Abstract

Background

   Monkeypox (MPOX) is a zoonotic disease caused by the MPOX virus (MPXV).
   MPOX resurfaced globally in May 2022, spreading throughout six WHO
   regions, resulting in nearly 87,000 cases and 112 deaths. Clinical
   symptoms include swollen lymph nodes, fever, joint pain and several
   neurological complications such as headache, encephalitis, myalgia,
   fatigue, photophobia and seizures. Despite these manifestations, the
   precise mechanisms of MPXV’s neurotropism remain elusive. This study
   aimed to explore the genetic underpinnings of MPOX-related neurological
   manifestations, including headache, myalgia, fatigue, and photophobia,
   using advanced bioinformatics tools.

Methods

   Data were sourced from the GeneCards database, which is an integrated
   database of human genes. Genes linked to MPOX and its neurological
   manifestations were identified and cross-referenced to uncover shared
   genes between these conditions. Network visualization was created using
   STRING, followed by topological analysis in Cytoscape to identify key
   genes based on degree and betweenness centrality. Functional enrichment
   analysis through ToppGene provided insights into molecular functions,
   biological processes, and cellular components associated with these
   target genes. Pathway analysis was performed using WikiPathways, and
   cell-type-specific enrichment was conducted using Enrichr.
   Additionally, we predicted functional microRNAs using mirTarbase and
   identified potential drug candidates via the Stitch database.

Results

   We identified 32 MPOX-associated genes and a large set of neurological
   manifestation-related genes. Ten hub genes, including CD55, CXCL1,
   NFKB1, CXCL8, CD4, IL6, MX1, CFH, KLRK1, and CD46 were shared between
   MPOX and its neurological manifestations. Five novel genes, including
   CFHR3, C5AR1, C3AR1, IFNA2, and CXCL3 were predicted to be associated
   with MPOX and its neurological complications. Gene ontology analysis
   highlighted biological processes such as immune regulation, viral life
   cycle, and lymphocyte activation, while pathway enrichment identified
   critical signaling mechanisms like prostaglandin signaling, toll-like
   receptor 4 (TLR4) signaling, complement activation, and
   neuroinflammation. Moreover, cell types such as T-helper cells, natural
   killer cells, and microglia were found to be significantly impacted by
   MPOX and its frequent neurological complications. We identified 11 key
   microRNAs associated with MPOX-neurological manifestations and
   repurposed eight potential drugs, offering promising therapeutic
   strategies.

Conclusion

   This study emphasizes the central role of the complement system,
   immunological responses, and inflammatory pathways in the neurological
   manifestations of MPOX. The identification of novel genes and predicted
   therapeutic targets paves the way for future research and therapeutic
   interventions. Experimental validation is required to confirm these
   findings and determine the effectiveness of the proposed treatments.

   Keywords: monkeypox, bioinformatics, natural killer cells, headache,
   T-helper cells, myalgia, photophobia, drug repurposing

1. Introduction

   Monkeypox (MPOX) is an emerging zoonotic disease caused by the
   monkeypox virus (MPXV), a member of the Orthopoxvirus genus within the
   Poxviridae family, closely related to the variola virus responsible for
   smallpox ([27]El Eid et al., 2022; [28]Karagoz et al., 2023).The virus
   was first identified in 1958 during research involving monkeys, hence
   its name. The first human case was reported in 1970 in a 9-month-old
   boy from the Democratic Republic of the Congo (DRC) ([29]Ladnyj et al.,
   1972; [30]Mileto et al., 2022). Since then, sporadic outbreaks have
   occurred primarily in endemic regions of Africa, with occasional cases
   reported outside the continent, including the United States in 2003 and
   cases in the United Kingdom, Singapore, and Israel between 2018 and
   2019 ([31]Ligon, 2004; [32]Reynolds et al., 2007; [33]Oladoye, 2021;
   [34]Adler et al., 2022).

   MPOX re-emerged in May 2022 and quickly spread over Europe, the
   Americas, and all six World Health Organization (WHO) regions, causing
   around 87,000 cases and 112 deaths in 110 countries ([35]Al-Tammemi
   et al., 2022; [36]Lim et al., 2023; [37]Mitjà et al., 2023). While the
   primary reservoir of MPXV has yet to be found, rodents are the most
   likely candidates. Transmission occurs through direct or indirect
   contact with respiratory droplets, infected skin lesions, or body
   fluids from infected animals or humans, as well as via placenta
   transfer and sexual interaction ([38]Guarner et al., 2004; [39]Heskin
   et al., 2022; [40]Riopelle et al., 2022).

   Clinically, MPOX presents with symptoms such as swollen lymph nodes,
   fever, back pain, muscle pain, and headache, resembling a milder form
   of smallpox ([41]Ahmed et al., 2023). Additionally, a wide range of
   neurological complications such as headache, encephalitis, myalgia,
   seizure, decreased hearing, visual changes, fatigue, photophobia,
   malaise, loss of appetite, and changes in consciousness have been
   observed sporadically in MPOX patients, which may be linked to the
   virus’s ability to penetrate brain tissue, as evidenced in certain
   infected animals ([42]Badenoch et al., 2022; [43]Billioux et al., 2022;
   [44]Slomski, 2022; [45]Khan et al., 2023). Neuroinvasion mechanisms
   remain poorly understood but may involve direct invasion via the
   olfactory epithelium and hematogenous spread via infected
   monocytes/macrophages have been suggested as two possible routes for
   viral neuroinvasion. Despite these hypotheses, the precise mechanisms
   of neurotropism remain unknown ([46]Sepehrinezhad et al., 2023).
   Studies have demonstrated that the MPXV is capable of infecting human
   astrocytes, microglia, and neurons. Animal studies have also reported
   that replication of the virus is possible in brain parenchyma ([47]Earl
   et al., 2012; [48]Hutson et al., 2015; [49]Sergeev et al., 2016;
   [50]Chailangkarn et al., 2022; [51]Miranzadeh Mahabadi et al., 2024;
   [52]Bauer et al., 2023; [53]Schultz-Pernice et al., 2023). Moreover,
   elevated levels of inflammatory markers such as interleukin-6 (IL-6),
   interleukin-1B (IL-1B), tumor necrosis factor alpha (TNFα) and C-X-C
   motif chemokine ligand 8 (CXCL8) in MPOX patients further support the
   hypothesis of an immune-mediated neurological pathogenesis
   ([54]Johnston et al., 2015; [55]Wong et al., 2018; [56]Agrati et al.,
   2023).

   Although more cases are now reporting neurological complications linked
   to MPOX, we still know very little about how the virus actually causes
   these manifestations at the molecular level. There are currently no
   treatments specifically designed to address the neurological
   complications of MPOX. While antivirals like tecovirimat and cidofovir
   have been approved for smallpox and are sometimes used in MPOX cases,
   it’s unclear how effective they are against viruses that can affect the
   brain ([57]Das et al., 2023; [58]Prévost et al., 2024). This gap in
   knowledge highlights the urgent need to better understand how MPXV
   interacts with the nervous system and to explore new therapeutic
   strategies that can help protect patients from these potentially
   serious complications.

   In this context, our study utilizes advanced bioinformatics approaches
   to identify shared genes and molecular mechanisms between MPOX and its
   most prevalent neurological manifestations (headache, myalgia, fatigue,
   and photophobia). Through integrative analysis including gene network
   construction, pathway enrichment, cell-types and drug repurposing, we
   aim to uncover key immune and inflammatory pathways that may underlie
   MPOX-associated neurological manifestations and suggest viable
   intervention options for therapy development.

2. Methods

2.1. Dataset selection and collection of relevant genes

   All genes utilized in this study were sourced from the GeneCards
   database ([59]https://www.genecards.org/) ([60]Stelzer et al., 2016;
   [61]Grissa et al., 2022). GeneCards is an integrated database that
   contains a variety of proteomic, genomic, and transcriptomic
   information about human genes. We discovered after doing a thorough
   literature review that headache, myalgia, fatigue, and photophobia were
   the most common neurological manifestations of MPOX ([62]Badenoch
   et al., 2022; [63]Khan et al., 2023). Genes associated with these
   manifestations and MPOX were cross-referenced from GeneCards and
   literature on MPOX. Subsequently, we exported all gene sets to an Excel
   file to uncover shared genes between MPOX and neurological
   manifestation’s linked genes. All subsequent analyses focused on the
   shared genes between MPOX-related and neurological
   manifestations-associated genes (target genes).

2.2. Network visualization of target genes and predicting novel associated
genes

   To visualize and predict protein-protein interactions in the Homo
   sapiens organism, we submitted our target genes to STRING
   ([64]https://string-db.org/). The resulting network was then imported
   into Cytoscape version 3.10.1 for analysis of the key topological
   features, such as degree, and betweenness centrality. These
   characteristics aid in identifying the most influential genes in the
   network: nodes with the most connections (degree) and genes in the most
   central positions (betweenness). According to gene-gene interaction
   data in STRING we also predicted some novel genes for MPOX-neurological
   manifestations shared genes.

2.3. Functional analysis, microRNA prediction and drug repurposing

   To identify and predict potentially relevant functional mechanisms for
   our target genes, we performed an integrative enrichment analysis with
   ToppGene ([65]https://toppgene.cchmc.org/). Different functional
   enrichment was carried out using the ToppFun panel in the ToppGene
   online tool ([66]Ghanbarzehi et al., 2023). Each gene set was
   individually enriched for gene ontology (GO), which includes molecular
   function, biological activity, and cellular component. Pathway analysis
   was performed through WikiPathways in ToppGene. Cell-type specific
   enrichment analysis was also conducted through Cell-marker 2024 section
   in Enrichr ([67]https://maayanlab.cloud/Enrichr/). Importantly, we also
   predicted several functional miRNAs for the MPOX-neurological
   manifestations associated genes using mirTarbase in ToppGene.
   Subsequently, we repurposed some potentially effective drugs for our
   target genes using stitch database in ToppGene. We used a p-value
   cut-off of ≤0.05 in all statistical analyses. All results were
   presented as -log (p-value) to enable visualization of both high and
   low significance values on a linear scale and displayed using GraphPad
   Prism version 9 or [68]Biorender.com.

3. Results

3.1. Identified gene sets and reconstructed genetic network

   According to extracted data from GeneCards, 32 genes were detected to
   be linked to the MPOX ([69] Supplementary Table S1 ). Furthermore, for
   neurological manifestations gene sets, 6650, 2829, 6842, and 3956 genes
   were associated with headache ([70] Supplementary Table S2 ), myalgia
   ([71] Supplementary Table S3 ), fatigue ([72] Supplementary Table S4 ),
   and photophobia ([73] Supplementary Table S5 ), respectively. To
   determine shared genes between MPOX-associated and neurological
   manifestations-related genes, we inserted all genes in an excel file
   that concluded 10 genes (Hub genes) including CD55 molecule (CD55),
   C-X-C motif chemokine ligand 1 (CXCL1), nuclear factor kappa B (NFKB1),
   subunit 1, CXCL8, CD4 molecule (CD4), IL-6, MX dynamin-like GTPase 1
   (MX1), complement factor H (CFH), killer cell lectin-like receptor K1
   (KLRK1), CD46 molecule (CD46) were shared among totally 20309 genes in
   five gene sets ([74] Figure 1A, B and [75]Table 1 ). Network analyzer
   using Cytoscape revealed main network features of the reconstructed
   genetic network as 27 edges, average number of neighbors: 5.400,
   network diameter: 2, clustering coefficient: 0.779, network
   heterogeneity: 0.333, and network centralization: 0.500 ([76] Figure 1B
   ). In this network, genes such as IL6, CD4, CXCL8, MX1 and NFKB1 had
   the highest degree besides IL6, CD4, CXCL8, CFH, MX1 and NFKB1 had
   greatest betweenness centrality ([77] Table 1 ). Based on gene-gene
   interaction data in STRING, we also predicted five novel genes such as
   complement factor H related 3 (CFHR3), complement C5a receptor 1
   (C5AR1), complement C3a receptor 1 (C3AR1), interferon alpha 2 (IFNA2),
   and C-X-C motif chemokine ligand 3 (CXCL3) for MPOX-neurological
   manifestations associated genes ([78] Figure 1C ). According to the
   literature and primary experimental studies, some of these human hub
   genes (i.e., CFH, NFKB1, CXCL8) interact with MPXV-specific genes ([79]
   Supplementary Table S6 ). The MOPICE (monkeypox virus inhibitor of
   complement enzymes) gene, which is mainly present in the Congo Basin
   clade (clade I) of MPXV, plays an important role in modulating the host
   complement system. The MOPICE protein binds directly to complement
   components C3b and C4b, thereby inhibiting activation through both
   classical and alternative complement pathways ([80]Liszewski et al.,
   2006; [81]Estep et al., 2011; [82]Hudson et al., 2012). A46R, expressed
   by both clade I and clade II (West African) strains, inhibits TLR/NF-kB
   signaling by targeting the NFKB1 pathway ([83]Chen et al., 2005). C23L,
   which primarily present in clade I strains, is a homolog of host
   chemokines such as CXCL8, and may modulate host immune responses via
   chemokine mimicry ([84]Reading et al., 2003).

Figure 1.

   [85]A three-part figure depicting A) a Venn diagram with overlapping
   areas for Monkeypox, Headache, Myalgia, Photophobia, and Fatigue,
   showing intersections and numbers of occurrences; B and C) two network
   diagrams showing protein interactions with nodes labeled by proteins
   such as KLRK1, MX1, and IL6, connected by lines of different colors,
   illustrating complex interactions between proteins.
   [86]Open in a new tab

   Hub genes associated with MPOX and its neurological manifestations and
   its reconstructed genetic networks. (A) Venn diagram represents the
   distribution of genes associated with MPOX and its neurological
   manifestations in five distinct categories. (B) Genetic network of hub
   genes associated with MPOX and its neurological manifestations. (C)
   Genetic network of hub genes associated with MPOX and its neurological
   manifestations and predicted five novel associated genes (white nodes).
   Edge colors indicating interaction types: light blue, known
   interactions from curated databases; pink, known interactions from
   experimentally determined; green, predicted interactions from gene
   neighborhood analysis; dark blue, predicted interactions from gene
   co-occurrence; yellow, interactions inferred from text mining; black,
   co-expression-based interactions.

Table 1.

   Network parameters for MPOX and neurological manifestations shared
   genes.
   Index Gene name Gene full name Degree Betweenness centrality
   1 IL6 Interleukin 6 9 0.21527777777
   2 CD4 CD4 molecule 8 0.15277777777
   3 CXCL8 C-X-C motif chemokine ligand 8 7 0.08564814814
   4 MX1 MX dynamin like GTPase 1 5 0.00694444444
   5 NFKB1 Nuclear factor kappa B subunit 1 5 0.00694444444
   6 CFH Complement factor H 4 0.01851851851
   7 CD55 CD55 molecule 4 0.00694444444
   8 CD46 CD46 molecule 4 0.00694444444
   9 CXCL1 C-X-C motif chemokine ligand 1 4 0.0
   10 KLRK1 Killer cell lectin like receptor K1 4 0.0
   [87]Open in a new tab

3.2. Result of gene ontology and pathway analysis for MPOX and its common
neurological manifestations

   The results of gene ontology analysis indicated that response to other
   organism (GO:0051707), response to external biotic stimulus
   (GO:0043207), regulation of immune system process (GO:0002682), humoral
   immune response (GO:0006959), cellular response to lipid (GO:0071396),
   leukocyte cell-cell adhesion (GO:0007159), positive regulation of T
   cell activation (GO:0050870), regulation of complement activation
   (GO:0030449), viral life cycle (GO:0019058), and positive regulation of
   lymphocyte activation (GO:0051251) were primary affected biological
   processes for MPOX-neurological associated genes ([88] Figure 2 ). The
   most enriched molecular functions for our target shared genes included
   interleukin-8 receptor binding (GO:0005153), virus receptor activity
   (GO:0001618), exogenous protein binding (GO:0140272), CXCR chemokine
   receptor binding (GO:0045236), cytokine activity (GO:0005125),
   chemokine activity (GO:0008009), MHC protein binding (GO:0042287),
   interleukin 16 receptor activity (GO:0042012), signaling receptor
   activator activity (GO:0030546), immune receptor activity (GO:0140375),
   signaling receptor regulator activity (GO:0030545), interleukin 6
   receptor binding (GO:0005138), complement component C3b binding
   (GO:0001851), and glycosaminoglycan binding (GO:0005539). Furthermore,
   the significantly enriched cellular component in MPXV and its
   neurological manifestations were primarily associated with external
   side of plasma membrane (GO:0009897), secretary vesicles (GO:0099503),
   interleukin 6 receptor complex (GO:0005896), I-kappaB/NF-kappaB complex
   (GO:0033256), inner acrosomal membrane (GO:0002079) and
   serine-endopeptidase complex (GO:1905286) ([89] Figure 2 ).

Figure 2.

   [90]Bar chart titled “Homo Sapiens Gene Ontology Enrichment” displaying
   biological processes, molecular functions, and cellular components. The
   y-axis shows negative logarithm of P values, indicating significance,
   while the x-axis lists various biological terms like “defense response
   to other organism,” “MHC protein binding,” and “specific granule
   lumen.” Bars are color-coded, with purple for biological processes,
   blue for molecular functions, and green for cellular components,
   reflecting their enrichment significance.
   [91]Open in a new tab

   Gene ontology enrichment of shared genes between MPOX and its
   neurological manifestations. From left to right purple, blue, and green
   bars represent biological process, molecular function, and cellular
   component respectively.

   Our pathway enrichment analysis also predicted various inflammatory
   signaling pathways such as prostaglandin signaling (M42532),
   Lactoferrin (LTF) danger signal response pathway (M39701), cytokines
   and inflammatory response (M39711), TLR4 signaling and tolerance
   (M39561), overview of proinflammatory and profibrotic mediators
   (M42533), fibrin complement receptor 3 signaling pathway (MM15897),
   complement and coagulation cascades (M39649), TH17 cell differentiation
   pathway (M45541), complement system in neuronal development and
   plasticity (M42535), cells and molecules involved in local acute
   inflammatory response (M39733), LDL influence on CD14 and TLR4
   (M45556), and neuroinflammation and glutamatergic signaling (M42572)
   for MPOX-neurological manifestations associated genes ([92] Figure 3 ).
   It was revealed that prostaglandin signaling pathways were matched with
   MPOX-neurological manifestations associated genes, which were enriched
   with four proteins IL-6, NFKB1, CXCL1, and CXCL8 (IL-8; [93]Figure 4 ).
   Interestingly, two more major enriched pathways were LTF danger signal
   response pathway and TLR4 signaling and tolerance, which were predicted
   based on three MPOX-neurological manifestations associated proteins
   IL6, NFKB1, and CXCL8 (IL-8; [94]Figure 5A, B ). It also resulted that
   the complement system in neuronal development and plasticity pathway
   was enriched according to three important hub genes including CFH,
   CD46, and CD55 ([95] Figure 6 ). Two common genes between MPOX and its
   neurological complications, such as IL-6 and NFKB1, also predict
   another crucial signaling pathway, namely neuroinflammation and
   glutamatergic signaling ([96] Figure 7 ).

Figure 3.

   [97]Bar chart titled “Homo Sapiens Wikipathway Enrichment” displaying
   various biological pathways on the x-axis with corresponding negative
   log p-values on the y-axis. Prostaglandin signaling has the highest
   p-value at 8.26. Other pathways include L1TF danger signal response and
   Cytokines with values decreasing to Neuroinflammation and glutamatergic
   signaling at 2.36. Each bar includes a hit count in the query list.
   [98]Open in a new tab

   Biological pathway enrichment results for MPOX and its common
   neurological manifestations.

Figure 4.

   [99]Pathway diagram illustrating prostaglandin E2 signaling and its
   interactions with different cellular processes. It includes modules on
   IFN, IL6, NK cell, T cell, and inflammasome signaling. Various
   proteins, metabolites, and complexes are represented, with interactions
   such as activation, stimulation, conversion, and inhibition. The
   diagram features a color-coded legend, highlighting gene/protein,
   metabolite, and complex symbols.
   [100]Open in a new tab

   Prostaglandin signaling as a significantly enriched pathway for MPOX
   and its neurological manifestations. Genes associated with both MPOX
   and its common neurological manifestations are shown in red boxes.
   WikiPathways database.

Figure 5.

   [101]Diagram illustrating cellular signaling pathways in response to
   trauma or infection. Panel A depicts a pathway involving receptors
   RAGE, TLR2, TLR4, CD14, and TREM, leading to activation of MYD88,
   MAPK1, IRAK1/4, and TRAF6, culminating in NF-κB activation and cytokine
   production of IL1A, IL1B, IL6, IL8, TNF, IFNA, and IFNB. Panel B
   demonstrates TLR4 signaling through MYD88-dependent and independent
   pathways. The MYD88-dependent pathway involves TRAF6, NEMO, IKKα/β, and
   NF-κB, leading to IL6 production. The independent pathway utilizes
   TRIF, RIP1, and NAP1, signaling through IRF3/7 for IFNB production.
   Enhanced signaling is indicated by red arrows.
   [102]Open in a new tab

   Biological pathway enrichment results for MPOX and its common
   neurological manifestations. (A) LTF danger signal response pathway and
   (B) TLR4 signaling and tolerance. Genes associated to both MPOX and its
   common neurological manifestations are shown in red boxes. WikiPathways
   database.

Figure 6.

   [103]Complex biochemical pathway diagram detailing hippocampal
   neurogenesis, migration, synaptic pruning, and apoptosis. Includes
   labeled components like neurons, proteins, and molecules interconnected
   by directional arrows. Specific processes like phagocytosis,
   inflammation, and chemotaxis are highlighted. Color-coded legend
   explains symbols. Diagram sections focus on neuronal progenitor cell
   proliferation and cellular interactions within pathways.
   [104]Open in a new tab

   Complement system in neuronal development and plasticity pathway as a
   significantly enriched pathway for MPOX and its neurological
   manifestations. Genes associated with both MPOX and its common
   neurological manifestations are shown in red boxes. WikiPathways
   database.

Figure 7.

   [105]Complex biochemical pathway diagram illustrating interactions
   among various proteins, metabolites, and cellular processes in neurons
   and astrocytes. Key sections include glutamate-glutamine metabolism,
   D-serine metabolism, and signaling pathways. The diagram includes
   labeled nodes for genes, metabolites, and pathways, with directional
   arrows indicating interactions. A legend explains the symbols used for
   genes, proteins, metabolites, and pathways.
   [106]Open in a new tab

   Neuroinflammation and glutamatergic signaling are outstanding pathways
   for MPOX and its neurological manifestations. Genes associated with
   both MPOX and its common neurological manifestations are shown in red
   boxes. WikiPathways database.

3.3. Cell-type specific enrichment analysis

   To gain a better understanding of the types of cells impacted by both
   MPOX and neurological manifestations, we conducted a cell-type-specific
   analysis of their common genes using Enrichr. The enrichment results
   revealed that MPOX and its neurological manifestations predominantly
   impact immune cells [T helper 17 (Th17) cell, T helper 2 (Th2), natural
   killer cell, naive CD4^+ T cell, classical monocyte, natural killer
   cell, macrophage, T helper 1 (Th1) cell, effector memory T cell,
   conventional dendritic cell 1 (cDC1), conventional dendritic cell 2
   (cDC2), CD8^+ T cell lymphocyte], megakaryocyte erythroid cell, and
   central nervous system (CNS)-resident cells, microglia, ([107] Figure 8
   ).

Figure 8.

   [108]Diagram of Homo sapiens enriched cell types organized in a
   circular format with a brain illustration at the center. Various cell
   types are displayed in segments, such as Regulatory T cells, CD4+ T
   cells, and Natural Killer cells, with associated p-values listed. Each
   segment includes a visual representation of the cell type, highlighting
   the diversity and significance of different cell types in human
   biology.
   [109]Open in a new tab

   Cell-specific enrichment results for MPOX and its common neurological
   manifestations. Created with [110]BioRender.com.

3.4. Predicted microRNAs and repurposed drugs

   To identify key microRNAs linked with MPOX and its prevalent
   neurological manifestations, mirTarbase was used. We identified 11
   important microRNAs including hsa-miR-146a-5p (IL6, CFH, NFKB1, CXCL8),
   hsa-let-7a-5p (IL6, CD55, NFKB1, CXCL8), hsa-miR-9-5p (IL6, KLRK1,
   NFKB1), hsa-miR-124-3p (IL6, CD55, CXCL1, CXCL8), hsa-miR-146b-5p (IL6,
   NFKB1), hsa-miR-98-5p (IL6, CD46, CXCL8), hsa-miR-136-5p (IL6, CD55),
   hsa-miR-155-5p (IL6, NFKB1, CXCL8), hsa-miR-1-3p (IL6, CXCL1, CXCL8),
   hsa-miR-203a-3p (IL6, CXCL8), and hsa-miR-340-5p (CD46, CD55) for
   MPOX-neurological manifestations shared genes ([111] Figure 9 ).

Figure 9.

   [112]Circular diagram illustrating Homo Sapiens enriched microRNA with
   p-values. Each segment lists a microRNA, such as hsa-miR-9-5p, with
   corresponding p-values like 4.583859. Center features a brain icon and
   cell imagery, highlighting neural association.
   [113]Open in a new tab

   MicroRNA predicting enrichment results for MPOX and its common
   neurological manifestations. The values for the inner ring are the -log
   (P value) of enrichment for each miRNA which represents the statistical
   significance of association with MPOX-neurological manifestation genes.
   Created with [114]BioRender.com.

   Importantly, based on gene-drug interactions in the Stitch database, we
   repurposed eight potentially effective drugs including resiquimod (IL6,
   NFKB1, CXCL1, MX1, CXCL8, CD4), chloroquine (IL6, CD46, NFKB1, CXCL1,
   CXCL8, CD4), polyinosinic-polycytidylic acid (IL6, KLRK1, NFKB1, CXCL1,
   MX1, CXCL8), zithromac (IL6, NFKB1, CXCL8, CD4), methylene
   dimethanesulfonate (IL6, NFKB1, CXCL1, MX1), neopterin (IL6, MX1,
   CXCL8, CD4), betamethasone-d5 (IL6, CFH, CD46, NFKB1, CXCL1, CXCL8,
   CD4), and loxoribine (IL6, KLRK1, CXCL8) for MPOX-related and its
   common neurological manifestations-associated genes ([115] Figure 10 ).

Figure 10.

   [116]Illustration of various chemical structures displayed around a
   central image of a brain. Each segment contains a different molecule:
   Chloroquine, Polyinosinic-polycytidylic acid, Zithromax (Azithromycin),
   Methylene dimethanesulfonate, Neopterine, Betamethasone-d5, Loxoribine,
   and Resiquimod. Associated biological pathways and numerical values are
   listed alongside each structure, denoting potential interactions or
   effects on neural pathways.
   [117]Open in a new tab

   Repurposed drug results for MPOX and its common neurological
   manifestations. The Enriched drugs are presented based on –Log
   (p-value) and their primary inputted genes. Created with
   [118]BioRender.com.

4. Discussion

   Since the World Health Organization’s statement in May 2022, the
   outbreak of MPOX has expanded across countries, and a wide spectrum of
   neurological complications have also been documented ([119]Luna et al.,
   2022; [120]Shafaati and Zandi, 2022; [121]Sepehrinezhad et al., 2023).
   Furthermore, since August 2024, more than 21,000 new cases were
   reported in the Democratic Republic of the Congo ([122]Desai et al.,
   2024; [123]Duarte et al., 2024). This is despite the fact that the
   potential mechanisms of MPXV neurovirulence and the development of
   neurological manifestations remain unknown. In the present study, a
   series of computational methods were used to understand the underlying
   mechanisms of neurological manifestations of MPOX. Among the key
   findings, we identified ten shared genes between MPOX and its common
   neurological manifestations, including inflammatory (IL6, CXCL8,
   NFKB1), chemotactic (CXCL, CD4), antiviral (MX1, KLRK1), and
   complement-regulating genes (CFH, CD55, CD46). These genes were
   functionally enriched in inflammatory processes, leukocyte adhesion,
   immunological responses, and regulation of the complement system which
   demonstrates the multi-faceted host immune engagement during MPXV
   infection. This gene overlap emphasizes the possibility of a shared
   inflammatory and complement-mediated pathologic process affecting both
   systemic and neurological manifestations of MPOX. MPXV infection has
   been associated with systemic inflammation, cytokine storm, impaired
   leukocyte function, increased circulatory leukocyte count, aberrant
   immunological responses, and suppression of the host complement system
   ([124]Anderson et al., 2003; [125]Liszewski et al., 2006;
   [126]Hammarlund et al., 2008; [127]Bourquain et al., 2013; [128]Song
   et al., 2013a; [129]Tree et al., 2015; [130]Xuan et al., 2022;
   [131]Zandi et al., 2023; [132]Guo et al., 2024).

   The network analysis revealed important interactions related to hub
   genes shared between MPOX and its neurological manifestations ([133]
   Figure 1B , [134]Table 1 ). Topological metrics indicated that IL6,
   CD4, and CXCL8 were influential nodes with high connectivity (degree)
   and bridging (betweenness centrality) roles in the protein-protein
   interaction network. IL6 is a strong pro-inflammatory mediator in MPOX
   infection that contributes to acute-phase response and cytokine storm
   ([135]Johnston et al., 2015; [136]Agrati et al., 2023; [137]Meem
   et al., 2024; [138]Wang et al., 2024). The levels of this cytokine were
   also correlated with microglia activation and the onset of
   neuroinflammation ([139]Erta et al., 2012; [140]Bobbo et al., 2019;
   [141]Kummer et al., 2021). CD4^+ cells are involved in the recruitment
   of T-cells, and when this activity is suppressed, it leads to inhibited
   viral clearance ([142]Swain et al., 2012; [143]Lamens et al., 2025).
   Patients with MPXV infection exhibited reduced numbers of functional
   CD4^+ T-cells in peripheral blood, which correlated with increased
   disease severity, prolonged fever duration and higher viral loads
   ([144]Caldrer et al., 2025). CXCL8 is a potent chemotactic factor for
   neutrophils and other granulocytes that induce local inflammatory
   responses ([145]Cambier et al., 2023). MX1 and NFKB1 were determined to
   act as central hubs in our network analysis, despite their moderate
   connectivity. MX1 inhibits viral mRNA synthesis and disrupts viral
   particles assembly to exert its antiviral effects ([146]Haller and
   Kochs, 2011; [147]Verhelst et al., 2012), whereas NFKB1 is the master
   regulator of inflammatory responses ([148]Karin and Greten, 2005;
   [149]Bernal-Mizrachi et al., 2006; [150]Liu et al., 2017). This
   indicates that these two genes provide bottlenecks for antiviral
   response and neuroinflammation. The identified predicted novel genes
   generated secondary hubs, indicating the involvement of complement
   system as a potential mediator of MPXV neuropathology supported by
   pathway enrichment in [151]Figure 6 . This network structure
   demonstrated how MPXV could take advantage of immune hubs in the host
   to increase neurological damage.

   The present study predicted five novel genes for MPOX and its
   neurological manifestations, including CFHR3, C5AR1, C3AR1
   (complement-associated genes), IFNA2, and CXCL3. One of the main arms
   of innate immunity in viral infections, the complement system mediates
   numerous important antiviral activities, including the recruitment of
   neutrophils, the activation of leukocytes, the neutralization of
   viruses, inflammation, and the formation of membrane attack complexes
   on infected cells ([152]Mellors et al., 2020; [153]Afzali et al., 2022;
   [154]Ostrycharz and Hukowska-Szematowicz, 2022). Research has
   demonstrated that when MPXV infections are present, the host complement
   system’s activity is significantly inhibited ([155]Estep et al., 2011;
   [156]Li et al., 2023). The Central African MPXV clade encodes the
   MOPICE, which inhibits the activity of the complement system by
   interaction with C4b, C3b, and C5 ([157]Chen et al., 2005;
   [158]Liszewski et al., 2006). In prairie dogs, deletion of MOPICE from
   the Congo Basin strain reduced morbidity and mortality of animals 30
   days after intranasal inoculation of MPXV ([159]Hudson et al., 2012).

   In pathway analysis, prostaglandin signaling was considerably enriched
   for MPOX and its neurological manifestations. Prostaglandin E2 (PGE[2])
   is an arachidonic acid-derived molecule that is produced in response to
   inflammation or viral infections. Produced PGE2 affects immunological
   responses in the setting of viral infections ([160]Steer and Corbett,
   2003; [161]Pollara et al., 2012; [162]Kim et al., 2017; [163]Sander
   et al., 2017). As shown in [164]Figure 4 , PGE[2] through genes such as
   NFKB1, IL-6, and CXCL8 (IL-8) may be responsible for inducing cytokine
   storm and systemic inflammation after MPXV infection. Also, PGE[2] may
   affect the activity of macrophages, monocytes, and neutrophils via
   CXCL1 genes in the context of MPXV infection. The LTF danger signal
   response signaling pathway may be involved in the development of
   neurological manifestations of MPOX ([165] Figure 5A ). Following MPXV
   infections, LTF may activate NFKB1 via danger signal receptors such as
   TLR2/4 and CD14 as well as TREM1 and RAGE, leading to the production of
   IL-6 and IL-8 ([166] Figure 5A ). Following MPOX infection, activation
   of TLR4 via the MYD88/IRAK1/IRAK4-TRAF6-TAK1/TAB1/TAB2 signaling
   pathway may result in nuclear translocation of NFKB1, leading to the
   production of pro-inflammatory cytokines such as IL-6 and TNFα ([167]
   Figure 5B ). The TLR family, particularly TLR2 and TLR4, can activate
   antiviral immune responses by detecting viral capsid proteins
   ([168]Sartorius et al., 2021; [169]Zhou et al., 2021;
   [170]Gerber-Tichet et al., 2024). In a silico vaccine design study
   against MPOX, TLR4 was considered the primary target for docking with
   the proposed vaccine ([171]Lahimchi et al., 2024). As previously noted,
   the complement system may get involved during the development of
   MPOX-related neurological complications. [172]Figure 6 shows that
   various MPOX-neurological manifestations related genes, including CFH,
   CD55, and CD46, can block C3b, CFBb, and CFP genes, which catalyze the
   formation of C3a and C3b from C3. This suppresses the phagocytosis
   activity of macrophages and dendritic cells to remove invading
   infections (inhibit opsonization). A decrease in C3b production also
   reduces neurogenesis and migration of neural progenitor cells in the
   hippocampus, as well as the phagocytosis activity of brain microglial
   cells ([173] Figure 6 ). Furthermore, activation of the NFKB1 signaling
   pathway following MPXV infection may create pro-inflammatory cytokines
   such as IL-6 and TNFα, as well as oxidative indicators such as NOS1 and
   nitric oxide in brain astrocytes (neuroinflammation). NFKB1 activation
   can also have an inhibitory effect on the activity of plasma membrane
   proteins EAAT2 (SCLC1A2) and EAAT1 (SCLC1A3), which are crucial for
   astrocytes’ uptake of the excitatory neurotransmitter glutamate from
   extracellular space. In addition, the predominant phenotype of brain
   microglia in response to viral derivatives is M1 type, which secretes
   oxidative markers and produces pro-inflammatory cytokines such as TNFα,
   IL-1β, IL-1A, and IL-12 through an NFKB1-dependent signaling pathway
   (neuroinflammation). Human astrocytes and, to a lesser extent,
   microglia have been demonstrated to be susceptible to MPXV infection
   and replication ([174]Miranzadeh Mahabadi et al., 2024). These results
   offer functional insight into how MPXV-induced inflammation may impair
   neurovascular unit and induce glial dysfunction, and ultimately onset
   of encephalitic symptoms such as headache, and photophobia observed
   clinically. The enrichment of the complement system in neuronal
   development and plasticity, supports studies showing that MPXV encodes
   MOPICE which can inhibit C3b/C4b binding ([175]Chen et al., 2005;
   [176]Liszewski et al., 2006), so that could inhibit microglial
   phagocytosis and neurogenesis, giving a credible hypothesis as to how
   MPXV could change synaptic pruning (elimination of excess synapses) or
   immune surveillance in the CNS.

   The current study also predicted eleven regulatory hub miRNAs, which
   may play an important role in the development of neurological
   manifestations following MPOX infection. The generation of
   pro-inflammatory miR-146a-5p was activated upon activation of NFKB1 in
   neural cells, which played essential roles in the course of
   neurological disorders in the context of viral infections ([177]Taganov
   et al., 2006; [178]Hill et al., 2015; [179]Lukiw and Pogue, 2020;
   [180]Pogue and Lukiw, 2021). There is growing evidence that the tumor
   suppressor and pro-inflammatory microRNA let-7 has a role in antiviral
   responses and virus replication ([181]Qiu et al., 2017; [182]Zhou
   et al., 2017; [183]Masyeni et al., 2018; [184]Letafati et al., 2022).
   Moreover, the regulatory miR-9 inhibited the replication of Herpes
   simplex virus 1 in mouse primary neurons ([185]Deng et al., 2024).
   Also, miR-9-5p inhibited apoptosis of mouse dopaminergic neurons via
   activating β-catenin-SCRIB in vitro and in vivo ([186]Xiao et al.,
   2022). These miRNAs, which may modulate immune and inflammatory
   pathways, may consider as both diagnostic biomarkers and therapeutic
   targets for mitigating neurological manifestations associated with
   MPOX.

   The enrichment of Th17 cells in the study aligns with their role in
   driving neuroinflammation via IL-17 production, which activates
   dendritic cells (DCs), disrupts the integrity of the blood-brain
   barrier and induces immune cell recruitment ([187]Kebir et al., 2007;
   [188]Shichita et al., 2009; [189]Huppert et al., 2010; [190]Setiadi
   et al., 2019). IL-17 causes neuronal injury through both direct pathway
   as well as indirect mechanisms involving immune cell recruitment and
   inflammation ([191]Wang et al., 2009; [192]Siffrin et al., 2010;
   [193]Aghbash et al., 2021). Upregulation of the Th17 pathway has been
   reported in rhesus macaques infected with MPXV ([194]Aid et al., 2023).
   IL-17-ativated DCs and viral infections trigger natural killer (NK)
   cells to secret pro-inflammatory cytokines (i.e. TNFα and IFNγ)
   ([195]Milovanovic et al., 2020). NK cells are critical for early MPXV
   control, and their dysfunction may allow for viral dissemination to the
   CNS. In MPXV-infected rhesus macaques, an increased number of
   dysfunctional NK cells have been identified in blood and lymph nodes
   ([196]Song et al., 2013b). Importantly, microglia were the only
   CNS-specific cell type found, indicating their prominent role in
   MPXV-associated neuroinflammation. In vitro and ex vivo studies
   indicate that MPXV can penetrate and replicate within human glial
   cells, particularly astrocytes and microglia ([197]Miranzadeh Mahabadi
   et al., 2024; [198]Bauer et al., 2023). In response to viral
   infections, activated microglia secrete pro-inflammatory cytokines that
   exacerbate neuronal damage and may cause onset of symptoms like
   headache and photophobia ([199]Chhatbar and Prinz, 2021; [200]O’Brien
   et al., 2022; [201]Xu et al., 2024). Moreover, microglia can serve as
   reservoirs for viral entry into the CNS ([202]Ismail et al., 2024;
   [203]Miranzadeh Mahabadi et al., 2024). These findings reinforce the
   ability of MPXV to disrupt immune homeostasis at the neurovascular
   interface, most likely by dysregulating cytokine signaling and pathways
   involving complement.

   The MPXV infection is primarily self-limited, and there are no approved
   treatments for it. However, several medicines, such as tecovirimat,
   cidofovir, and brincidofovir, have strong antiviral activity against
   MPXV in cell cultures, animals, and humans ([204]Andrei and Snoeck,
   2010; [205]Merchlinsky et al., 2019; [206]Harris, 2022;
   [207]O’Laughlin, 2022; [208]Warner et al., 2022; [209]Imran et al.,
   2023; [210]Shamim et al., 2023). The eight novel medications predicted
   in this study may be useful in treating MPOX and related neurological
   manifestations. Resiquimod is a TLR7 and TLR8 agonist that has
   antiviral and antitumor properties ([211]Bernstein et al., 2001;
   [212]Pockros et al., 2007; [213]Lee et al., 2014; [214]Gupta et al.,
   2020). The beneficial effects of Resiquimod on skin lesions have been
   proven following viral infections ([215]Meyer et al., 2013). A new
   research team found that resiquimod enhances immunological response in
   human neuroblastoma cell cultures via TLR7-NFκB-C-C motif chemokine
   ligand 2 signaling, providing a novel possible therapy approach for
   neurotropism due to viruses ([216]Kaizuka et al., 2024). Resiquimod
   modulates the immune response by changing the expression levels of
   different genes related to inflammation and immunity. Although some
   specific studies in respect to IL6, NFKB1, CXCL1, MX1, CXCL8, and CD4
   might be slight, given the known mechanism of action of Resiquimod and
   general effects of TLR agonists on immune cells, we can deduce expected
   changes in the expression of these immune-related genes. Toll-like
   receptor agonists can activate NF-Kβ and increase the expression of
   pro-inflammatory cytokines such as IL-6 and TNFα ([217]Dockrell and
   Kinghorn, 2001; [218]Medzhitov, 2001; [219]Kirtland et al., 2020). TLR
   agonists can also activate immune cells to produce pro-inflammatory
   chemokines, including CXCL1 and CXCL8 ([220]Medzhitov, 2001).
   Chloroquine is an antimalarial drug that also has antiviral properties.
   Chloroquine inhibited the release of viral genetic material into host
   cells via reducing pH ([221]Al-Bari, 2017; [222]Manuja et al., 2023).
   Chloroquine also inhibited the production of pro-inflammatory cytokines
   IL-6, TNFα and IL-1β in activated human monocyte/macrophage cultures
   ([223]Jang et al., 2006). Moreover, anti-tumor agent
   polyinosinic-polycytidylic acid, a synthetic double-stranded RNA,
   mimics the features of TLR3 ligands (like viral particles) and binds to
   this receptor, initiating immunological responses and causes
   inflammation ([224]Manetti et al., 1995; [225]Fortier et al., 2004;
   [226]Forte et al., 2012). Polyinosinic-polycytidylic acid has also
   shown positive effects on wound healing in humans and mice ([227]Lin
   et al., 2012). Zithromac (azithromycin) is a synthetic macrolide
   antibiotic that has shown antiviral activity against Enteroviruses,
   Coronaviruses, Ebola viruses, Picornavirus, Orthomyxovirus, and
   Flavivirus ([228]Gielen et al., 2010; [229]Madrid et al., 2015;
   [230]Retallack et al., 2016; [231]Lee et al., 2017; [232]Kawamura
   et al., 2018; [233]Zeng et al., 2019; [234]Doan et al., 2020;
   [235]Khoshnood et al., 2022). Neopterin is an organic molecule formed
   from guanosine triphosphate and is created by macrophages and monocytes
   upon activation with interferon-gamma that results in an inflammatory
   state ([236]Murr et al., 2002; [237]Eisenhut, 2013; [238]Heneberk
   et al., 2023). Neopterin levels in cerebrospinal fluid increased after
   viral infections of the CNS ([239]Fredrikson et al., 1987; [240]Fuchs
   et al., 1989; [241]Bociąga-Jasik et al., 2011; [242]Yilmaz et al.,
   2013; [243]Miyaue et al., 2022). Loxoribine acts as a TLR7 agonist,
   initiating antiviral responses through the TLR7-MyD88-P50/P65-NFκB
   signaling pathway ([244]Heil et al., 2003; [245]Dzopalic et al., 2010).
   The antiviral activity of loxoribine has been reported in various viral
   infections ([246]Heil et al., 2003; [247]Petro, 2005; [248]Stewart
   et al., 2012; [249]Aparicio et al., 2014; [250]Enosi Tuipulotu et al.,
   2018; [251]Girkin et al., 2022; [252]Kayesh et al., 2023). While these
   candidates require experimental validation, our results provide a basis
   for rapid therapeutic development for MPOX-associated
   neuroinflammation.

5. Conclusion

   In conclusion, the hub genes between MPOX and its main neurological
   manifestations (i.e. headache, myalgia, fatigue, and photophobia)
   included complement system, inflammatory, chemotactic receptor, and
   antiviral-related genes. Also, the predicted novel genes such as CFHR3,
   C5AR1, C3AR1, IFNA2, and CXCL3 confirmed the importance of the
   complement system and inflammatory pathways in the development of
   neurological manifestations of MPOX. This study suggests that immune
   cells and glial cells, particularly cortical microglia cells, play an
   essential role in the development of neurological complications
   associated with MPXV infection. Moreover, our findings suggest that
   prostaglandin signaling, LTF danger signal response pathway, TLR4
   signaling, complement system in neuronal development and plasticity,
   and neuroinflammation and glutamatergic signaling all play crucial
   roles in the pathogenesis of MPXV infection in the CNS. The study sheds
   new light on the pathophysiology of MPOX and implies that targeting the
   complement system and immunotherapy could be a promising treatment
   strategy for managing the neurological consequences of MPOX disease.
   The lack of experimental confirmation of computational conclusions is
   seen as the study’s key limitation, necessitating additional research
   in future basic and clinical studies. Furthermore, our work predicted
   some regulatory microRNAs, which could serve as biomarkers and
   therapeutic targets for MPOX and its neurological manifestations. We
   also recommended several drugs that potentially have antiviral
   properties against MPXV and are useful in treating the primary
   neurological consequences of MPOX. However, these findings will require
   in-depth validation in future investigations.

Funding Statement

   The author(s) declare that no financial support was received for the
   research and/or publication of this article.

Data availability statement

   The original contributions presented in the study are included in the
   article/[253] Supplementary Material . Further inquiries can be
   directed to the corresponding author.

Author contributions

   AB: Data curation, Investigation, Writing – original draft, Writing –
   review & editing. HL: Data curation, Investigation, Writing – original
   draft, Writing – review & editing. AS: Conceptualization, Data
   curation, Investigation, Methodology, Project administration,
   Supervision, Validation, Visualization, Writing – original draft,
   Writing – review & editing.

Conflict of interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Generative AI statement

   The author(s) declare that no Generative AI was used in the creation of
   this manuscript.

Publisher’s note

   All claims expressed in this article are solely those of the authors
   and do not necessarily represent those of their affiliated
   organizations, or those of the publisher, the editors and the
   reviewers. Any product that may be evaluated in this article, or claim
   that may be made by its manufacturer, is not guaranteed or endorsed by
   the publisher.

Supplementary material

   The Supplementary Material for this article can be found online at:
   [254]https://www.frontiersin.org/articles/10.3389/fcimb.2025.1506687/fu
   ll#supplementary-material
   [255]DataSheet1.pdf^ (13MB, pdf)

References