Multiple introductions of monkeypox virus to Ireland during the international mpox outbreak, May 2022 to October 2023.

BackgroundMpox, caused by monkeypox virus (MPXV), was considered a rare zoonotic disease before May 2022, when a global epidemic of cases in non-endemic countries led to the declaration of a Public Health Emergency of International Concern. Cases of mpox in Ireland, a country without previous mpox reports, could reflect extended local transmission or multiple epidemiological introductions.AimTo elucidate the origins and molecular characteristics of MPXV circulating in Ireland between May 2022 and October 2023.MethodsWhole genome sequencing of MPXV from 75% of all Irish mpox cases (182/242) was performed and compared to sequences retrieved from public databases (n = 3,362). Bayesian approaches were used to infer divergence time between sequences from different subclades and evaluate putative importation events from other countries.ResultsOf 242 detected mpox cases, 99% were males (median age: 35 years; range: 15-60). All 182 analysed genomes were assigned to Clade IIb and, presence of 12 distinguishable subclades suggests multiple introductions into Ireland. Estimation of time to divergence of subclades further supports the hypothesis for multiple importation events from numerous countries, indicative of extended and sustained international spread of mpox. Further analysis of sequences revealed that 92% of nucleotide mutations were from cytosine to thymine (or from guanine to adenine), leading to a high number of non-synonymous mutations across subclades; mutations associated with tecovirimat resistance were not observed.ConclusionWe provide insights into the international transmission dynamics supporting multiple introductions of MPXV into Ireland. Such information supported the implementation of evidence-informed public health control measures.

Evaluation of analytical performance of the STANDARDTM M10 MPX/OPX assay for the simultaneous DNA detection and clade attribution of Monkeypox virus.

Monkeypox virus (MPXV) infection confirmation needs reliable polymerase chain reaction (PCR) assays; in addition, viral clade attribution is a key factor in containment measures, considering a more severe syndrome in clade I and the possibility of simultaneous circulation. This study evaluates the performance of all-in-one STANDARD M10 MPX/OPX (SD BIOSENSOR, South Korea - M10). Frozen samples from 205 subjects were selected and stratified according to routine test results (RealStar® Orthopoxvirus PCR Kit 1.0, Altona DIAGNOTICS, Germany - RS; RS-1): in detail, 100 negative skin lesions (SL) and 200 positive samples at the variable stage of infection were analysed. Positive samples were retested with RS (RS-2). Positive and Negative Percent Agreements (PPA, NPA) were calculated. The median (IQR) Ct values of RS and M10 (OPXV target) assays were highly similar. The PPA of M10 compared to RS-1 was 89.5% considering system interpretation, and 96.0% when the operator classified results as positive if any target was detected; NPA was 100%. Comparing the RS-2 run and M10, an overall concordance of 95.3% between assays was found; however, considering operator interpretation, M10 returned more positive results than RS-2. The occurrence of False-Negative results was likely associated with the influence of thawing on low viral concentration; no False-Positive tests were observed. All samples collected at the time of Mpox diagnosis were positive and M10 correctly attributed the clade (West-Africa/II). The M10 MPX/OPX assay demonstrated high reliability in confirming MPXV infection and clade attribution.

Nanopore adaptive sampling of a metagenomic sample derived from a human monkeypox case.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misannotated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic.

Monkeypox virus genomic accordion strategies.

The 2023 monkeypox (mpox) epidemic was caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. Person-to-person transmission appears higher than for clade I or subclade IIa MPXV, possibly caused by genomic changes in subclade IIb MPXV. Key genomic changes could occur in the genome's low-complexity regions (LCRs), which are challenging to sequence and are often dismissed as uninformative. Here, using a combination of highly sensitive techniques, we determine a high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This reveals significant variation in short tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of single-nucleotide polymorphisms (SNPs) and that LCRs are not randomly distributed. In silico analyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs), including OPG153, OPG204, and OPG208, could be affected in a manner consistent with the established "genomic accordion" evolutionary strategies of orthopoxviruses. We posit that genomic studies focusing on phenotypic MPXV differences should consider LCR variability.

A high-throughput DNA analysis method based on isothermal amplification on a suspension microarray for detecting mpox virus and viruses with comparable symptoms.

BACKGROUND: Mpox is a zoonotic disease caused by mpox virus (MPXV) infection. Since May 2022, there has been a marked increase in human mpox cases in different regions. Rash, fever, and sore throat are typical signs of mpox. However, other viruses, such as the B virus (BV), herpes simplex virus types 1 (HSV-1), herpes simplex virus types 2 (HSV-2), and varicella zoster virus (VZV), can also infect people and cause comparable symptoms. Therefore, clinical symptoms and signs alone make distinguishing MPXV from these viruses difficult. RESULTS: In this study, we combined suspension microarray technology with recombinase-aided amplification technology (RAA) to establish a high-throughput, sensitive, and quantitative method for detecting MPXV and other viruses that can cause similar symptoms. The experimental results confirmed that the technique has outstanding sensitivity, with a minimum detection limit (LOD) of 0.1 fM and a linear range of 0.3 fM to 20 pM, spanning five orders of magnitude. The approach also exhibits exquisite selectivity, as the amplified signal can only be detected when the target virus nucleic acid is present. Additionally, serum recoveries ranging from 80.52% to 119.09% suggest that the detection outcomes are generally considered reliable. Moreover, the time required for detection using this high-throughput method is very short. After DNA extraction, the detection signal amplified by isothermal amplification on the bead array can be obtained in just 1 h. SIGNIFICANCE AND NOVELTY: Our research introduces a new technique that utilizes suspension microarray technology and isothermal amplification to create a high-throughput nucleic acid assay. This innovative method offers multiple benefits compared to current techniques, such as being cost-effective, time-efficient, highly sensitive, and having high throughput capabilities. Furthermore, the assay is applicable not only for detecting MPXV and viruses with similar symptoms, but also for clinical diagnostics, food safety, and environmental monitoring, rendering it an effective tool for screening harmful microorganisms.

A comprehensive review of monkeypox virus and mpox characteristics.

Monkeypox virus (MPXV) is the etiological agent of monkeypox (mpox), a zoonotic disease. MPXV is endemic in the forested regions of West and Central Africa, but the virus has recently spread globally, causing outbreaks in multiple non-endemic countries. In this paper, we review the characteristics of the virus, including its ecology, genomics, infection biology, and evolution. We estimate by phylogenomic molecular clock that the B.1 lineage responsible for the 2022 mpox outbreaks has been in circulation since 2016. We interrogate the host-virus interactions that modulate the virus infection biology, signal transduction, pathogenesis, and host immune responses. We highlight the changing pathophysiology and epidemiology of MPXV and summarize recent advances in the prevention and treatment of mpox. In addition, this review identifies knowledge gaps with respect to the virus and the disease, suggests future research directions to address the knowledge gaps, and proposes a One Health approach as an effective strategy to prevent current and future epidemics of mpox.

Fast and Ultrasensitive Detection of Monkeypox by a Pyrococcus furiosus Argonaute System Coupled with a Short Amplification.

Monkeypox virus (MPXV), the pathogen responsible for the infectious disease monkeypox, causes lesions on the skin, lymphadenopathy, and fever. It has posed a global public health threat since May 2022. Highly sensitive and specific detection of MPXV is crucial for preventing the spread of the disease. Pyrococcus furiosus Argonaute (PfAgo) is an artificial DNA-guided restriction cleavage enzyme programmable with 5'-phosphorylated ssDNA sequences, which can be developed to specifically detect nucleic acids of pathogens. Here, a PfAgo-based system was established for the detection of MPXV-specific DNA targeting the F3L gene. A short amplicon of 79 bp could be obtained through a fast PCR procedure, which was completed within 45 min. Two 5'-phosphorylation guide DNAs were designed to guide PfAgo to cleave the amplicon to obtain an 18 bp 5'-phosphorylation sequence specific to MPXV, not to other orthopoxviruses (cowpox, variola, and vaccinia viruses). The 18 bp sequence guided PfAgo to cleave a designed probe specific to MPXV to emit fluorescence. With optimized conditions for the PfAgo-MPXV system, it could be completed in 60 min for the detection of the extracted MPXV DNA with the limit of detection (LOD) of 1.1 copies/reaction and did not depend on expensive instruments. Successful application of the PfAgo-MPXV system in sensitively detecting MPXV in simulated throat swabs, skin swabs, sera, and wastewater demonstrated the system's good performance. The PfAgo platform, with high sensitivity and specificity established here, has the potential to prevent the spread of MPXV.

Human monkeypox virus: Epidemiologic review and research progress in diagnosis and treatment.

Monkeypox virus (MPXV) is responsible for causing a zoonotic disease called monkeypox (mpox), which sporadically infects humans in West and Central Africa. It first infected humans in 1970 and, along with the variola virus, belongs to the genus Orthopoxvirus in the poxvirus family. Since the World Health Organization declared the MPXV outbreak a "Public Health Emergency of International Concern" on July 23, 2022, the number of infected patients has increased dramatically. To control this epidemic and address this previously neglected disease, MPXV needs to be better understood and reevaluated. In this review, we cover recent research on MPXV, including its genomic and pathogenic characteristics, transmission, mutations and mechanisms, clinical characteristics, epidemiology, laboratory diagnosis, and treatment measures, as well as prevention of MPXV infection in light of the 2022 and 2023 global outbreaks. The 2022 MPXV outbreak has been primarily associated with close intimate contact, including sexual activity, with most cases diagnosed among men who have sex with men. The incubation period of MPXV infection usually lasts from 6 to 13 days, and symptoms include fever, muscle pains, headache, swollen lymph nodes, and a characteristic painful rash, including several stages, such as macules, papules, blisters, pustules, scabs, and scab shedding involving the genitals and anus. Polymerase chain reaction (PCR) is usually used to detect MPXV in skin lesion material. Treatment includes supportive care, antivirals, and intravenous vaccinia immune globulin. Smallpox vaccines have been designed with four givens emergency approval for use against MPXV infection.

Co-Circulating Monkeypox and Swinepox Viruses, Democratic Republic of the Congo, 2022.

In September 2022, deaths of pigs manifesting pox-like lesions caused by swinepox virus were reported in Tshuapa Province, Democratic Republic of the Congo. Two human mpox cases were found concurrently in the surrounding community. Specific diagnostics and robust sequencing are needed to characterize multiple poxviruses and prevent potential poxvirus transmission.

Mpox virus Clade IIb infected Cynomolgus macaques via mimic natural infection routes closely resembled human mpox infection.

Generating an infectious non-human primate (NHP) model using a prevalent monkeypox virus (MPXV) strain has emerged as a crucial strategy for assessing the efficacy of vaccines and antiviral drugs against human MPXV infection. Here, we established an animal model by infecting cynomolgus macaques with the prevalent MPXV strain, WIBP-MPXV-001, and simulating its natural routes of infection. A comprehensive analysis and evaluation were conducted on three animals, including monitoring clinical symptoms, collecting hematology data, measuring viral loads, evaluating cellular and humoral immune responses, and examining histopathology. Our findings revealed that initial skin lesions appeared at the inoculation sites and subsequently spread to the limbs and back, and all infected animals exhibited bilateral inguinal lymphadenopathy, eventually leading to a self-limiting disease course. Viral DNA was detected in post-infection blood, nasal, throat, rectal and blister fluid swabs. These observations indicate that the NHP model accurately reflects critical clinical features observed in human MPXV infection. Notably, the animals displayed clinical symptoms and disease progression similar to those of humans, rather than a lethal outcome as observed in previous studies. Historically, MPXV was utilized as a surrogate model for smallpox. However, our study contributes to a better understanding of the dynamics of current MPXV infections while providing a potential infectious NHP model for further evaluation of vaccines and antiviral drugs against mpox infection. Furthermore, the challenge model closely mimics the primary natural routes of transmission for human MPXV infections. This approach enhances our understanding of the precise mechanisms underlying the interhuman transmission of MPXV.

Rapid identification of A29L antibodies based on mRNA immunization and high-throughput single B cell sequencing to detect Monkeypox virus.

With the large number of atypical cases in the mpox outbreak, which was classified as a global health emergency by the World Health Organization (WHO) on 23 July 2022, rapid diagnosis of mpox and diseases with similar symptoms to mpox such as chickenpox and respiratory infectious diseases in the early stages of viral infection is key to controlling the spread of the outbreak. In this study, antibodies against the monkeypox virus A29L protein were efficiently and rapidly identified by combining rapid mRNA immunization with high-throughput sequencing of individual B cells. We obtained eight antibodies with a high affinity for A29L validated by ELISA, which were was used as the basis for developing an ultrasensitive fluorescent immunochromatographic assay based on multilayer quantum dot nanobeads (SiTQD-ICA). The SiTQD-ICA biosensor utilizing M53 and M78 antibodies showed high sensitivity and stability of detection: A29L was detected within 20 min, with a minimum detection limit of 5 pg/mL. A specificity test showed that the method was non-cross-reactive with chickenpox or common respiratory pathogens and can be used for early and rapid diagnosis of monkeypox virus infection by antigen detection. This antibody identification method can also be used for rapid acquisition of monoclonal antibodies in early outbreaks of other infectious diseases for various studies.

Ongoing mpox outbreak in Kamituga, South Kivu province, associated with monkeypox virus of a novel Clade I sub-lineage, Democratic Republic of the Congo, 2024.

Since the beginning of 2023, the number of people with suspected monkeypox virus (MPXV) infection have sharply increased in the Democratic Republic of the Congo (DRC). We report near-to-complete MPXV genome sequences derived from six cases from the South Kivu province. Phylogenetic analyses reveal that the MPXV affecting the cases belongs to a novel Clade I sub-lineage. The outbreak strain genome lacks the target sequence of the probe and primers of a commonly used Clade I-specific real-time PCR.

A multivalent mRNA monkeypox virus vaccine (BNT166) protects mice and macaques from orthopoxvirus disease.

In response to the 2022 outbreak of mpox driven by unprecedented human-to-human monkeypox virus (MPXV) transmission, we designed BNT166, aiming to create a highly immunogenic, safe, accessible, and scalable next-generation vaccine against MPXV and related orthopoxviruses. To address the multiple viral forms and increase the breadth of immune response, two candidate multivalent mRNA vaccines were evaluated pre-clinically: a quadrivalent vaccine (BNT166a; encoding the MPXV antigens A35, B6, M1, H3) and a trivalent vaccine (BNT166c; without H3). Both candidates induced robust T cell responses and IgG antibodies in mice, including neutralizing antibodies to both MPXV and vaccinia virus. In challenge studies, BNT166a and BNT166c provided complete protection from vaccinia, clade I, and clade IIb MPXV. Furthermore, immunization with BNT166a was 100% effective at preventing death and at suppressing lesions in a lethal clade I MPXV challenge in cynomolgus macaques. These findings support the clinical evaluation of BNT166, now underway (NCT05988203).

Genomic and transcriptomic analysis of the recent Mpox outbreak.

The Mpox (formerly named Monkeypox) virus is the etiological cause of a recent multi-country outbreak, with thousands of distinct cases detected outside the endemic areas of Africa as of December 2023. In this article, we analyze the sequences of full genomes of Mpox virus from Europe and compare them with all available Mpox sequences of historical relevance, annotated by year and geographic origin, as well as related Cowpox and Variola (smallpox) virus sequences. Our results show that the recent outbreak is most likely originating from the West African clade of Mpox, with >99 % sequence identity with sequences derived from historical and recent cases, dating from 1971 to 2017. We analyze specific mutations occurring in viral proteins between the current outbreak, previous Mpox and Cowpox sequences, and the historical Variola virus. Genome-wide sequence analysis of the recent outbreak and other Mpox/Cowpox/Variola viruses shows a very high conservation, with 97.9 % (protein-based) and 97.8 % (nucleotide-based) sequence identity. We identified significant correlation in human transcriptional responses as well, with a conserved immune pathway response induced in human cell cultures by the three families of Pox virus. The similarities identified between the major strains of Pox viruses, as well as within the Mpox clades, both at the genomic and transcriptomic levels, provide a molecular basis for the observed efficacy of Variola vaccines in other Poxviruses.

Transcriptome and proteomic analysis of mpox virus F3L-expressing cells.

Background: Monkeypox or mpox virus (mpox) is a double-stranded DNA virus that poses a significant threat to global public health security. The F3 protein, encoded by mpox, is an apoenzyme believed to possess a double-stranded RNA-binding domain (dsRBD). However, limited research has been conducted on its function. In this study, we present data on the transcriptomics and proteomics of F3L-transfected HEK293T cells, aiming to enhance our comprehension of F3L. Methods: The gene expression profiles of pCAGGS-HA-F3L transfected HEK293T cells were analyzed using RNA-seq. Proteomics was used to identify and study proteins that interact with F3L. Real-time PCR was used to detect mRNA levels of several differentially expressed genes (DEGs) in HEK293T cells (or Vero cells) after the expression of F3 protein. Results: A total of 14,822 genes were obtained in cells by RNA-Seq and 1,672 DEGs were identified, including 1,156 up-regulated genes and 516 down-regulated genes. A total of 27 cellular proteins interacting with F3 proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and 19 cellular proteins with large differences in abundance ratios were considered to be candidate cellular proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were significantly enriched in immune-related pathways, including type I interferon signaling pathway, response to virus, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, etc. Moreover, some selected DEGs were further confirmed by real-time PCR and the results were consistent with the transcriptome data. Proteomics data show that cellular proteins interacting with F3 proteins are mainly related to RNA splicing and protein translation. Conclusions: Our analysis of transcriptomic and proteomic data showed that (1) F3L up-regulates the transcript levels of key genes in the innate immune signaling pathway, such as RIGI, MDA5, IRF5, IRF7, IRF9, ISG15, IFNA14, and elicits a broad spectrum of antiviral immune responses in the host. F3L also increases the expression of the FOS and JNK genes while decreasing the expression of TNFR2, these factors may ultimately induce apoptosis. (2) F3 protein interacts with host proteins involved in RNA splicing and protein translation, such as SNRNP70, POLR2H, HNRNPA1, DDX17, etc. The findings of this study shed light on the function of the F3 protein.

Concurrent Clade I and Clade II Monkeypox Virus Circulation, Cameroon, 1979-2022.

During 1979-2022, Cameroon recorded 32 laboratory-confirmed mpox cases among 137 suspected mpox cases identified by the national surveillance network. The highest positivity rate occurred in 2022, indicating potential mpox re-emergence in Cameroon. Both clade I (n = 12) and clade II (n = 18) monkeypox virus (MPXV) were reported, a unique feature of mpox in Cameroon. The overall case-fatality ratio of 2.2% was associated with clade II. We found mpox occurred only in the forested southern part of the country, and MPXV phylogeographic structure revealed a clear geographic separation among concurrent circulating clades. Clade I originated from eastern regions close to neighboring mpox-endemic countries in Central Africa; clade II was prevalent in western regions close to West Africa. Our findings suggest that MPXV re-emerged after a 30-year lapse and might arise from different viral reservoirs unique to ecosystems in eastern and western rainforests of Cameroon.

Review of virological methods for laboratory diagnosis and characterization of monkeypox virus (MPXV): lessons learned from the 2022 Mpox outbreak.

Monkeypox virus (MPXV), originally endemic in West Africa (Clade II) and Central Africa (Clade I), has recently emerged worldwide and has reinforced the need for rapid and accurate MPXV diagnostics. This review presents and critically discusses the range of virological methods for laboratory diagnosis and characterization of MPXV as well as related lessons learned and practical experience gained from the 2022 Mpox global outbreak. Real-time PCR is currently considered the diagnostic gold standard and ensures accurate and timely confirmation of suspected Mpox cases based on suspicious skin lesions, and digital PCR improves the precision of MPXV DNA quantification. Whole genome sequencing reveals the diversity within the Clade IIb outbreak and highlights the role of microevolution in the adaptation of the virus to the human host. Continuous genomic surveillance is important for better understanding of human-to-human transmission and prevention of the emergence of variola virus-like strains. Traditional virological methods such as electron microscopy and virus isolation remain essential for comprehensive virus characterization, particularly in the context of vaccine and antiviral drug development. Despite the current challenges, serological tests detecting a range of anti-MPXV antibodies are important adjunct diagnostic and research tools for confirmation of late-presenting or asymptomatic MPXV cases, contact tracing, epidemiological studies, seroepidemiological surveys, and better understanding of the role of IgG and neutralizing antibodies in the immune response to infection and vaccination. A multidisciplinary approach combining advanced molecular techniques with traditional virological methods is important for rapid and reliable diagnosis, surveillance, and control of the outbreak.

Differences and similarities between Monkeypox and Chickenpox in children during an outbreak.

INTRODUCTION: Herein, we described cases of children under 16 years old suspected to be infected with Monkeypox virus (MKPV) and diagnosed with chickenpox in public hospitals of Marseille, south of France. MATERIAL AND METHODS: We conducted a retrospective study from March 23rd, 2022 to October 20th, 2022 in our institution of results of MKPV DNA and varicella-zoster virus (VZV) DNA detection by PCR performed on cutaneous lesions swabs collected from children <16 years old. RESULTS: None of the cutaneous swabs collected from 14 children were positive for MKPV DNA. In contrast, 30/168 (17 %) cutaneous swabs collected from children were positive for VZV DNA. Of these 30 VZV-positive children, 7 had been suspected of MKPV infection because of their atypical rash, due to the location of the lesions and the chronology of their appearance. DISCUSSION: As in our cohort, pediatric cases of the 2022 Monkeypox outbreak in non-endemic developed countries have been very rare. This variant of MKPV does not normally spread easily and requires very close physical contact between an infected person (skin lesions, bodily fluids or respiratory droplets) and another person to be transmitted. It will nevertheless be a question of remaining vigilant as not to ignore the possibility of close contact or sexual transmission of Monkeypox in a child, or the possibility of a new and more contagious variant. CONCLUSION: It is difficult to differentiate Monkeypox infection from other infections associated with rashes, it is important to remember that viruses change as well as their forms of presentation.

Effect of chemical and physical agents on monkeypox virus infectivity and downstream research applications.

The 2022 global spread of Monkeypox Virus (MPXV) underlined the need to investigate safe-handling procedures of clinical and research samples. Here we evaluated the efficiency in reducing MPXV infectious titer of Triton X-100 (0.1 and 0.2%), UV-C irradiation (15 or 30 min), and heat (56 °C 30 min or 70 °C 5 min). The treatment of MPXV at 70 °C resulted in the strongest decrease of MPXV infectious titer (5.4 Log TCID50/mL), 56 °C and UV-C had a lighter impact (3.9 and 4.3Log), Triton X-100 was less efficient (1.8-2.5Log). Notably, SARS-CoV-2 was much more susceptible to Triton X-100 (4.0 Log decrease). UV-C had the highest impact on MPXV DNA detection by PCR (2.2-4.3 Ct value increase); protein detection by ELISA was dramatically impaired by heating. Overall, UV-C and heating were more effective in lowering MPXV infectious titer but their impact on nucleic acids or protein detection assays must be considered.