap in the upregulated genes compared to downregulated genes between the cerebellum and spinal cord may be explained by a higher similarity in the immune responses, but a site-specific impairment of distinct neural functions carried by these two CNS structures. In addition, we show that WNV infection alters transcriptional regulation of developmental and repair processes in the CNS, which are associated with upregulation of the immune system development/wound healing and downregulation of the neuronal/axonal regenerative processes. However, further dissection of the molecular environment associated with axonal regeneration also indicated a trend that was more skewed to the axon growth permissive rather than inhibitory environment, suggesting initiation of the neuronal network repair programs.To further analyze the magnitude of transcriptional dysregulation within the CNS caused by WNV infection, we employed a statistical functional enrichment method that takes into account the fold changes associated with DEGs . This meThese results demonstrate that WNV infection induces coordinated shifts in the transcriptional regulation of the CNS homeostasis and that these shifts differ in magnitude and are large when associated with the immune system and subtle when related to the nervous system.Since the subtle coordinated transcriptional shifts in WNV-ND were associated with neurons and their functions , but larAnalysis of the genes that were downregulated in the cerebellum or spinal cord did not reveal any significantly enriched terms related to cell death, apoptosis, necroptosis, or necrosis. In contrast, analysis of the genes that were upregulated in the cerebellum or spinal cord showed significant (FDR < 0.05) enrichment of terms associated with regulation of cell death. Further dissection of the transcriptional regulation of cell death processes in WNV-ND revealed that: (i) cell death processes in WNV-ND were regulated bi-directionally  ; (ii) neTaken together, these results demonstrate that activation of transcriptional regulation of the cell death in WNV-ND is (i) bi-directional , (ii) region-specific , and (iii) cell-type-specific . Importantly, the magnitude of cell death regulation in neurons was about twofold less than that in lymphocytes, suggesting that neurons are not a major cell type with increased transcriptional regulation of cell death processes. Instead, it appears that the major cell type with increased regulation of cell death processes was represented by the lymphocytes . StrikiBased on the identified large coordinated shifts in transcriptional regulation of the CNS homeostasis in WMV-ND .Immunoreactivity for glial fibrillary acidic protein (GFAP) was increased in the cerebellum and spinal cord in advanced WNV-ND and showed hypertrophy of somata and processes of astrocytes and their neuron-centripetal migration and perineuronal topology, all consistent with reactive astrocytosis  was observed in the cerebellum and spinal cord in advanced WNV-ND, consistent with microglial/macrophage activation. Activated/reactive microglial cells displayed neuron-centripetal migration and perineuronal topology . These cImmunoreactivity for the lymphocytic protein markers CD20, CD4, and CD8 was detected in the cerebellum and spinal cord in advanced WNV-ND, consistent with infiltration of the CNS by peripheral CNS-extrinsic immune cells . CD20 B Based on the identified subtle coordinated shifts in transcriptional regulation of the CNS homeostasis in WMV-ND .To identify the specific neuronal cell types supporting WNV replication in the cerebellum and spinal cord, we used brightfield immunohistochemistry for WNV-antigens followed by double immunofluorescent staining for WNV-antigens and appropriate cell type markers. Colorimetric immunoreactivity for WNV-antigens (brown) was detected in Purkinje cells in the cerebellum  and motoStrikingly, double immunofluorescent staining for WNV-antigens and a calcium-binding protein calbindin D28k  revealed that immunoreactivity for CALB was diminished in Purkinje cells that were WNV-infected , compared to highly intense CALB-immunoreactivity in non-infected Purkinje cells  . CALB-imSimilar to the staining pattern with WNV-antigens and a neuron-specific marker observed in the Purkinje cells, double immunofluorescent staining for WNV-antigens and spinal motor neuron-specific marker choline acetyltransferase (ChAT) revealed that ChAT-immunoreactivity was markedly reduced in the somata and processes of spinal motor neurons that were WNV-infected , compared to highly intense ChAT-immunoreactivity in non-infected spinal motor neurons  . StrikinImmunoreactivity for the synaptic marker protein synaptophysin (SYP) was greatly depleted in advanced WNV-ND compared to mock, encompassing all layers of the cerebellar cortex   and grayImmunoreactivity for microtubule-associated protein 2  was also markedly reduced, confirming the loss of neuronal somatodendritic integrity in Purkinje cells and granule cells in the cerebellum  and spinTogether, these alterations in somatodendritic and synaptic integrity of infected neurons corresponded to the significantly enriched GO terms related to the neuronal cell body, dendrites, microtubule cytoskeleton organization, and synapse .Based on these findings, we were interested in identifying a cellular compartment of neurons that was a major target of differential transcriptional regulation in advanced WNV-ND. Ranking the neuronal CC GO terms based on their enrichment values, we identified the synapse as the most significantly dysregulated neuronal cell compartment .Topography of the observed loss of the presynaptic compartments  and postTaken together, these results reveal numerous alterations of the structural integrity and function of infected neurons and suggest that the synapse is a major target of transcriptional dysregulation in WNV-infected cerebellar and spinal neurons, which is supported by evidence of changes in synaptic integrity at the level of protein expression.Having identified the synapse as the top transcriptionally dysregulated neuronal cell compartment in advanced WNV-ND, we next asked what specific synaptic subcompartments (hereafter referred as synaptic location) and BP (hereafter referred as function) were affected. We also sought to infer whether there was a loss or gain of synaptic functions based on the synaptic genes that were upregulated or downregulated during the symptomatic stages of WNV-ND. For this, we used the genomic analysis tool SynGO, a knowledge base that accumulates research about the synapse biology and includes about 3000 expert-curated GO annotations for 1112 synaptic genes . SynGO sSynGO enrichment analysis of the DEGs annotated to the CC GO term synapse (GO:0045202) . In the Analysis of upregulated versus downregulated synaptic genes in the cerebellum at the early-symptomatic stage of WNV-ND showed that 80% of synaptic genes were upregulated and 20% were downregulated . The uprAt the advanced-symptomatic stage of WNV-ND, the number of downregulated synaptic genes increased relative to upregulated genes in both the cerebellum and spinal cord, with a higher percentage of downregulated genes in the spinal cord (71.5%). Nonetheless, about one third of the differentially expressed synaptic genes were upregulated at this stage in both the cerebellum and spinal cord . The upregulated and downregulated synaptic genes shared their annotated location and function suggesting a bidirectional dysregulation of transcriptional synaptic homeostasis in WNV-ND, affecting the spinal cord to a higher degree compared to the cerebellum . Full reTaken together, these findings suggest that WNV infection disrupts transcriptional homeostasis of the CNS synapses and impairs neurotransmission.MX1 and STAT1) have a dual immune-neural functionality , we next sought to determine whether the number of such genes increased as infection progressed. With a focus on the advanced-symptomatic stage of WNV-ND, that had the highest number of dysregulated genes, we identified overlaps in DEGs annotated to the immune or nervous system   stage of WNV-ND showed that some of genes (ctively) .Relative to the total number of WNV-induced DEGs, the cerebellum had a higher percentage of immune DEGs (33.1%), compared to the spinal cord (18.6%), even though the absolute number of immune DEGs in the spinal cord was higher. Strikingly, despite more than twice the number of the neural DEGs induced in the spinal cord compared to the cerebellum (642 vs. 252), the percentage of neural DEGs relative to the total number of DEGs was almost identical in these two CNS structures (14.6% vs. 14.5%) . These dWe next identified 94 and 179 common immune-neural DEGs that were induced in the cerebellum and spinal cord, respectively . This inWe next asked what neuronal locations and functions were under transcriptional control by the WNV-induced pleiotropic genes. Somatodendritic compartment, neuron projection, and synapse were identified as the main neuronal targets of regulation by pleiotropic genes  . Neuron Since functional characterization of WNV-induced pleiotropic immune-neural DEGs suggested that the synapse is one of their major targets in the neuron, we next determined the overlap between these pleiotropic DEGs and WNV-induced synaptic DEGs identified . These pC1QA that encodes a major constituent of the complement system subcomponent C1, was upregulated and mapped to significantly enriched GO terms associated with (i) immune/defense response to virus infection, (ii) glial cell response, (iii) neuron differentiation, and (iv) synapse . Taken together, these results validate the transcriptome data by the qPCR and provide a strong additional support to the functional genomics of the WNV-ND in NHPs reported here.We performed the validation of the WNV-ND transcriptome in NHPs at the level of select transcript expression by the qPCR. The genes for qPCR validation were selected based on their involvement in the regulation of several BPs that were found to be affected in this study: cellular calcium ion homeostasis (CALB1); excitatory synapses (GRIA1); inhibitory synapses (GABRA2 and GABBR1); perineuronal/perisynaptic extracellular matrix (TNC); astrocyte activation (GFAP); complement activation (C3); antigen processing and presentation via MHC class I (B2M); and immune cell chemotaxis/migration (CXCL10). qPCR analysis of these select transcripts had confirmed their significant  dysregulation during WNV infection in the cerebella of NHPs  as the immune-neural-synaptic pleiotropic gene in this study supports a growing evidence of the multiple roles of its protein product in the immune system and in elimination of synapses during development and disease , we also provide evidence of strict transcriptional regulation of the cell death processes in neurons. In fact, it appears that at the advanced-symptomatic stage of WNV-ND, the magnitude of regulation of cell death processes in neurons does not exceed that in lymphocytes, suggesting that neurons are not a major cell type targeted by increased transcriptional regulation of cell death processes. Instead, it appears that the major cell types with increased regulation of cell death processes were lymphocytes . Thus, Since WNV can spread trans-synaptically , changes10PFU/g [at or below the limit of detection] at 3 dpi; 6.4 ± 0.3 and 5.6 ± 0.4 log10PFU/g at 7 dpi; and 6.9 ± 0.1 and 5.8 ± 0.2 log10PFU/g at 9 dpi, respectively) , compared to the cerebellum. The changes in gene expression also became much stronger in the spinal cord, compared to the cerebellum, as infection progressed. At least three pathophysiological components should be considered to explain this phenomenon: (i) mode of the virus spread to the target neurons in the cerebellum  and spinal cord  from the site of intracerebral inoculation  by axonal transport ; (ii) ability of the virus to establish productive replication in specific neuronal cell types , and (iii) CNS-region specificity of host responses to infection. Our previous reconstruction of the directionality of trans-synaptic virus spread based on the neuroanatomical connectivity  suggestectively) , suggestctively) . Thus, tctively) . FurtherThis study may have important translational implications associated with the use of NHPs that have a high level of genetic homology to humans, parallels in functioning of the immune system, similar to humans organization of neuroanatomical pathways and skilled motor behavior and hand dexterity, all of which are coupled with their outbred nature . HoweverBesides an immunocompromised state, age-related alterations in immune responses are the most well-defined risk factors for increased susceptibility to severe WNV-ND , but howThe course of WNV-ND in our animals was abruptly interrupted at the height of neurological signs (9 dpi) due to humane animal care requirements. Therefore, there is no way to know if the disease would lead to a death or recovery, and how the phenotypic features uncovered in this study would change with time. The rapid course of WNV-ND in our NHP model is consistent with limited data showing that acute WNV encephalitis in NHPs leads to death or a moribund state that requires euthanasia 9–15 days after infection . HoweverIn summary, our findings add a new dimension to understanding of regulation of the immune-neural-synaptic axis and how its homeostasis is altered during virus infection in primates. Induction of pleiotropic genes with distinct functions in each component of the immune-neural-synaptic axis suggests an unintended off-target negative impact of virus-induced immune responses on neurotransmission in the CNS. Since activation of expression of the pleiotropic genes reported here may be a part of conserved host immune responses to many viral infections, our data may serve as a resource in the search for new therapeutic approaches to restore homeostasis in interactions between the nervous and immune system at the time when virus has been cleared from the CNS.Macaca mulatta; 2–3-year-old; seven males and two females) inoculated intrathalamically  with a dose of 5.0 log10 PFU of wild-type WNV strain NY99-35262 (hereafter WNV) that were used as a positive control in our prior study of the WNV vaccine safety  that were mock-inoculated intrathalamically  with an identical to virus inoculum volume (0.25 ml)  and other Federal statutes and regulations relating to animals. The NIAID DIR Animal Care and Use Program is guided by the ‘U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training’ (http://oacu.od.nih.gov/regs/USGovtPrncpl.htm).Tissue samples from the cerebellum and spinal cord were selected from nine rhesus monkeys (e safety  and from0.25 ml)  of dilueThree WNV-infected and one mock-inoculated monkeys were euthanized at 3, 7, and 9 dpi. Detailed clinical, virological, and histopathological information about these animals can be found in our prior publications . Tissue https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122798).Microarray experiments were performed at the Research Technologies Branch, Rocky Mountain Laboratories . The miRNeasy Mini kit (Qiagen) was used to extract total RNA via the QIAcube robot (Qiagen). To prepare target, 50 ng of each RNA sample was used as template for the Ovation V2 RNA Amplification System  to make amplified cDNA, which was purified using QIAquick 96-well (Qiagen) protocol. The cDNA (3.75 μg) was fragmented and labeled using the Encore Biotin Labeling Kit , hybridized onto the Rhesus Macaque GeneChip , washed, and scanned according to manufacturer’s instructions. Microarray data were normalized using Affymetrix Expression Console Software and gene expression analyzed using Affymetrix Transcriptome Analysis Console . Differentially expressed transcripts identified by ANOVA were arithmetically averaged and compared ratiometrically to average expression in control tissue for visualization using Spotfire Analyst  and fold changes (FC) ≤−2 and ≥2.0, false discovery rate (FDR) < 0.05 were used as cut-offs to define the significantly DEGs for subsequent functional genomic analyses. The NHP gene expression data have been deposited in NCBI's Gene Expression Omnibus  and are https://build.ngchm.net/NGCHM-web-builder/View_HeatMap.html?adv=Y. The Reactome Knowledgebase . Resulting cDNAs were purified using the QIAquick 96 PCR Purification Kit according to the manufacturer’s protocol . These purified cDNAs were used in the TaqMan qPCR validation assay. cDNAs were analyzed by qPCR using the reference gene TAF11 primers and probe in duplex with primers and probe for each select gene. Linear gene-to-reference gene ratios were calculated for each gene and sample. Normalized microarray values were used to calculate gene-to-reference ratios for each sample and gene. Spearman correlation  was calculated between qPCR and microarray values. The list of primers is provided in  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your studies of West Nile Virus infection in the primate brain provide novel insights into the devastating consequences of this disease in particular and perhaps that of other viral infections more broadly. The detailed analysis of gene expression changes at multiple time points after infection reveals a complex network of responses and will provide valuable information for other researchers pursuing additional modes of analysis.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Tadatsugu Taniguchi as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Alan Barrett (Reviewer #2).Thank you for submitting your article "Virus infection disrupts the immune-neural-synaptic axis via induction of pleiotropic gene regulation of host responses" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:Maximova et al. report on studies using a non-human primate model of encephalitis due to West Nile virus (WNV). WNV is the leading cause of arboviral neuroinvasive disease in the US with post-infectious neurologic sequelae including movement disorders and memory impairments. The authors performed intracerebral inoculation of a virulent strain of WNV and performed extensive analyses of WNV-induced alterations in gene expression on cerebellar and spinal cord tissues at 3, 7, and 9 days post-infection (dpi) compared with mock-infected animals. They also performed immunohistochemical studies examining astrocytes, microglia and T cells and neuronal infection, loss and synapse elimination.The paper is very well-written, supports findings in murine models and human disease, which have shown that presynaptic termini are eliminated in the WNV-infected hippocampus. That these processes may occur in multiple CNS regions is clinically relevant and supports the need for translation of these findings into new targets that might address multiple neurologic symptoms and sequelae.This is a largely descriptive study on the transcriptional response of two brain regions, the cerebellum and spinal cord, to i.c.v. infusion of West Nile virus. The strength of the study is the use of non-human primates, analysis at two time-points post infection and the importance of the topic in light of the current global viral pandemic. Analytics that focus on particular cellular components, thereby identifying genes associated with pre- and post-synaptic compartments as particularly strongly regulated targets, is another strength. The use of IHC to confirm predicted changes in protein levels under girding astrocytosis, microglia and T-cell reactivity and cell death is a further strength.Essential revisions:1) It is important to remember that WNV only causes disease in NHPs when the virus is directly inoculated into the brain (as pointed out by the authors). This raises important questions about the contribution of putting a needle directly in the brain and how this affects gene regulation. Unfortunately, the authors do not tell us if the control animals also received a needle injection into the brain too . This is an important point to consider in terms of the validity of the data. It is interesting that changes were detected in the spinal cord before the cerebellum in virus infected animals plus no gene changes in the cerebellum at day 3pi supporting the possibility that the needle injection does not contribute to the data obtained. However, this needs to be formally addressed in the revision.2) Related to the above, there is limited information as to how well the i.c.v. infusion of virus replicates WNV-ND in humans and this too should be more formally addressed. The Discussion provides a justification for the system being used. The reviewers note this is an artificial system. Humans get WN disease following a mosquito bite and the virus crosses the periphery into the brain. Humans do not get the virus infection by direct injection into the brain. I think this paragraph needs some revisions.3) The manuscript would benefit from more cellular analyses to localize synaptic proteins within microglia or astrocytes within the CB and SC, and their relationship with clinical findings. Presumably the authors have existing tissue which could be further analyzed to address, at least in part, some of the following issues:a) Figure 4. The authors show extensive astrocyte and microglial activation in both CNS regions of infected NHPs. Given the microglial nodules observed, can they also determine whether there was any observed neuronophagia in these tissues?b) Figure 5. The loss of synaptophysin is very striking. Can the authors also look at post-synaptic markers to determine whether both sides of the synapse are affected? It would also be important to determine whether synaptic elements can be detected within microglia, as has been observed in murine models of WNV CNS infection.c) Figure 6. The data demonstrating differential regulation of pre- and post-synaptic genes is very striking. As mRNAs for synaptic genes are localized in neurites, it would be important to determine whether the pre-synaptic termini are eliminated more efficiently and the post-synaptic termini spared.d) Figure 7. As a follow-up, it would be important to examine locations of synapse elimination within both CNS regions and relate this to motor findings in the animals.Also, given the role of IL-1 in neurotoxicity , it would be interesting to determine cellular sources of this cytokine in the NHP model.4) Figure 1. The authors mention that animals are euthanized when they develop severe neurologic symptoms. Please comment on whether the model would lead to universal death in NHPs if this were not performed. This is important because it is unclear whether patient survivors exhibit the same loss of neurons observed in those who succumb during acute CNS infection. Can the authors report on whether expression of the same genes is altered in both CNS regions during symptomatic stages of disease? Also, as many genes designated as “immune systems” function in the CNS during development and repair, it would be important to categorize them as “ׅneuroimmune systems”, which is consistent with increasing evidence of ancestral functions of neural genes as immune genes. It would also be helpful to highlight those known to function during development as they may be involved in repair.5) Figure 2. This figure shows upregulated immune system genes and down-regulated nervous system genes. It would be important to look at both up and down in both systems for the two regions. It is interesting that the CB seems to lag behind the spinal cord with regard to innate immune responses such as activation of TLRs. Can the authors comment on the time-frame of appearance of symptoms in the animals? That is, do spinal cord deficits precede cerebellar deficits? It also appears that cytokine signaling is quite different between the two regions. Studies in mice suggest that the CB is far less targeted by WNV and has constitutive expression of many PRRs. Is this the case in NHPs?6) Figure 3. The results of these analyses are consistent with published studies showing persistent activation of innate and adaptive immune responses after viral clearance. The more subtle changes in nervous system genes are also consistent with results suggesting that survivors of WNV may not have extensive neural cell damage but instead exhibit alterations in neuronal networks. Can the authors provide any additional analyses to evaluate this such as examining cell death pathways in neurons?7) That the genes which are being regulated have multiple functions, i.e. are pleiotropic, does not seem particularly surprising. The authors argue this leads to unintended deleterious effects but the logical connection there is not clear, why could it not be the opposite? Actually providing a benefit? Essential revisions:1) It is important to remember that WNV only causes disease in NHPs when the virus is directly inoculated into the brain (as pointed out by the authors). This raises important questions about the contribution of putting a needle directly in the brain and how this affects gene regulation. Unfortunately, the authors do not tell us if the control animals also received a needle injection into the brain too . This is an important point to consider in terms of the validity of the data. It is interesting that changes were detected in the spinal cord before the cerebellum in virus infected animals plus no gene changes in the cerebellum at day 3pi supporting the possibility that the needle injection does not contribute to the data obtained. However, this needs to be formally addressed in the revision.over mock-infected and protein expression in virus-infected CNS compared to mock-infected at the same time point after inoculation).The control animals used for this study were mock-infected . That is, the procedure for intracerebral  was identical  for all animals used in the study. The only difference between the virus-infected and mock-infected animals was that the inoculum injected into the thalami of the mock-control animals contained only a diluent but not the virus (1) (detailed procedure is described by us previously (2)). Thus, a potential artifact of needle insertion and injection of the inoculum volume can be excluded since we designed the study to be well-controlled and all analyses are based on the comparisons of the virus-infected animals and mock-infected animals  to formally address and clarify this in the Results and Materials and methods sections:Results:“The mock control cerebellum or spinal cord samples were from NHPs (n=4) that were inoculated intracerebrally in an identical manner to the virus-inoculated animals, except that the inoculum contained only diluent and not virus (1) (detailed procedure is described by us previously (2)). These four mock-inoculated control animals were used for normalization of gene expression and one animal (euthanized at 10 dpi) was used as a normal control for immunohistochemistry that examined the advanced-symptomatic stage of WNV-ND (9 dpi)”.Materials and methods:Macaca mulatta; 2-3-year-old; 7 males and 2 females) inoculated intrathalamically  with a dose of 5.0 log10 PFU of wild-type WNV strain NY99-35262 (hereafter WNV) that were used as a positive control in our prior study of the WNV vaccine safety (1) and from the cerebellum and spinal cord of four rhesus monkeys  that were mock-inoculated intrathalamically  with an identical to virus inoculum volume (0.25 ml) (1) of diluent without the virus  ).”“Tissue samples from the cerebellum and spinal cord were selected from nine rhesus monkeys (2) Related to the above, there is limited information as to how well the i.c.v. infusion of virus replicates WNV-ND in humans and this too should be more formally addressed. The Discussion provides a justification for the system being used. The reviewers note this is an artificial system. Humans get WN disease following a mosquito bite and the virus crosses the periphery into the brain. Humans do not get the virus infection by direct injection into the brain. I think this paragraph needs some revisions.To address these comments, we removed lines from the Discussion and revised the paragraphs where we discuss the limitations of our animal model, as follows:“This study may have important translational implications associated with the use of nonhuman primates that have a high level of genetic homology to humans, parallels in functioning of the immune system, similar to humans organization of neuroanatomical pathways and skilled motor behavior and hand dexterity, all of which are coupled with their outbred nature . […] Therefore, future studies may address the impact of WNV infection on the immune-neural-synaptic axis in older animals using the intracerebral route of infection”.3) The manuscript would benefit from more cellular analyses to localize synaptic proteins within microglia or astrocytes within the CB and SC, and their relationship with clinical findings. Presumably the authors have existing tissue which could be further analyzed to address, at least in part, some of the following issues:We strongly agree that our understanding of the roles of microglia and astrocytes in the pathogenesis of WNV infection of the CNS would benefit from further studies. However, we believe that the suggestion to address the outstanding issues listed below would require extensive analyses which should encompass methodologies that are more comprehensive that suggested by reviewers in order to get meaningful insights. Main focus of our current study was to reveal the transcriptional dysregulation of the CNS homeostasis and alterations in regulation of neural functions when the viral infection of neurons is introduced into the equation. Clearly, as we believe could be seen below from our responses, the investigation of the mechanisms of synaptic loss/elimination and identification of major cell players involved would be not a trivial task and is well beyond the scope of this study.a) Figure 4. The authors show extensive astrocyte and microglial activation in both CNS regions of infected NHPs. Given the microglial nodules observed, can they also determine whether there was any observed neuronophagia in these tissues?In our opinion, neuronophagia is an old and not very meaningful term since it can only describe the dying neurons surrounded by other cells, presumably phagocytic, in the routinely histologically stained  sections. We do not describe the microglial nodules  but went further in analyzing the topographical relation of the activated and phagocytic microglial cells to infected neurons and presented a detailed results in Figure 6B and Table 1, showing that at the advanced-symptomatic stage of WNV-ND, activated microglia migrated toward infected Purkinje and spinal motor neurons  and assumed the perineuronal topology. However, a close apposition of the activated microglia to neurons does not necessarily mean that the sole action of these cells is to phagocytose the neurons and/or their synapses. In fact, ascribing such roles to microglia can be a case of guilt by association (5) until proven otherwise. We acknowledge that more studies are needed to decipher the roles of microglia in various viral infections of the CNS, but stress that the goal of current study was to find out whether the microglia were activated at the protein level (CD68 immunostaining) and whether their cell morphology, migration pattern, and topology corresponded to the functional states revealed by changes in gene expression.b) Figure 5. The loss of synaptophysin is very striking. Can the authors also look at post-synaptic markers to determine whether both sides of the synapse are affected? It would also be important to determine whether synaptic elements can be detected within microglia, as has been observed in murine models of WNV CNS infection.c) Figure 6. The data demonstrating differential regulation of pre- and post-synaptic genes is very striking. As mRNAs for synaptic genes are localized in neurites, it would be important to determine whether the pre-synaptic termini are eliminated more efficiently and the post-synaptic termini spared.We also found the pattern of loss of synaptophysin immunoreactivity in relation to the topography of WNV-infected neurons very striking. We elected to probe for the synaptophysin in order to validate our most striking finding showing that the synapse was the most transcriptionally dysregulated cellular compartment in neurons during acute symptomatic WNV infection . Advantages of using this protein marker to confirm synaptic dysfunction at the gene expression level in this study were twofold: (i) synaptophysin is an integral component of synaptic vesicles and the most established protein marker used to assess the synaptic integrity; and (ii) synaptophysin is a pan-synaptic protein which is present in virtually all types of synapses . In addition, during the revision, we added the results of immunostaining for the choline acetyltransferase which, in addition to being an established protein marker for the spinal motor neurons, also provided a means to assess the integrity of one of the largest and numerous synaptic terminals innervating these neurons – cholinergic C-boutons. Together, the dramatic decrease of immunoreactivity for these proteins provide a strong support to the extensive transcriptional dysregulation of synaptic compartments revealed in this study.However, given the enormous number of currently known synaptic proteins ), straining for any single presynaptic molecule (number of currently known presynaptic genes: 482) or postsynaptic molecule (number of currently known presynaptic genes: 592) would neither give a full picture of synaptic dysregulation, nor it would answer the question whether the pre- or postsynaptic compartments are preferentially eliminated or spared and by which mechanisms.In our opinion, addressing the issue of presynaptic terminals and/or their postsynaptic partners loss or retention, as well as synaptic elimination by microglia (and/or astrocytes) by immunostaining for a single pre- or postsynaptic protein ) and conventional fluorescent microscopy does not provide a sufficient resolution to draw definitive conclusions. Not only the loss of a single protein expression from each synaptic compartment should not be judged as a loss of entire compartment (see our argument above for the enormous numbers of currently established proteins in each compartment), presence of microglia in close proximity to the synapses should not be judged as evidence of active stripping of synapses by microglial cells (5). Indeed, a high-resolution correlative light and electron microscopy have already challenged such conclusions (8)). Therefore, we believe the above issues should be better addressed by the highest resolution methods such as electron microscopy at the morphological level and a high throughput proteomics at a functional level. Notably, previously published attempts with using the electron microscopy (7) does not, in our opinion, provide a convincing evidence of synapse elimination by microglia, but rather show microglial cells and their processes only “adjacent” or “surrounding” the synapse, again, consistent with a case of a guilt by association (5).On the other hand, presence of synaptic proteins within the microglia may reflect a secondary effort by these cells that acquired an activated phagocytosis phenotype (confirmed in this study by detecting upregulation of phagocytosis at gene expression level and high expression of phagocytosis protein CD68) to clean up already dysfunctional and detached synapses and thus should be more appropriately viewed as a consequence rather than a cause. In line with this, we identified a dysregulation of protein-protein interactions at synapse and downregulation of synapse adhesion molecules (neurexins and neuroligins) between pre- and post-synapse in this study ; Tables 2 and 3), suggesting the detachment of the presynaptic terminals from their postsynaptic partners as possible mechanism of synaptic dysfunction and disruption of the neurotransmission. Whether the neurons orchestrate their own synapse elimination (9) by glial cells , infiltrated cytotoxic lymphocytes, or any combination of these cells in specific developmental or pathological conditions , or such elimination represents a secondary clean up events after the presynaptic terminal were already detached from their postsynaptic partners, is an open question.Clearly, addressing all these outstanding questions is beyond of the scope of our current manuscript, but we enthusiastically agree with reviewers’ comments that such insights are highly desired and should be a focus of the dedicated follow up studies. Adding to a complexity of such future research, the virus infection induced changes must be studied in the context of each specific neural circuitry since their synaptic type composition and respective functions will be unique.d) Figure 7. As a follow-up, it would be important to examine locations of synapse elimination within both CNS regions and relate this to motor findings in the animals.We agree that this will have to be deciphered in the future follow-up studies, but current staining patterns for the synaptophysin and choline acetyltransferase suggest the loss of synaptic connections that innervate specific neuronal types infected by WNV – Purkinje cells in the cerebellar cortex and spinal motor neurons in the gray matter of ventral horns of the spinal cord. We added the lists of putatively affected synapses innervating the Purkinje cells and spinal motor neurons in Table 1 and following text:“Topography of the observed loss of the presynaptic compartments  and postsynaptic cellular targets  in WNV-ND suggests that (i) putatively affected synapses in the cerebellar cortex may include synapses innervating Purkinje cells  putatively affected synapses in the spinal cord may include synapses innervating the spinal motor neurons  (Table 1).”Also, given the role of IL-1 in neurotoxicity , it would be interesting to determine cellular sources of this cytokine in the NHP model.In this work, we did not cherry-pick the molecules to analyze. However, our transcriptome data will provide an extensible resource for future data mining and validation of cell sources and the data will be publicly available at the time of publication.4) Figure 1. The authors mention that animals are euthanized when they develop severe neurologic symptoms. Please comment on whether the model would lead to universal death in NHPs if this were not performed. This is important because it is unclear whether patient survivors exhibit the same loss of neurons observed in those who succumb during acute CNS infection.We commented on this point in the original Discussion:“The course of WNV-ND in our animals was abruptly interrupted at the height of neurological signs (9 days postinfection) due to humane animal care requirements. […] However, a non-fatal encephalitis in NHPs, possibly associated with immunosuppression, can take a subacute course followed by virus persistence (18)”.Can the authors report on whether expression of the same genes is altered in both CNS regions during symptomatic stages of disease?We added the Venn diagram comparisons of the genes that were upregulated or downregulated in the cerebellum and spinal cord during symptomatic stages of disease . This analysis showed that:“Similarity between the genes that were differentially expressed in the WNV-infected cerebellum and spinal cord was higher for the upregulated genes (40 – 42%), compared to overlaps in the downregulated genes (9 – 12%)”.Also, as many genes designated as “immune systems” function in the CNS during development and repair, it would be important to categorize them as “neuroimmune systems”, which is consistent with increasing evidence of ancestral functions of neural genes as immune genes.As mentioned in the discussion, our main goal was to “to deconvolute the complex changes in CNS physiology that occur during flavivirus infection by examining differential regulation of biological processes related to the immune and nervous systems”. To this end, we relied on the categorization of differentially expressed genes established by their respective immune or neural gene ontologies. To further clarify this, we added the following to conclusions in the Results section:“Together, these results show that WNV infection induces progressive and CNS-structure-dependent gene expression changes that manifest in transcriptional upregulation of biological pathways ascribed to the immune system gene ontologies and downregulation of functions related to the neuronal system biological domain.”It would also be helpful to highlight those known to function during development as they may be involved in repair.To address this suggestion, we performed additional study and added new results and discussion:Results:“Since the top largest enriched GO BP term for downregulated genes was “nervous system development“, we further dissected transcriptional regulation of the developmental and repair processes in the CNS that may be altered by WNV infection using the gProfiler (19). […] Taking into account the fold changes for these 22 axon growth genes, when (i) downregulation of permissive or upregulation of inhibitory molecules would indicate the inhibitory molecular environment for axonal regeneration, while (ii) downregulation of inhibitory or upregulation of permissive molecules would indicate the permissive environment, we found a progressive trend which was skewed to the axon growth permissive environment.”Results:“In addition, we show that WNV infection alters transcriptional regulation of developmental and repair processes in the CNS, which are associated with upregulation of the immune system development/wound healing and downregulation of the neuronal/axonal regenerative processes. However, further dissection of the molecular environment associated with axonal regeneration also indicated a trend that was more skewed to the axon growth permissive rather than inhibitory environment, suggesting initiation of the neuronal network repair programs.”Discussion:“It is important to underscore that even during the acute course of WNV-ND in this study, several developmental processes such as response to wounding, gliogenesis, and extracellular matrix/tissue remodeling became transcriptionally upregulated. […] This may provide valuable insights and inform on how to harness potentially beneficial processes that lead to resolution of infection and neural repair. “5) Figure 2. This figure shows upregulated immune system genes and down-regulated nervous system genes. It would be important to look at both up and down in both systems for the two regions.Both upregulated and downregulated genes for the two CNS regions  at both the early- and advanced-symptomatic stages of WNV-ND were analyzed for enrichment using the Reactome Knowledgebase. Original Figure 2 presented only statistically significant enrichment for both the immune and neuronal systems. We clarified this by adding two sentences:Sentence #1: “As expected based on the identification of the immune system as the top biological system significantly enriched for the upregulated genes , Reactome pathways associated with this system showed significant enrichment only for upregulated genes , but not for the downregulated genes .”Sentence #2: “As expected based on the identification of the neuronal system as the top biological system significantly enriched for the downregulated genes , Reactome pathways associated with this system showed significant enrichment only for downregulated genes , but not for upregulated genes .”It is interesting that the CB seems to lag behind the spinal cord with regard to innate immune responses such as activation of TLRs. Can the authors comment on the time-frame of appearance of symptoms in the animals? That is, do spinal cord deficits precede cerebellar deficits?The clinical time course in rhesus monkeys after bilateral intrathalamic inoculation of 5 log PFU of the wild type WNV was very rapid, with (i) onset of the clumsiness and lethargy on day 4 postinoculation, (ii) shaky movements, incoordination, and limb weakness on day 6 postinoculation; and (iii) worsening of the above neurological signs leading to a moribund state by day 9-10 postinoculation (1). Given such a rapid development of neurological deficits, constrains of the level-3 biological containment, and the fact that it is unpractical to administer a comprehensive neurological exam to rhesus monkeys to test for specific cerebellar and spinal cord deficits, we cannot judge whether the deficits associated with the cerebellar or spinal neural networks preceded one another. The observed neurological signs such clumsiness, shaky movements, incoordination, and limb weakness can be attributed to the deficits in the cerebellar and/or spinal functions. Interestingly, we previously showed that WNV spread from the thalamus (site of inoculation) occurs most probably transsynaptically by both anterograde and retrograde axonal transport (20). Both the cerebellar neurons  and spinal motor neurons represent the second order of neurons reached by WNV ). However, virus spread to the Purkinje cells in the cerebellum could occur only by the retrograde axonal transport, while virus most likely used the anterograde axonal transport to spread to the spinal cord motor neurons. Adding to a complexity, the speed of these modes of axonal transport and the length of neuroanatomical pathways used by the virus to reach the cerebellum and spinal cord  should be considered. Nevertheless, the kinetics of viral replication in the cerebellum and lumbar spinal cord was similar and virus titers reached were among the highest compared to all other CNS regions studied ). Thus, the cerebellar lagging behind the spinal cord cannot be simply explained and awaits further elucidation.It also appears that cytokine signaling is quite different between the two regions. Studies in mice suggest that the CB is far less targeted by WNV and has constitutive expression of many PRRs. Is this the case in NHPs?The “Cytokine signaling in Immune System” node  became progressively significantly enriched  over time course of symptomatic WNV-ND in both the cerebellum and spinal cord. This was also the true for the ”child” nodes including the “Interferon Signaling”  and “Signaling by Interleukins” . Pathways in the “Interferon Signaling” nodes  were also enriched in both cerebellum and spinal cord during symptomatic WNV-ND. Slightly less enrichment in specific interleukin family pathways in the cerebellum most likely does not constitute a crucial difference in the cytokine signaling between these two CNS regions.Since no specifics provided in referring to the studies in mice that suggest that the cerebellum is less targeted by WNV in mouse models, it is difficult to comment on that. However, we aware that the granule cell neurons in the cerebellum  have been studied ex vivo and in vivo in mice and shown to have unique innate immune signatures (high expression and epigenetic regulation of the interferon-stimulated genes), that correlated with enhanced antiviral response in this type of the cerebellar neurons and rendered them less permissive to infection (21). This underscores the fact that the cerebellum is not “far less targeted by WNV”, and that the Purkinje cells but not granule cell neurons are major targets of WNV in the cerebellum in NHPs (20) and humans (22) and that Purkinje cells but not granule cell neurons are responsible for very high virus loads in the cerebellum of NHPs .6) Figure 3. The results of these analyses are consistent with published studies showing persistent activation of innate and adaptive immune responses after viral clearance. The more subtle changes in nervous system genes are also consistent with results suggesting that survivors of WNV may not have extensive neural cell damage but instead exhibit alterations in neuronal networks. Can the authors provide any additional analyses to evaluate this such as examining cell death pathways in neurons?We performed additional analyses to examine transcriptional regulation of the cell death in the cerebellum and spinal cord during the advanced-symptomatic stage of WNV-ND with a particular focus on neurons.The results are described in the new section: “Cell death processes in WNV-ND are regulated bi-directionally and magnitude of neuron cell death regulation does not exceed that of lymphocytes”.In summary, these results demonstrated that “activation of transcriptional regulation of the cell death in WNV-ND is (i) bi-directional , (ii) region-specific , (iii) cell-type-specific . […] Strikingly, regulation of the apoptotic processes in lymphocytes was a region-specific, with increased regulation of T-cell apoptosis in the cerebellum but not in the spinal cord and vice versa – increased regulation of B-cell apoptosis in the spinal cord but not in the cerebellum, suggesting differential lymphocytic responses to WNV infection in these two CNS regions.”Thus, a closer examination of transcriptional regulation of the cell death appear to support the reviewers’ view in regards to a lack of extensive activation of cell death pathways in neurons, especially when compared to other cell types in WNV-infected CNS . In addition, a CNS-region-specific activation of the programmed cell death in infiltrated lymphocytes would suggest the initiation of the resolution phase of the acute cellular inflammatory responses in WNV-infected CNS. This is consistent with our previously published results with NHP model of other flavivirus infections of the CNS (23), where we showed that the perivascular infiltrated lymphocytes rather than neurons were undergoing apoptosis.Discussions of the above findings were added to the Discussion.7) That the genes which are being regulated have multiple functions, i.e. are pleiotropic, does not seem particularly surprising. The authors argue this leads to unintended deleterious effects but the logical connection there is not clear, why could it not be the opposite? Actually providing a benefit?If the concept of pleiotropy does not seem particularly surprising, the concept of an unintended detrimental effect of induction of the pleiotropic genes that regulate immune/defense responses to virus infection but also have distinct functions in neurons, their synapses, and neurotransmission, should be clear. We argue that WNV infection of the CNS disrupts normal homeostasis of the immune-neural-synaptic axis via induction of pleiotropic gene regulation of host responses with possible off-target effects of virus-induced host immune responses on neural functions and neurotransmission. We believe that our Discussion provides a sufficient logical explanation of these concepts.“CNS neurons are terminally differentiated cells and cannot be replenished, thus virus infection of neurons present a challenge for both the nervous and immune system to coordinate virus clearance while protecting neurons and preserving neural function. […] Remarkably, some pleiotropic proteins, such as proinflammatory cytokines and proteins in the complement system and major histocompatibility complex are essential for the establishment, organization, function, and removal of synapses between neurons (24).”“We propose a scenario in which changes in expression of pleiotropic genes that are intended to activate and maintain immune responses to virus infection would have unintended and potentially devastating off-target effects on neuron-to-neuron synapses and chemical neurotransmission”.“Since WNV can spread transsynaptically (20), changes in synaptic homeostasis identified in this study may indicate either a direct damaging impact of infection on neurotransmission or an attempt by the host to arrest virus dissemination and compensate for changes in function. More studies will be required to understand to what extent these synaptic changes represent pathological, protective, and/or compensatory mechanisms, as well as whether they are reversible”.“In summary, our findings add a new dimension to understanding of regulation of the immune-neural-synaptic axis and how its homeostasis is altered during virus infection in primates. […] Since activation of expression of the pleiotropic genes reported here may be a part of conserved host immune responses to many viral infections, our data may serve as a resource in the search for new therapeutic approaches to restore homeostasis in interactions between the nervous and immune system at the time when virus has been cleared from the CNS”.References:1) Maximova OA, et al. (2014) Assurance of neuroattenuation of a live vaccine against West Nile virus: A comprehensive study of neuropathogenesis after infection with chimeric WN/DEN4Delta30 vaccine in comparison to two parental viruses and a surrogate flavivirus reference vaccine. Vaccine 32(26):3187-3197.2) Maximova OA, et al. (2008) Comparative neuropathogenesis and neurovirulence of attenuated flaviviruses in nonhuman primates. Journal of virology 82(11):5255-5268.3) Messaoudi I, Estep R, Robinson B, and Wong SW (2011) Nonhuman primate models of human immunology. Antioxidants and redox signaling 14(2):261-273.4) Lemon RN and Griffiths J (2005) Comparing the function of the corticospinal system in different species: organizational differences for motor specialization? Muscle and nerve 32(3):261-279.135) Perry VH and O'Connor V (2010) The role of microglia in synaptic stripping and synaptic degeneration: a revised perspective. ASN neuro 2(5):e00047.6) Koopmans F, et al. (2019) SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse. Neuron.7) Vasek MJ, et al. (2016) A complement-microglial axis drives synapse loss during virus-induced memory impairment. Nature 534(7608):538-543.8) Weinhard L, et al. (2018) Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction. Nature communications 9(1):1228.9) Di Liberto G, et al. (2018) Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping. Cell 175(2):458-471.e419.10) Perry VH and O'Connor V (2008) C1q: the perfect complement for a synaptic feast? Nature reviews. Neuroscience 9(11):807-811.11) Eroglu C and Barres BA (2010) Regulation of synaptic connectivity by glia. Nature 468(7321):223-231.12) Verkhratsky A and Nedergaard M (2014) Astroglial cradle in the life of the synapse. Philosophical transactions of the Royal Society of London. Series B, Biological sciences 369(1654):20130595.13) Chung WS, et al. (2013) Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways. Nature 504(7480):394-400.14) Troscher AR, et al. (2019) Microglial nodules provide the environment for pathogenic T cells in human encephalitis. Acta neuropathologica 137(4):619-635.+ T cell-mediated dendrite and synapse loss. The Journal of experimental medicine 210(10):2087-2103.15) Kreutzfeldt M, et al. (2013) Neuroprotective intervention by interferon-gamma blockade prevents CD816) Stephan AH, Barres BA, and Stevens B (2012) The complement system: an unexpected role in synaptic pruning during development and disease. Annual review of neuroscience 35:369-389.17) Garber C, et al. (2019) T cells promote microglia-mediated synaptic elimination and cognitive dysfunction during recovery from neuropathogenic flaviviruses. Nature neuroscience 22(8):1276-1288.18) Pogodina VV, et al. (1983) Study on West Nile virus persistence in monkeys. Archives of virology 75(1-2):71-86.19) Raudvere U, et al. (2019) g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic acids research 47(W1):W191-w198.20) Maximova OA, Bernbaum JG, and Pletnev AG (2016) West Nile Virus Spreads Transsynaptically within the Pathways of Motor Control: Anatomical and Ultrastructural Mapping of Neuronal Virus Infection in the Primate Central Nervous System. PLoS neglected tropical diseases 10(9):e0004980.21) Cho H, et al. (2013) Differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded RNA viruses. Nature medicine 19(4):458-464.22) Omalu BI, Shakir AA, Wang G, Lipkin WI, and Wiley CA (2003) Fatal fulminant pan-meningo-polioencephalitis due to West Nile virus. Brain pathology  13(4):465-472.23) Maximova OA, Faucette LJ, Ward JM, Murphy BR, and Pletnev AG (2009) Cellular inflammatory response to flaviviruses in the central nervous system of a primate host. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 57(10):973-989.24) Boulanger LM (2009) Immune proteins in brain development and synaptic plasticity. Neuron 64(1):93-109.


This article comments on:Rozier F, Riglet L, Kodera C, Bayle V, Durand E, Schnabel J, Gaude T, Fobis-Loisy I. 2020. Live-cell imaging of early events following pollen perception in self-incompatible Arabidopsis thaliana. Journal of Experimental Botany 71, 2513–2526. The first stage of male–female recognition in flowering plants takes place when pollen lands on the surface of the stigma. In Brassicaceae, compatible pollen will hydrate and germinate, leading to successful fertilization, while these processes are severely impaired in incompatible pollen, preventing fertilization. Successful fertilization in flowering plants involves multiple communication events between the pollen and the pistil . In BrasAlthough we have good knowledge of the SI-induced signalling events leading to self-pollen rejection in the Brassicaceae  and the self-incompatible pathway (B).Brassica) in the papilla cells, which is essential for the hydration of pollen grains (A).Signalling factors in the stigma that trigger the compatible pathway remain largely unknown (A). PCP-B pollen coat proteins could act as ligands for triggering the compatibility pathway that initiates water transfer from stigmatic papilla cells. EXO70A1 regulates polarized secretion of vesicles (in Arabidopsis) or MVBs (in S-locus CRP SCR/SP11 (B). This interaction results in the phosphorylation of SRK, which in turn phosphorylates two other proteins: M-locus protein kinase (MLPK) and Armadillo repeat containing 1 (ARC1) with E3 ubiquitin ligase activity. The activated ARC1 facilitates the proteasomal degradation of the exocyst subunit EXO70A1. EXO70A1 therefore acts at the interconnection of the two signalling pathways, while it appears that these two pathways are separated upstream of EXO70A1. The functional role of the increase in cytosolic Ca2+ observed after a compatible pollination (A), and even more dramatic after an incompatible pollination (B) remains to be resolved. Note that the ACA13-mediated export of Ca2+ from the papilla cells to the pollen, only induced by compatible pollination, is not shown here.The self-incompatibility pathway leading to self-pollen rejection is initiated by the interaction of the stigma-expressed SRK receptor with the cognate pollen 2+ and other factors altering the activity of ABPs) initiated by the compatibility response.Reorganization and focalization of the actin cytoskeleton (shown as orange dashed lines) have been observed in the stigma at the contact site with compatible pollen and the pollen tube . It remains to be confirmed if this actin reorganization is triggered by the mechanical pressure produced by pollen tubes at the site of cell wall penetration and/or if it is a direct consequence of a signalling cascade  suggests that this penetration step may represent an additional checkpoint (besides the pollen hydration step) to help ensure that incompatible pollen cannot achieve fertilization. This penetration step may require the regulated secretion of enzymes along the pollen–papilla contact sites to facilitate pollen tube growth through the cell wall matrix (C).Several studies have used imaging techniques to investigate changes in the stigmatic papilla cells during self- and cross-pollination e.g. . The newin vivo assays using live-cell imaging to record early pollination events. Using the ratio of the long and wide pollen axis (L/W) as a proxy for pollen hydration, they showed that compatible pollen need to pass below a threshold value of L/W=1.4 before they start germinating. As shown before in Brassica  is a pollen coat-derived CRP involved in controlling recognition of incompatible pollen. SCR/SP11 acts as the ligand in Brassicaceae SI, binding to the allelic S-locus receptor kinase (SRK) in the stigmatic papillae to activate a signalling cascade that inhibits hydration and germination of incompatible pollen (Secreted cysteine-rich proteins (CRPs) found in the pollen coat have been shown to be involved in cellular recognition/communication during the earliest stages of pollen–stigma interaction . This further illustrates the extent of vesicle trafficking and targeted secretion required at the papilla–pollen contact site to accommodate the rapid pollen hydration process in Brassicaceae.Exocyst-mediated polarized secretion in stigmatic papilla cells was shown to be important for compatible pollen hydration ; Box 1. Brassica (2+, just below the contact site, with the highest increase observed when the pollen tube penetrates the papilla cell wall (2+ from papilla cells to germinating pollen, a process promoting successful fertilization and mediated by ACA13, a calmodulin-activated calcium pump, accumulating at the plasma membrane near the pollen attachment site (Remodelling of the actin cytoskeleton, leading to actin focalization at the contact site with compatible pollen, appears to be a hallmark feature in compatible pollen–stigma interactions in Arabidopsis and Brassica  and may,2+ levels and polarized secretion in papilla cells, the level of integration and crosstalk between these events remains largely unknown (see The function of actin focalization remains to be determined. The fact that the actin cytoskeleton is important for the delivery of secretory vesicles and knowledge that there is polarized delivery of secretory vesicles in the papilla cells to the pollen contact site to promote localized exocyst tethering and membrane fusion (nown see .Early electron microscopy studies have shown that, prior to pollen tube penetration, a compatible pollen–stigma interaction triggers an expansion of the outer papilla cell wall layer at the site of interaction . FollowiBrassica SI system, along with other genetic tools that can be combined with live-cell imaging approaches utilizing the set-up described by Two interacting signalling pathways have been suggested to operate in the stigma of Brassicaceae that exhibit SI: the ‘basal compatible pollen response pathway’ regulating the acceptance of compatible pollen, and the extensively studied self-incompatible pathway leading to self-pollen rejection . Future


Respiratory viruses are a major cause of infectious disease and mortality worldwide. Among respiratory viruses, emerging and well-known coronaviruses and influenza viruses cause seasonal epidemics and pandemics and rank as very important pathogens. The clinical signs of viral respiratory infections range from asymptomatic forms to life-threatening acute respiratory distress syndrome . Another reported cause of mortality is multiple organ dysfunction. The ongoing pandemic of coronavirus disease 19 ) is a serious public health problem. SARS-CoV-2 infection induces to a clinical signature distinct from that associated with other types of pneumonia, and SARS-CoV-2 differs from influenza viruses with regard to target cells and replicative properties. The morbidity and mortality rates are higher for COVID-19 than for seasonal influenza.Immunity, Zhu et al. described the dynamic immune landscape in patients with SARS-CoV-2 or influenza A virus (IAV) infections.1 Using single-cell RNA sequencing, the researchers compared transcriptomic changes during the course of infection in peripheral blood mononuclear cells (PBMCs). This approach enabled the researchers to report that the gene expression pattern in patients with COVID-19 differs from that in IAV-infected patients.When considering community-acquired viral pneumonia caused by different pathogens, it is essential to compare the respective host immune signatures. This comparison can highlight conserved or specific immune pathways involved in the pathogenesis. In a recent issue of PRDM1 and IRF4 in the B cells). This indicates that on admission, patients have already started to mount a virus-specific immune response. Compared to IAV-infected patients, the counts of XCL1+ NK cells and CD8+ T cells were significantly increased in patients with COVID-19. Conversely, IAV-infected patients had an increased count of activated CD4+ T cells. Regarding the molecular components of the immune response, the expression of proinflammatory cytokines (TNF) and cytokine receptors  was higher in multiple cell types in patients with COVID-19 than in IAV-infected patients. Of note, many receptors involved in the activation of cytokines were more strongly expressed in activated CD4+ T cells, indicating a particular role of this subset in the cytokine-driven inflammatory response reported in COVID-19 patients. The expression levels of hallmark genes in the type I IFN response  were also increased in the circulating immune cells of patients with COVID-19. This is of interest with regard to the controversial role of type I IFNs in the host response to SARS-CoV-2 infection.2 Conversely, IAV-infected patients had enhanced expression of transcription factors  involved in the regulation of the inflammatory immune response when compared with the expression in patients with COVID-19. Taken as a whole, Zhu et al.’s results suggest that patients with COVID-19 and patients with influenza have both common and specific systemic immune traits  was greater in all patients with viral infection than in healthy controls, suggesting an active mechanism of producing protective antibodies  would also be useful. Longitudinal analyses of well-characterized clinical cohorts (in terms of age and comorbidities) will be essential. Complementary high-throughput technologies  might also be very informative.6 Exploring the mechanisms that underlie the transition from respiratory virus-related pneumonia to ARDS and multiple organ dysfunction is critical for furthering our understanding of the pathogenesis of COVID-19 and flu. Therefore, studies of patients with various clinical courses and outcomes will be highly valuable. Since the inflammatory response in viral infection-induced ARDS (including COVID-19-driven ARDS) is compartmentalized within the lung,8 a comparative analysis of the immune landscape in the lung will be of great interest. It is noteworthy that single-cell omics of the upper respiratory tract9 and bronchoalveolar lavages10 has already revealed interesting clues. Finally, although scientists are usually conditioned to identify differences and to generate original data, highlighting common immune traits in distinct diseases might also be useful for advancing our understanding of the pathogenesis and developing therapeutic strategies. Moreover, immunologists should be encouraged to create a comprehensive and dynamic picture of the immune response during acute viral respiratory infections. This type of approach might yield valuable predictive markers of disease severity and outcomes and might pave the way for generic or more tailored treatment options.Although these data provide important insights and reveal differences in the immune landscapes in patients infected with SARS-CoV-2 and those infected with influenza viruses, they also raise new questions. The small number of patients in each group  might accentuate individual differences. Single-cell transcriptomics has revealed the heterogeneity of peripheral immune activation in patients with COVID-19.


The differential diagnosis of a regular, narrow QRS, long-R–P tachycardia includes atypical atrioventricular nodal reentry tachycardia, atrial tachycardia, and atrioventricular reentry tachycardia via a slowly conducting accessory pathway with decremental conduction properties. Almost all described diagnostic maneuvers in the electrophysiology laboratory have exceptions to their primary interpretation. The usual proviso is that the observation must be reproducible. A single spontaneous His-refractory PVC (Hr-PVC) during the tachycardia terminated the ongoing tachycardia . What is the possible mechanism of this termination response of tachycardia?A 27-year-old female with a history of narrow complex tachycardia was referred for radiofrequency ablation. Her tachycardia was easily induced by premature ventricular contractions (PVCs) during electrocardiography 6The differential diagnosis of a long-R–P supraventricular tachycardia with the earliest atrial activation in the His-bundle region includes atypical atrioventricular (AV) nodal reentry tachycardia (AVNRT), atrial tachycardia (AT), AV reentry tachycardia (AVRT) via a slowly conducting accessory pathway with decremental conduction properties, nodofasciular reentrant tachycardia (NFRT), or nodoventricular reentrant tachycardias (NVRT). and 2B for ease of initiation of AV node– dependent tachycardias.8 Adenosine-induced termination of this tachycardia, with an A-wave followed by A–H block, eliminated the diagnosis of AT .4 An Hr-PVC in Figure 3 stopped the tachycardia without retrograde conduction to the atrium, excluding the diagnosis of AT10 and leaving us with the possibility of AVNRT with a bystander nodofasciular pathway.14 Termination of the tachycardia with an Hr-PVC without affecting atrial depolarization is suggestive of AVRT or NFRT/NVRT15; however, the coincidental termination or atypical AVNRT with bystander pathways might also explain this response.16 In the current case, the termination response without A  was not reproducible. Almost all described diagnostic maneuvers in the EP laboratory have exceptions to their primary interpretation17; therefore, the usual proviso is that the observation must be reproducible.Post-PVC prolongation of the P–R interval and tachycardia initiating can be explained by concealed conduction. The partial penetration of an impulse into the AV node after a PVC might infer the behavior of the subsequent sinus impulse that is conducted through the AV node . However, the cycle length (CL) alternation during tachycardia was of such a large degree that it should be readily identifiable. Therefore, the Hr-PVC response in the current case was not applicable as an EP maneuver. As the durations of two H–H intervals were each equal to double the length of an H–H interval for both Hr-PVC cycles , both Hr-PVC responses were accepted as pseudo-resetting responses   and 5, which can be explained by a reciprocal CL alternans during AVNRT. Ho et al. reported that Hr-PVC assessment is the only viable maneuver by which to identify a concealed, bystander NFRT during atypical AVNRT18; these pseudo-resetting responses to Hr-PVCs also favor atypical AVNRT over NFRT/NVRT.18 Furthermore, the ventricular overdrive suppression did not entrain the tachycardia but rather dissociated the ventricle from the ongoing tachycardia, ruling out the AVRT or nodoventricular pathway as a tachycardia mechanism .20 These results collectively seem sufficient to establish a diagnosis of atypical AVNRT. A longitudinal dissociation of one slow pathway or two separate slow pathways could explain the oscillation of two CLs.21 The degree of wobble in this example is about 40 ms, raising the possibility of atypical AVNRT using two separate slow pathways for retrograde conduction.22 Slow pathway ablation was successful in eliminating the arrhythmia. This case stresses that almost all described diagnostic maneuvers in the EP laboratory have exceptions to their primary interpretation.17It was interesting that both advancement-like and delay-like responses to Hr-PVCs were observed during the current case