Functional Analysis of Monkeypox and Interrelationship between Monkeypox and COVID-19 by Bioinformatic Analysis

The outbreak of monkeypox may be considered a novel and urgent threat after the coronavirus disease (COVID-19). No wide-ranging studies have been conducted on this disease since it was first reported. We systematically assessed the functional role of gene expression in cells infected with the monkeypox virus using transcriptome profiling and compared the functional relation with that of COVID-19. Based on the Gene Expression Omnibus database, we obtained 212 differentially expressed genes (DEGs) of GSE36854 and GSE21001 of monkeypox datasets. Enrichment analyses, including KEGG and gene ontology (GO) analyses, were performed to identify the common function of 212 DEGs of GSE36854 and GSE21001. CytoHubba and Molecular Complex Detection were performed to determine the core genes after a protein–protein interaction (PPI). Metascape/COVID-19 was used to compare DEGs of monkeypox and COVID-19. GO analysis of 212 DEGs of GSE36854 and GSE21001 for monkeypox infection showed cellular response to cytokine stimulus, cell activation, and cell differentiation regulation. KEGG analysis of 212 DEGs of GSE36854 and GSE21001 for monkeypox infection showed involvement of monkeypox in COVID-19, cytokine-cytokine receptor interaction, inflammatory bowel disease, atherosclerosis, TNF signaling, and T cell receptor signaling. By comparing our data with published transcriptome of severe acute respiratory syndrome coronavirus 2 infections in other cell lines, the common function of monkeypox and COVID-19 includes cytokine signaling in the immune system, TNF signaling, and MAPK cascade regulation. Thus, our data suggest that the molecular connections identified between COVID-19 and monkeypox elucidate the causes of monkeypox.


Introduction
Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), belonging to the Orthopoxvirus genus, which includes double-stranded DNA viruses [1,2]. Te MPXV has been reported in Western and Central African countries, the United States, and Europe [3]. MPXV penetrates the body through direct contact via the skin, airways, or mucous membranes such as nose, eyes, or mouth from an infected person, through respiratory droplets of face-to-face contact and through indirect contact via contaminated materials [4]. Te symptoms of MPXV infection are similar to those of a milder form of smallpox [5]. Te distinction is that lymphadenopathy is caused by MPXV infection rather than smallpox [6]. Te MPXV has split into two distinct clades: West Africa and the Congo Basin [7]. Te diseases caused by the two MPXV clades difer in clinical and epidemiological characteristics. Te Congo Basin strain has a 10% mortality rate [8]. West Africa has a fatality rate of about 1%, with patients with the human immunodefciency virus (HIV) coinfection having a higher mortality rate [9].
In comparison to the West African clade, the Congo Basin monkeypox clade selectively suppresses host responses such as apoptosis and growth factor responses [10]. By repressing cognate T cell activation, MPXV avoids antiviral CD4+ and CD8+ T cell responses [11]. Realegeno et al. have shown that the high-throughput genetic screen of MPXV infection in haploid cell is involved in Golgi-associated retrograde protein complex [12]. Monkeypox requires heat shock factor 1 (HSF1) for infection [13]. MPXV infection resulted in signifcant increases in the number of natural killer cells and cell proliferation [14].
Te outbreak of coronavirus disease  caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally in December 8, 2020, according to the World Health Organization [15]. COVID-19 has afected in various diseases such as infammatory response and nervous system symptoms such as ischemic stroke and delineation [16,17]. Molecular and immunological diagnostic techniques have been developed to identify SARS-CoV-2 infection [18]. Currently, the most reliable method for diagnosing SARS-CoV-2 is real-time PCR-based assays for viral RNA detection [18,19]. Furthermore, more advanced molecular approaches for detecting SARS-CoV-2 RNA are being developed, including next-generation sequencing (NGS), droplet-digital PCR (ddPCR), and clustered regularly interspaced short palindromic repeats (CRISPRs) [18,[20][21][22]. Recently, the number of monkeypox cases has increased in 2022 after the COVID-19 outbreak; however, the natural reservoir and function of monkeypox are unknown.
Several studies have reported the possible relationship between monkeypox and COVID-19. One connection is that patients with SARS-CoV-2 infection are more prone to coinfect with MPXV due to the reduced amount of circulating immune cells [23]. Another link between the SARS-CoV-2 and MPXV outbreaks is a lack of immunity in younger generations to the smallpox virus. Smallpox is in the same family as monkeypox, and the discontinuation of its vaccine contributes to increased monkeypox cases as well as spread of SARS-CoV-2 by lowering immunity among local residents [23,24]. It is still unclear whether the new monkeypox outbreak is a distinct phenomenon or if it is exacerbated by the COVID-19 pandemic [25]. Because of the current small patient sample size, the exact cause-efect relationship between SARS-CoV-2 and MPXV coinfection cannot yet be established [25,26].
In this study, we aimed to investigate the possible molecular function of monkeypox by performing bioinformatic analyses. In addition, by comparing our data of monkeypox infection with published transcriptome of SARS-CoV-2 infection in other cell lines, we found that the function of monkey pox and COVID-19 infection is similar in terms of cytokine signaling in the immune system and TNF signaling. Terefore, the potential functional link of monkeypox and COVID-19 is described in this bioinformatic study.

Te Collection of Databases and the Identifcation of DEGs.
To create a dataset of monkeypox infection, DEGs of monkeypox infection were obtained from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/ geo/) database. Two GEO datasets were collected to obtain the DEGs related to monkeypox, including GSE21001 and GSE36854, and the datasets were analyzed using the GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) web tool. GSE21001 was the database for GeneChip rhesus macaque genome microarrays on a 3-and 7-hour postinfection (hpi) time point after an MPXV infection on Macaca mulatta kidney epithelial cells (MK2) [27]. GSE36854 was the database for the gene expression profle at 6 hpi after infection with MPXV strain MSF#6 on HeLa cells [28]. Te cutof criteria were obtained for GSE21001 and GSE36854 using an adjusted P value <0.05.

Identify Hub Genes of MPXV Infection.
Te cytoHubba plugins of Cytoscape were used to identify the top 20 nodes (hub genes), ranked using the maximal clique centrality (MCC) algorithm of cytoHubba. Te Metascape tool [31] was to identify the Molecular Complex Detection (MCODE) components for functional gene clusters.

DisGeNET, PaGenBase, and TRRUST for Enrichment
Analyses. DisGeNET, PaGenBase, and TRRUST were used to analyze the monkeypox infection, and comparison between monkeypox and COVID-19 was performed through Metascape (https://metascape.org). DisGeNET is a platform for the genetic underpinning of human diseases [32,33]. PaGenBase is a database for obtaining information on pattern genes under various tissue and time specifc conditions [34]. TRRUST is a platform for transcriptional regulatory networks [35].

Design.
To determine the function of the MPXV infection, the public datasets GSE36854 and GSE21001 were used. Figure 1(a) shows a fowchart of this study. GEO2R was used to obtain DEGs from the GEO database. Adjusted P values <0.05 were considered as the criteria for the DEGs. Figure 1(b) shows the intersections of the two groups of GSE36854 and GSE21001, indicating the common DEGs.
To obtain the functional clusters of MPXV infection, a module analysis was carried out using the Metascape tool.
In the MCODE analysis by Metascape, 211 DEGs of GSE36854 and GSE21001 of MPXV infection were grouped into fve modules. Te pathways in module 1 were mainly associated with systemic lupus erythematosus, histone deacetylases (HDACs), and alcoholism. Interleukin -10, -4, -13 signaling, and overview of proinfammatory and profbrotic mediators were involved in module 2. Te MCODE3 module included TNFR2 noncanonical NF-kB pathway, TNF receptor superfamily (TNFSF) members mediating noncanonical NF-kB pathway, and NF-kappa B signaling pathway. Te MCODE4 module included EGFR downregulation, signaling by EGFR, and branch elongation of an epithelium. Finally, the MCODE5 module included protein ubiquitination and protein modifcation by small protein conjugation (Figure 2(b)).

KEGG and GO Analyses of MPXV Infection.
To determine the functional role of MPXV infection, the KEGG and GO analyses of 212 DEGs of GSE36854 and GSE21001 were carried out with the online tool SHINEY GO (https:// bioinformatics.sdstate.edu/go/). Te network of GO BP of 211 DEGs from the MPXV infection using Shiny Go was mainly involved in the regulation of multicellular organism development, regulation of phosphate metabolic process, regulation of cell diferentiation, and positive regulation (Figure 3(a)). As shown in Figure 3(b), KEGG analysis of 212 DEGs from GSE36854 and GSE21001 showed involvement in coronavirus disease, cytokine-cytokine receptor interaction, infammatory bowel disease, Chagas disease, lipid and atherosclerosis, T cell receptor signaling pathway, Ctype lectin receptor signaling pathway, rheumatoid arthritis, and malaria.

Predicted Drug-Target Relationship Using 212 DEGs of Monkeypox Infection.
We used the drug matador from the SHINEY GO online tool to predict drug-target relationships using 212 DEGs of GSE36854 and GSE21001 for MPXV infection. Te top ten drugs related to monkeypox include troglitazone, futicasone propionate, indomethacin, atorvastatin, nitroglycerin, cerivastatin, budesonide, 3morpholinosydnonimine, quinapril, and valsartan (Figure 3(c)).

DisGNET, PaGenBase, and TRRUST of 212 DEGs from MPXV Infection.
DisGeNET database disease enrichment analysis by using the Metascape tool demonstrated that the hub genes of 212 DEGs from GSE36854 and GSE21001 were associated with proliferative vitreoretinopathy, pneumonitis, juvenile arthritis, anemia, airway disease, and so on (Figure 4(a)). PaGenBase was used to perform a tissue characteristic enrichment analysis with the Metascape tool and showed that the hub genes of 212 DEGs from GSE36854 and GSE21001 were mainly enriched in the lung ( Figure 4(b)). Te TRRUST database analysis by using the Metascape tool demonstrated that RELA, SP1, and NF-κB1 are core transcription factors regulating the hub genes of 212 DEGs of MPXV infection (Figure 4(c)).

Te Functional Analysis of MPXV and SARS-CoV-2
Infections. KEGG analysis of 212 DEGs from GSE36854 and GSE21001 for MPXV infection revealed COVID-19 relationship with COVID-19. Terefore, to determine the relationship between MPXV and SARS-CoV-2 infections, the Coronascape COVID database (https://metascape.org/ COVID) was utilized. Te biological function of coexpressed genes between SARS-CoV-2 infection and 212 DEGs from GSE36854 and GSE21001 for MPXV infection included GO1902532 negative regulation of intracellular signal transduction, GO0010942 positive regulation of cell death, hsa05202 transcriptional misregulation in cancer, GO0045596 negative regulation of cell diferentiation, GO0043408 regulation of the MPAK cascade, hsa05417 NFkappa B signaling pathway, GO 0009617 response to bacterium, and hsa04668 TNF signaling pathway ( Figure 5(a)).  Te network was visualized using Cytoscape5, wherein each node represents an enriched term and was colored frst by its cluster ID and MCODE algorithm to see densely connected network components of genes coexpressed between SARS-CoV-2 and MPXV infections, as shown in Figure 5(b). Te PPI interactions of the coexpressed genes were subjected to pathway and process enrichment analyses. Te network of enriched term by clusters is involved in     Genetics Research cytokine signaling in the immune system, TNF signaling, response to growth factor, regulation of MAPK cascade, positive regulation of cellular components, COVID-19, and so on ( Figure 5(b)).

Discussion
Te outbreak of monkeypox has spread across several countries in 2022 after the COVID-19 pandemic. However, there is no information of the function of monkeypox and its relation with COVID-19. In the present study, the function of MPXV infection was identifed by bioinformatics analyses. In addition, key pathways between SARS-CoV-2 and monkeypox infections were suggested using the bioinformatics analyses tool. Although SARS-CoV-2 and MPXV are both enveloped viruses that replicate in the cytoplasm and require host machinery to replicate and make new viral proteins, there are distinctions between the two viruses [23]. MPXV is a double-stranded DNA virus, whereas SARS-CoV-2 is a single-stranded RNA virus. Within the last two years, SARS-CoV-2 has several diferent clades being mutated from its alpha form to beta, gamma, delta, and omicron clades [23,36]. MPXV has two clades such the Congo Basin and Nigerian clades [6]. MPXV is transmitted primarily through very close physical contact from human-human transmission and long-term contact with an infected animal, whereas COVID-19 illness is caused by droplet, direct contact, and airborne transmission of SARS-CoV-2 [23]. Te SARS-CoV-2 pandemic spreads in the form of tides and waves, whereas MPXV spreads more linearly [23]. MPXV enters cells via macropinocytosis, whereas SARS-CoV-2 penetrates the human body by attaching its spikes to endothelial cells angiotensin converting enzyme 2 receptors (ACE-2) [23].
In this study, the hub genes of 212 DEGs of MPXV infection by cytoHubba analysis were involved in cytokinerelated genes such IL-6, IL-10, TNF, CCL2, ICAM1, CXCL8, and HDAC-related genes. In addition, the MCODE analysis also associated with systemic lupus erythematosus, HDACs, and IL-10 and -4 signaling. HDACs and histone acetyl transferases play major roles in cell survival, growth, and proliferation as well as the regulation of gene transcription [37]. IL-10 plays an important role as an anti-infammatory cytokine by preventing pathogenic infection and excessive immune response [38,39], and IL-4 regulates the immune responses [40]. Systemic lupus erythematosus is well known as a chronic autoimmune disease that produces autoantibodies and afects multiple organs [41].
In the present study, KEGG analysis of 212 DEGs of GSE36854 and GSE21001 for MPXV infection showed its involvement in COVID-19, cytokine-cytokine receptor interaction, and TNF signaling. In addition, the biological function of coexpressed genes between SARS-CoV-2 and MPXV infections in the cell lines by Metascape/COVID-19 analysis was enriched in cytokine signaling in the immune system, TNF signaling, response to bacterium, response to growth factor, and regulation of the MAPK cascade. Tere is evidence that cytokine modulation is associated with the severity of the monkeypox disease in humans [42]. MPXV infection in Hela cells involves genes for proinfammatory cytokine and leukocyte chemotaxis [28] and that in MK2 cells enhanced the IL-8 gene [27,43]. Many studies have suggested that SARS-CoV-2 is associated with a dysregulated infammatory response, such as cytokine storm and infammatory molecules [44,45]. Terefore, monkeypox and COVID-19 might be associated with the infammatory immune response.
A previous study demonstrated that transcription factors play an important role in the regulation of transcription, metabolism, and immune response [43,46], and they are closely associated with diseases [47]. In this study, JUN, REL, NFKB1, and STAT3 are mainly involved transcriptional factor according to the TRRUST analysis when comparing MPXV and SARS-CoV-2 infections. Tere is evidence that JUN, REL, NFKB1, and STAT3 are involved in infammation [48][49][50][51].

Conclusions
In conclusion, the hub genes based on the cytoHubba and MCODE analyses of MPXV infection included cytokine and HDAC-related genes. KEGG analysis of MPXV infection showed involvement of MPXV in COVID-19, cytokinecytokine receptor interaction, and TNF signaling pathway. Our study data suggest that the function of monkeypox is similar to that COVID-19 in terms of cytokine signaling in the immune system and TNF signaling. Our study provides pathological insight of monkeypox and molecular connections between COVID-19 and monkeypox. Our study has some limitations due to the lack of experimental verifcation of computational data. It will inevitably need in-depth research in the future.

Data Availability
Data used in this study are available at the NCBI (National Center for Biotechnology Information Gene Expression Omnibus), https://www.ncbi.nlm.nih.gov/geo/.

Conflicts of Interest
Te author declares that there are no conficts of interest.