Molecular Aspects of the Monkeypox Virus and their Impact on the Virus's Change in Epidemiology.

OBJECTIVE: Monkeypox is a viral zoonotic disease endemic to West and Central Africa; it has been reported in more countries during the last decade than in the previous 40 years. In 2022 a multinational outbreak occurred. This change in the epidemiology of the virus may represent an evolutionary adaptation. The purpose of this study is to analyze the molecular aspects of Monkeypox virus (MPXV) disease that may explain the latter's change in epidemiology during the 2022 outbreak. METHODS: From July 2022 through December 2022, the period of the outbreak, a narrative review was conducted on the available literature, with a total of 271 articles published in the MEDLINE/PubMed and LILACS databases being examined. The chosen articles were organized using the search and reference manager Mendeley Desktop 1.19.4. Duplicates and articles that did not meet the study's objective were eliminated, resulting in the selection of 49 articles for the present review. DISCUSSION: MPXV resurgence poses challenges due to waning immunity and changing epidemiological patterns. Recent outbreaks show different transmission routes, affecting new demographics. Genomic evolution, vaccination history, and potential new animal reservoirs complicate containment efforts. Continued surveillance and vaccination are crucial for control. CONCLUSIONS: It seems possible that MPXV has (re-)emerged to occupy the ecological niche left by the smallpox virus. Mutations of the apolipoprotein B mRNA editing enzyme, catalytic subunit 3G motif, in MPXV clade IIb since 2017 may explain the epidemiological change that has occurred in recent years. This pattern could be due to sustained transmission in a new host or a new route of infection.

Antivirals for monkeypox virus: Proposing an effective machine/deep learning framework.

Monkeypox (MPXV) is one of the infectious viruses which caused morbidity and mortality problems in these years. Despite its danger to public health, there is no approved drug to stand and handle MPXV. On the other hand, drug repurposing is a promising screening method for the low-cost introduction of approved drugs for emerging diseases and viruses which utilizes computational methods. Therefore, drug repurposing is a promising approach to suggesting approved drugs for the MPXV. This paper proposes a computational framework for MPXV antiviral prediction. To do this, we have generated a new virus-antiviral dataset. Moreover, we applied several machine learning and one deep learning method for virus-antiviral prediction. The suggested drugs by the learning methods have been investigated using docking studies. The target protein structure is modeled using homology modeling and, then, refined and validated. To the best of our knowledge, this work is the first work to study deep learning methods for the prediction of MPXV antivirals. The screening results confirm that Tilorone, Valacyclovir, Ribavirin, Favipiravir, and Baloxavir marboxil are effective drugs for MPXV treatment.

Editorial: Reasons for Increasing Global Concerns for the Spread of Mpox.

On August 14, 2024, the Director General of the World Health Organization (WHO) declared that the increasing outbreaks of mpox (formerly monkeypox) should be regarded as an international public health emergency due to the growing number of cases in endemic and non-endemic geographical areas, and increasing disease severity. The latest update from the WHO and the alerts given regarding the status of mpox follows an upsurge in the incidence and severity of mpox in the Democratic Republic of the Congo (DRC) and an increasing number of African countries, with spread to other continents and countries This Editorial aims to provide an update on the current status of mpox and includes reasons for the increasing global concerns for the spread of the mpox virus (MPXV).

Multi-omics characterization of the monkeypox virus infection.

Multiple omics analyzes of Vaccinia virus (VACV) infection have defined molecular characteristics of poxvirus biology. However, little is known about the monkeypox (mpox) virus (MPXV) in humans, which has a different disease manifestation despite its high sequence similarity to VACV. Here, we perform an in-depth multi-omics analysis of the transcriptome, proteome, and phosphoproteome signatures of MPXV-infected primary human fibroblasts to gain insights into the virus-host interplay. In addition to expected perturbations of immune-related pathways, we uncover regulation of the HIPPO and TGF-ß pathways. We identify dynamic phosphorylation of both host and viral proteins, which suggests that MAPKs are key regulators of differential phosphorylation in MPXV-infected cells. Among the viral proteins, we find dynamic phosphorylation of H5 that influenced the binding of H5 to dsDNA. Our extensive dataset highlights signaling events and hotspots perturbed by MPXV, extending the current knowledge on poxviruses. We use integrated pathway analysis and drug-target prediction approaches to identify potential drug targets that affect virus growth. Functionally, we exemplify the utility of this approach by identifying inhibitors of MTOR, CHUK/IKBKB, and splicing factor kinases with potent antiviral efficacy against MPXV and VACV.

Real-time PCR assay to detect the novel Clade Ib monkeypox virus, September 2023 to May 2024.

Monkeypox virus (MPXV) is an emerging zoonotic pathogen with complex epidemiology necessitating rapid diagnosis and distinguishing between clades and subclades. The emerging Clade Ib lacks the genomic region used in the Clade I-specific assay from the Centers for Disease Control and Prevention. We report an MPXV real-time PCR to specifically detect Clade Ib. The assay demonstrated proficient sensitivity and specificity in 92 samples and can be included along other TaqMan-based assays to detect MPXV and distinguish between clades and subclades.

Computational analysis of pathogen-host interactome for fast and low-risk in-silico drug repurposing in emerging viral threats like Mpox.

Monkeypox (Mpox), a zoonotic illness triggered by the monkeypox virus (MPXV), poses a significant threat since it may be transmitted and has no cure. This work introduces a computational method to predict Protein-Protein Interactions (PPIs) during MPXV infection. The objective is to discover prospective drug targets and repurpose current potential Food and Drug Administration (FDA) drugs for therapeutic purposes. In this work, ensemble features, comprising 2-5 node graphlet attributes and protein composition-based features are utilized for Deep Learning (DL) models to predict PPIs. The technique that is used here demonstrated an excellent prediction performance for PPI on both the Human Integrated Protein-Protein Interaction Reference (HIPPIE) and MPXV-Human PPI datasets. In addition, the human protein targets for MPXV have been identified accurately along with the detection of possible therapeutic targets. Furthermore, the validation process included conducting docking research studies on potential FDA drugs like Nicotinamide Adenine Dinucleotide and Hydrogen (NADH), Fostamatinib, Glutamic acid, Cannabidiol, Copper, and Zinc in DrugBank identified via research on drug repurposing and the Drug Consensus Score (DCS) for MPXV. This has been achieved by employing the primary crystal structures of MPXV, which are now accessible. The docking study is also supported by Molecular Dynamics (MD) simulation. The results of our study emphasize the effectiveness of using ensemble feature-based PPI prediction to understand the molecular processes involved in viral infection and to aid in the development of repurposed drugs for emerging infectious diseases such as, but not limited to, Mpox. The source code and link to data used in this work is available at: https://github.com/CMATERJU-BIOINFO/In-Silico-Drug-Repurposing-Methodology-To-Suggest-Therapies-For-Emerging-Threats-like-Mpox .

Characterization of Human Immortalized Keratinocyte Cells Infected by Monkeypox Virus.

Monkeypox virus (MPXV) can induce systemic skin lesions after infection. This research focused on studying MPXV proliferation and the response of keratinocytes. Using transmission electron microscopy (TEM), we visualized different stages of MPXV development in human immortalized keratinocytes (HaCaT). We identified exocytosis of enveloped viruses as the exit mechanism for MPXV in HaCaT cells. Infected keratinocytes showed submicroscopic changes, such as the formation of vesicle-like structures through the recombination of rough endoplasmic reticulum membranes and alterations in mitochondrial morphology. Transcriptome analysis revealed the suppressed genes related to interferon pathway activation and the reduced expression of antimicrobial peptides and chemokines, which may facilitate viral immune evasion. In addition, pathway enrichment analysis highlighted systemic lupus erythematosus pathway activation and the inhibition of the Toll-like receptor signaling and retinol metabolism pathways, providing insights into the mechanisms underlying MPXV-induced skin lesions. This study advances our understanding of MPXV's interaction with keratinocytes and the complex mechanisms leading to skin lesions.

A Comparative Evaluation of Three Diagnostic Assays for the Detection of Human Monkeypox.

Accurate and early diagnosis of monkeypox virus (MPXV) is crucial for controlling epidemics and treating affected individuals promptly. This study aimed to assess the analytical and clinical performance of the MolecisionTM Monkeypox Virus qPCR Assay, Biorain Monkeypox Virus ddPCR Assay, and MAGLUMI® Monkeypox Virus Ag (chemiluminescence immunoassay, CLIA) Assay. Additionally, it aimed to compare the clinical application of antigen and nucleic acid assays to offer insights into using commercial monkeypox assay kits. Specimens from 117 clinical patients, serial diluted virus cell culture supernatant, and artificially created positive samples were tested to evaluate the performance of these assay kits for MPXV diagnostics. The Biorain Monkeypox Virus ddPCR Assay had a limit of detection (LoD) of 3.89 CCID50/mL, while the MolecisionTM Monkeypox Virus qPCR Assay had an LoD of 15.55 CCID50/mL. The MAGLUMI® Monkeypox Virus Ag (CLIA) Assay had an LoD of 0.500 pg/mL. The accuracy of the MolecisionTM Monkeypox Virus qPCR Assay was comparable to the Biorain Monkeypox Virus ddPCR Assay, and the MAGLUMI® Monkeypox Virus Ag (CLIA) Assay demonstrated high sensitivity. The specificity of all three MPXV diagnostic assays for clinical specimens with potential cross-reacting substances was 100%. In conclusion, this study provides valuable insights into the clinical application of monkeypox assays, supporting efforts to mitigate and control the spread of monkeypox.

A Combined Transcriptomic and Proteomic Analysis of Monkeypox Virus A23 Protein on HEK293T Cells.

Monkeypox virus (MPXV) is a cross-kingdom pathogen infecting both humans and wildlife, which poses a significant health risk to the public. Although MPXV attracts broad attention, there is a lack of adequate studies to elucidate pathogenic mechanisms associated with viral infections. In this study, a high-throughput RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used to explore the transcriptional and metabolic responses of MPXV A23 protein to HEK293T cells. The protein-protein interactions and signaling pathways were conducted by GO and KEGG analyses. The localization of A23 protein in HEK293T cells was detected by immunofluorescence. A total of 648 differentially expressed genes (DEGs) were identified in cells by RNA-Seq, including 314 upregulated genes and 334 downregulated genes. Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected 115 cellular proteins that interact with the A23 proteins. Transcriptomic sequencing analysis revealed that transfection of MPXV A23 protein modulated genes primarily associated with cellular apoptosis and DNA damage repair. Proteomic analysis indicated that this protein primarily interacted with host ribosomal proteins and histones. Following the identification of the nuclear localization sequence RKKR within the A23 protein, a truncated mutant A23ΔRKKR was constructed to investigate the subcellular localization of A23 protein. The wild-type A23 protein exhibits a significantly higher nuclear-to-cytoplasmic ratio, exceeding 1.5, in contrast to the mutant A23ΔRKKR, which has a ratio of approximately 1. Immunofluorescence assays showed that the A23 protein was mainly localized in the nucleus. The integration of transcriptomics and proteomics analysis provides a comprehensive understanding of the interaction between MPXV A23 protein and the host. Our findings highlight the potential role of this enzyme in suppressing host antiviral immune responses and modulating host gene expression.

Role of Direct Sexual Contact in Human Transmission of Monkeypox Virus, Italy.

The 2022 global mpox outbreak was driven by human-to-human transmission, but modes of transmission by sexual relationship versus sexual contact remain unclear. We evaluated sexual transmission of mpox by using monkeypox virus (MPXV) G2R-mRNA as a marker of ongoing viral replication through in vitro experiments. We analyzed clinical samples of 15 MPXV-positive patients in Italy from different biological regions by using the setup method. The presence of MPXV DNA, MPXV G2R-mRNA, or both in all analyzed lesion swab samples, independent of viral load, confirmed a higher infectivity risk from skin lesions. Positivity for MPXV G2R-mRNA in nasopharyngeal swabs was associated with high MPXV load, whereas positive results for MPXV G2R-mRNA were obtained only in the 2 semen samples with the lowest MPXV loads. Our results suggest that close or skin-to-skin contact during sexual intercourse is the main route of sexual transmission and that semen is a minor driver of infection, regardless of MPXV load.

Computational investigations of potential inhibitors of monkeypox virus envelope protein E8 through molecular docking and molecular dynamics simulations.

The World Health Organization (WHO) has declared the monkeypox outbreak a public health emergency, as there is no specific therapeutics for monkeypox virus (MPXV) disease. This study focused on docking various commercial drugs and plant-derived compounds against the E8 envelope protein crucial for MPXV attachment and pathogenesis. The target protein structure was modeled based on the vaccinia virus D8L protein. Notably, maraviroc and punicalagin emerged as potential ligands, with punicalagin exhibiting higher binding affinity (- 9.1 kcal/mol) than maraviroc (- 7.8 kcal/mol). Validation through 100 ns molecular dynamics (MD) simulations demonstrated increased stability of the E8-punicalagin complex, with lower RMSD, RMSF, and Rg compared to maraviroc. Enhanced hydrogen bonding, lower solvent accessibility, and compact motions also attributed to higher binding affinity and stability of the complex. MM-PBSA calculations revealed van der Waals, electrostatic, and non-polar solvation as principal stabilizing energies. The binding energy decomposition per residue favored stable interactions between punicalagin and the protein's active site residues (Arg20, Phe56, Glu228, Tyr232) compared to maraviroc. Overall study suggests that punicalagin can act as a potent inhibitor against MPXV. Further research and experimental investigations are warranted to validate its efficacy and safety.

The Generation and Characterization of Monoclonal Antibodies against the MPXV A29L Protein.

Mpox (formerly known as monkeypox) is a zoonotic disease caused by monkeypox virus (MPXV), a DNA virus belonging to the Orthopoxvirus genus, in the Poxviridae family. The disease constitutes a moderate risk to public health at the global level. The MPXV A29L protein plays a crucial role in coordinating virion assembly and facilitating important virus-host interactions. This study focused on the expression, purification, and recombinant protein synthesis of the A29L protein of MPXV using prokaryotic systems. Using hybridoma technology, we successfully generated the monoclonal antibodies (mAbs) 1E12 and 4B2, which specifically recognize the A29L protein. These mAbs were found to be suitable for use in indirect immunofluorescence assays (IFA), Western blotting, and immunoprecipitation (IP). Our investigation also revealed that mAbs 1E12 and 4B2 could detect the A27L protein, a homologous protein found in the vaccinia virus Western Reserve (VACV WR) strain, using IFA, Western blotting, and immunoprecipitation (IP). Using mAbs 1E12 and 4B2 as primary immunological probes, A27L protein expression was detected as early as 6 h postinfection with VACV WR, with increasing protein levels being observed throughout the infection. This study enhances our understanding of the protein structure and function of MPXV and contributes to the development of specific MPXV detection methods.

Computational discovery of tripeptide inhibitors targeting monkeypox virus A42R profilin-like protein.

Monkeypox is an infectious disease caused by the monkeypox virus (MPXV), a member of the Orthopoxvirus genus closely related to smallpox. The structure of the A42R profilin-like protein is the first and only available structure among MPXV proteins. Biochemical studies of A42R were conducted in the 1990s and later work also analyzed the protein's function in viral replication in cells. This study aims to screen tripeptides for their potential inhibition of the A42R profilin-like protein using computational methods, with implications for MPXV therapy. A total of 8000 tripeptides underwent molecular docking simulations, resulting in the identification of 20 compounds exhibiting strong binding affinity to A42R. To validate the docking results, molecular dynamics simulations and free energy perturbation calculations were performed. These analyses revealed two tripeptides with sequences TRP-THR-TRP and TRP-TRP-TRP, which displayed robust binding affinity to A42R. Markedly, electrostatic interactions predominated over van der Waals interactions in the binding process between tripeptides and A42R. Three A42R residues, namely Glu9, Ser12, and Arg38, appear to be pivotal in mediating the interaction between A42R and the tripeptide ligands. Notably, tripeptides containing two or three tryptophan residues demonstrate a pronounced binding affinity, with the tripeptide comprising three tryptophan amino acids showing the highest level of affinity. These findings offer valuable insights for the selection of compounds sharing a similar structure and possessing a high affinity for A42R, potentially capable of inhibiting its enzyme activity. The study highlights a structural advantage and paves the way for the development of targeted therapies against MPXV infections.

Rapid regional mobile laboratory response and genomic monkeypox virus (MPXV) surveillance in seven East African Community partner states, August 2024: preparedness activities for the ongoing outbreak.

The East African Community (EAC) is experiencing an unprecedented, emerging mpox outbreak since July 2024 in five of eight partner states. We highlight rapid regional response measures, initiated August 2024 coordinated by EAC: field deployment of six mobile laboratories in Burundi, Rwanda, Uganda, Tanzania, Kenya, South Sudan to high-risk areas, donation of one mobile laboratory to Democratic Republic of the Congo and genomic monkeypox virus (MPXV) surveillance support. These interventions aim to limit local mpox spread and support international containment.

Innate Immune Response to Monkeypox Virus Infection: Mechanisms and Immune Escape.

BACKGROUND: The reemergence of monkeypox virus (Mpox, formerly monkeypox) in 2022 in non-endemic countries has raised significant concerns for global health due to its high transmissibility and mortality rate. A major challenge in combating Mpox is its ability to evade the host's innate immune system, the first line of defense against viral infections. SUMMARY: Mpox encodes various proteins that interfere with key antiviral pathways and mechanisms, such as the nuclear factor kappa B signaling, cytokine production, complement and inflammasome activation, and chemokine binding. These proteins modulate the expression and function of innate immune mediators, such as interferons, interleukins, and Toll-like receptors, and impair the recruitment and activation of innate immune cells, such as natural killer cells. By suppressing or altering these innate immune responses, Mpox enhances its replication and infection in the host tissues and organs, leading to systemic inflammation, tissue damage, and organ failure. KEY MESSAGES: This study reveals new insights into the molecular and cellular interactions between Mpox and the host's innate immune system. It identifies potential targets and strategies for antiviral interventions, highlighting the importance of understanding these interactions to develop effective treatments and improve global health responses to Mpox outbreaks.

Global perspectives on smallpox vaccine against monkeypox: a comprehensive meta-analysis and systematic review of effectiveness, protection, safety and cross-immunogenicity.

A large outbreak of monkeypox occurred in 2022, and most people lack immunity to orthopoxvirus. Smallpox vaccination is essential for preventing further smallpox outbreaks. This study evaluated the effectiveness, protection, safety, and cross-immunogenicity of smallpox vaccine in preventing monkeypox infection. PubMed, Embase, Scopus, and Web of Science were searched from database inception to 10 March 2024. We included studies involving "monkeypox virus" and "vaccinations", and excluded reviews, animal studies, and articles with missing or duplicate data. A total of 37 studies with 57,693 participants were included in the final analysis. The effectiveness data showed that monkeypox infection rates were lower in the smallpox-vaccinated group than in the unvaccinated group (risk ratio [RR]: 0.46; 95% confidence interval [CI]: 0.31-0.68). The protection data showed that smallpox vaccination effectively reduced the risk of severe monkeypox infection (RR: 0.61; 95% CI: 0.42-0.87). Third-generation vaccines showed greater efficacy (RR: 0.36, 95% CI: 0.22-0.56) than first-generation vaccines. The number of doses of smallpox vaccine has no significant effect on monkeypox. Safety data showed that adverse reactions after smallpox vaccination were mainly mild and included local erythema, swelling, induration, itching, and pain. Meanwhile, we found that smallpox vaccination could induce the production of neutralizing antibodies against monkeypox. Our findings offer compelling evidence supporting the clinical application of the smallpox vaccine for preventing monkeypox and advocate that high-risk groups should be prioritized for receiving one dose of the smallpox vaccine if the vaccine stockpile is low.

Profiling of viral load, antibody and inflammatory response of people with monkeypox during hospitalization: a prospective longitudinal cohort study in China.

BACKGROUND: The dynamics of viral shedding and the specific humoral response against monkeypox virus (MPXV) have not been well characterized in patients across their disease course during hospitalisation. The aim of this study was to determine the viral load and the levels of antibodies against MPXV using longitudinal paired-collected samples from hospitalized patients. METHODS: Patients who were hospitalised with mpox were recruited at Beijing Ditan Hospital Capital Medical University in China between June 2 and September 23, 2023. Paired samples, including samples from skin lesions, the oropharynx, saliva, faeces, urine, plasma, and serum, were serially collected at days 1, 3, 7, and 14 after admission until discharge. Not all of the patients had samples obtained at all of the timepoints. All the samples were analysed via quantitative PCR. Virus isolation was performed by using clinical samples and Vero cells. The presence of IgM, IgA, IgG, and neutralising antibodies (NAbs) against MPXV was evaluated. The first collected plasma sample was taken when the patient was hospitalised, and the levels of cytokines and chemokines were measured in the sample. The demographic data, smallpox vaccination status, history of known exposure to MPVX, HIV status and other clinical data were collected using a standard case report form. FINDINGS: A total of 510 specimens were serially collected from 39 recruited people with mpox. Among all the samples, the skin lesions had the highest viral DNA detection rates and viral loads, and the saliva samples had the second highest rates and viral loads. One day before discharge, 85% of the dry scrabs (median Ct 28.2, range 19.0-38.3) and 70% of the saliva samples (median Ct 32.4, range 24.5-38.1) were positive for viral DNA, Of which, 23.1% of dry scrabs were positive in viral culture. The rate of viral DNA detection in the oropharyngeal, saliva, and faecal samples decreased with time, while the rates in the plasma, serum, and urine samples increased quickly before 10 days post symptom onset (PSO). The median days of appearance of MPXV-IgM, MPXV-IgA, MPXV-IgG, and NAb were at 8 (interquartile range [IQR] 7-9), 9 (7-10), 12 (9-15), and 12 (9-15) PSO, respectively. The IgM, IgA, IgG, and NAb titres increased with time. Between days 11 and 21 PSO, the NAb titres were lower in people living with HIV (PWH) than in people living without HIV (PWOH). Increased NAb titres were associated with decreased viral loads in the saliva (r = 0.28, p = 0.025), faeces (r = 0.35, p = 0.021), plasma (r = 0.30, p = 0.0044), and serum samples (r = 0.37, p = 0.001). Compared with PWOH, PWH had higher plasma levels of MIP-1α, MIP-1ß, G-CSF, IL-4, and FGF-basic. INTERPRETATION: The high positive viral culture rate of clinical samples of patients when they are discharged from the hospital indicates that effective public health management strategies are needed for people with mpox. The low NAb titres and high levels of cytokines in PWH shows that earlier treatment is needed to control inflammation in high-risk populations. FUNDING: National Natural Science Foundation of China, Chinese Academy of Medical Sciences, Fundamental Research Funds for the Central Universities for Peking Union Medical College, National Key R&D Program of China.

Clinical characteristics, viral dynamics, and antibody response of monkeypox virus infections among men with and without HIV infection in Guangzhou, China.

Background: Monkeypox virus (MPXV) is spreading globally and nearly half of the infected people were human immunodeficiency virus (HIV) positive. Therefore, an in-depth understanding of the effects of HIV infection on the outcomes of MPXV infection is urgently needed. This study aimed to explore the clinical features, viral dynamics, and antibody response to MPXV infections in men who had sex with men (MSM) with and without HIV co-infection. Design or methods: MPXV-infected patients diagnosed by PCR were recruited in this study and were divided into MPXV and MPXV + HIV groups based on whether they were co-infected with HIV. Clinical data and samples were collected during of the hospital stay and follow up interviews. The symptoms and signs, laboratory examinations, viral shedding in various body fluids or swabs, antibody dynamics were tracked and compared between the two groups. Results: A total of 41 MPXV patients were recruited through June 2023 to September 2023 in Guangzhou. The MPXV group and MPXV + HIV group comprised 20 and 21 MSM, respectively. Patients in the two groups exhibited similar clinical characteristics except for pruritus and eschar, both were significantly fewer in MPXV + HIV group than in MPXV only group. Among the 355 clinical samples collected, MPXV DNA was detected in 100% of scabs, 97.4% of skin swabs, and 92.3% of exudate swabs from lesions, while the positive rate was 87.5% from oropharyngeal swabs, 59% from saliva, 51.3% from anal swabs, 50% from feces, 30.6% from urine samples, 37.5% of semen, and 28.2% from sera. Dynamics analysis revealed that viral DNA was undetectable in most patients 20 days after symptom onset. IgM and IgG antibodies to MPXV were detected in all patients with 3-5 days earlier in the MPXV group than in the MPXV + HIV group. Conclusion: This cohort analysis based on a large outbreak among MSM in Guangzhou indicated no obvious differences in clinical symptoms, viral DNA data, but antibody responses were 3-5 days later in mpox patients with HIV infection.