Structural basis of the monkeypox virus mRNA cap N7 methyltransferase complex.

The global outbreak of Mpox, caused by the monkeypox virus (MPXV), has attracted international attention and become another major infectious disease event after COVID-19. The mRNA cap N7 methyltransferase (RNMT) of MPXV methylates the N7 position of the added guanosine to the 5'-cap structure of mRNAs and plays a vital role in evading host antiviral immunity. MPXV RNMT is composed of the large subunit E1 and the small subunit E12. How E1 and E12 of MPXV assembly remains unclear. Here, we report the crystal structures of E12, the MTase domain of E1 with E12 (E1CTD-E12) complex, and the E1CTD-E12-SAM ternary complex, revealing the detailed conformations of critical residues and the structural changes upon E12 binding to E1. Functional studies suggest that E1CTD N-terminal extension (Asp545-Arg562) and the small subunit E12 play an essential role in the binding process of SAM. Structural comparison of the AlphaFold2-predicted E1, E1CTD-E12 complex, and the homologous D1-D12 complex of vaccinia virus (VACV) indicates an allosteric activating effect of E1 in MPXV. Our findings provide the structural basis for the MTase activity stimulation of the E1-E12 complex and suggest a potential interface for screening the anti-poxvirus inhibitors.

MPXV DNA kinetics in bloodstream and other body fluids samples.

Since spring 2022, the global epidemiology of the monkeypox virus (MPXV) has changed. The unprecedented increase of human clade II MPXV cases worldwide heightened concerns about this emerging zoonotic disease. We analysed the positivity rates, viral loads, infectiousness, and persistence of MPXV DNA for up to 4 months in several biological samples from 89 MPXV-confirmed cases. Our data showed that viral loads and positivity rates were higher during the first two weeks of symptoms for all sample types. Amongst no-skin-samples, respiratory specimens showed higher MPXV DNA levels and median time until viral clearance, suggesting their usefulness in supporting MPXV diagnosis, investigating asymptomatic patients, and monitoring viral shedding. Infectious virus was cultured from respiratory samples, semen, and stools, with high viral loads and collected within the first 10 days. Notably, only one saliva and one semen were found positive for viral DNA after 71 and 31 days from symptoms, respectively. The focus on bloodstream samples showed the best testing sensitivity in plasma, reporting the overall highest MPXV DNA detection rate and viral loads during the 3-week follow-up as compared to serum and whole-blood. The data here presented can be useful for MPXV diagnostics and a better understanding of the potential alternative routes of its onward transmission.

Molecular Evolution of Protein Sequences and Codon Usage in Monkeypox Viruses.

The monkeypox virus (mpox virus, MPXV) epidemic in 2022 has posed a significant public health risk. Yet, the evolutionary principles of MPXV remain largely unknown. Here, we examined the evolutionary patterns of protein sequences and codon usage in MPXV. We first demonstrated the signal of positive selection in OPG027, specifically in the Clade I lineage of MPXV. Subsequently, we discovered accelerated protein sequence evolution over time in the variants responsible for the 2022 outbreak. Furthermore, we showed strong epistasis between amino acid substitutions located in different genes. The codon adaptation index (CAI) analysis revealed that MPXV genes tended to use more non-preferred codons compared to human genes, and the CAI decreased over time and diverged between clades, with Clade I > IIa and IIb-A > IIb-B. While the decrease in fatality rate among the three groups aligned with the CAI pattern, it remains unclear whether this correlation was coincidental or if the deoptimization of codon usage in MPXV led to a reduction in fatality rates. This study sheds new light on the mechanisms that govern the evolution of MPXV in human populations.

Exploring monkeypox virus proteins and rapid detection techniques.

Monkeypox (mpox) is an infectious disease caused by the mpox virus and can potentially lead to fatal outcomes. It resembles infections caused by viruses from other families, challenging identification. The pathogenesis, transmission, and clinical manifestations of mpox and other Orthopoxvirus species are similar due to their closely related genetic material. This review provides a comprehensive discussion of the roles of various proteins, including extracellular enveloped virus (EEV), intracellular mature virus (IMV), and profilin-like proteins of mpox. It also highlights recent diagnostic techniques based on these proteins to detect this infection rapidly.

Understanding the biology of monkeypox virus to prevent future outbreaks.

Historically, monkeypox (mpox) was a zoonotic disease endemic in Africa. However, in 2022, a global outbreak occurred following a substantial increase in cases in Africa, coupled with spread by international travellers to other continents. Between January 2022 and October 2023, about 91,000 confirmed cases from 115 countries were reported, leading the World Health Organization to declare a public health emergency. The basic biology of monkeypox virus (MPXV) can be inferred from other poxviruses, such as vaccinia virus, and confirmed by genome sequencing. Here the biology of MPXV is reviewed, together with a discussion of adaptive changes during MPXV evolution and implications for transmission. Studying MPXV biology is important to inform specific host interactions, to aid in ongoing outbreaks and to predict those in the future.

Experimental inoculation of pigs with monkeypox virus results in productive infection and transmission to sentinels.

Monkeypox virus (MPXV) is a re-emerging zoonotic poxvirus responsible for producing skin lesions in humans. Endemic in sub-Saharan Africa, the 2022 outbreak with a clade IIb strain has resulted in ongoing sustained transmission of the virus worldwide. MPXV has a relatively wide host range, with infections reported in rodent and non-human primate species. However, the susceptibility of many domestic livestock species remains unknown. Here, we report on a susceptibility/transmission study in domestic pigs that were experimentally inoculated with a 2022 MPXV clade IIb isolate or served as sentinel contact control animals. Several principal-infected and sentinel contact control pigs developed minor lesions near the lips and nose starting at 12 through 18 days post-challenge (DPC). No virus was isolated and no viral DNA was detected from the lesions; however, MPXV antigen was detected by IHC in tissue from a pustule of a principal infected pig. Viral DNA and infectious virus were detected in nasal and oral swabs up to 14 DPC, with peak titers observed at 7 DPC. Viral DNA was also detected in nasal tissues or skin collected from two principal-infected animals at 7 DPC post-mortem. Furthermore, all principal-infected and sentinel control animals enrolled in the study seroconverted. In conclusion, we provide the first evidence that domestic pigs are susceptible to experimental MPXV infection and can transmit the virus to contact animals.

Development of a multiplex real-time PCR assay for the simultaneous detection of mpox virus and orthopoxvirus infections.

Since May 2022, the multi-country outbreak of monkeypox (mpox) has raised a great concern worldwide. Early detection of mpox virus infection is recognized as an efficient way to prevent mpox transmission. Mpox specific detection methods reported up to now are based on the SNPs among mpox virus and other orthopoxviruses. We have therefore developed a real-time PCR based mpox detection method targeting mpox virus specific sequences (N3R and B18Rplus). We have also optimized an orthopoxvirus detection system which targets the highly conserved E9L and D6R genes. The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction) and specificity. Mpox viral DNA and clinical samples from mpox patients are detected with the mpox detection system. Furthermore, we have established a multiplex real-time PCR detection system allowing simultaneous and efficient detection of mpox and orthopoxvirus infections.

Reverse Zoonotic Transmission of SARS-CoV-2 and Monkeypox Virus: A Comprehensive Review.

Reverse zoonosis reveals the process of transmission of a pathogen through the human-animal interface and the spillback of the zoonotic pathogen. In this article, we methodically demonstrate various aspects of reverse zoonosis, with a comprehensive discussion of SARS-CoV-2 and MPXV reverse zoonosis. First, different components of reverse zoonosis, such as humans, different pathogens, and numerous animals (poultry, livestock, pets, wild animals, and zoo animals), have been demonstrated. Second, it explains the present status of reverse zoonosis with different pathogens during previous occurrences of various outbreaks, epidemics, and pandemics. Here, we present 25 examples from literature. Third, using several examples, we comprehensively illustrate the present status of the reverse zoonosis of SARS-CoV-2 and MPXV. Here, we have provided 17 examples of SARS-CoV-2 reverse zoonosis and two examples of MPXV reverse zoonosis. Fourth, we have described two significant aspects of reverse zoonosis: understanding the fundamental aspects of spillback and awareness. These two aspects are required to prevent reverse zoonosis from the current infection with two significant viruses. Finally, the One Health approach was discussed vividly, where we urge scientists from different areas to work collaboratively to solve the issue of reverse zoonosis.

Expression analysis and mapping of Viral-Host Protein interactions of Poxviridae suggests a lead candidate molecule targeting Mpox.

BACKGROUND: Monkeypox (Mpox) is an important human pathogen without etiological treatment. A viral-host interactome study may advance our understanding of molecular pathogenesis and lead to the discovery of suitable therapeutic targets. METHODS: GEO Expression datasets characterizing mRNA profile changes in different host responses to poxviruses were analyzed for shared pathway identification, and then, the Protein-protein interaction (PPI) maps were built. The viral gene expression datasets of Monkeypox virus (MPXV) and Vaccinia virus (VACV) were used to identify the significant viral genes and further investigated for their binding to the library of targeting molecules. RESULTS: Infection with MPXV interferes with various cellular pathways, including interleukin and MAPK signaling. While most host differentially expressed genes (DEGs) are predominantly downregulated upon infection, marked enrichments in histone modifiers and immune-related genes were observed. PPI analysis revealed a set of novel virus-specific protein interactions for the genes in the above functional clusters. The viral DEGs exhibited variable expression patterns in three studied cell types: primary human monocytes, primary human fibroblast, and HeLa, resulting in 118 commonly deregulated proteins. Poxvirus proteins C6R derived protein K7 and K7R of MPXV and VACV were prioritized as targets for potential therapeutic interventions based on their histone-regulating and immunosuppressive properties. In the computational docking and Molecular Dynamics (MD) experiments, these proteins were shown to bind the candidate small molecule S3I-201, which was further prioritized for lead development. RESULTS: MPXV circumvents cellular antiviral defenses by engaging histone modification and immune evasion strategies. C6R-derived protein K7 binding candidate molecule S3I-201 is a priority promising candidate for treating Mpox.

Rapid and highly sensitive colorimetric LAMP assay and integrated device for visual detection of monkeypox virus.

BACKGROUND: The monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics. RESULTS: Here, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h. SIGNIFICANCE: The device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.

Monkeypox: Past, Present, and Future.

Monkeypox (Mpox) is a zoonotic disease caused by a virus (monkeypox virus-MPV) belonging to the Poxviridae family. In humans, the disease has an incubation period of 5-21 days and then progresses in two phases, the prodromal phase and the rash phase. The prodromal phase is characterized by non-specific symptoms such as fever, muscle pain, malaise, lymphadenopathy, headache, and chills. Skin lesions appear in the rash phase of the disease. These lesions progress through different stages (macules, papules, vesicles, and pustules). In May 2022, WHO reported an outbreak of human Mpox in several countries which were previously Mpox-free. As per the CDC report of March 01, 2023, a total of 86,231 confirmed cases of Mpox and 105 deaths have been reported from 110 countries and territories across the globe. Notably, more than 90% of these countries were reporting Mpox for the first time. The phylogenetic analysis revealed that this outbreak was associated with the virus from the West African clade. However, most of the cases in this outbreak had no evidence of travel histories to MPV-endemic countries in Central or West Africa. This outbreak was primarily driven by the transmission of the virus via intimate contact in men who have sex with men (MSM). The changing epidemiology of Mpox raised concerns about the increasing spread of the disease in non-endemic countries and the urgent need to control and prevent it. In this chapter, we present all the documented cases of Mpox from 1970 to 2023 and discuss the past, present, and future of MPV.

Zoonotic and Zooanthroponotic Potential of Monkeypox.

The current multicounty outbreak of monkeypox virus (MPXV) posed an emerging and continued challenge to already strained public healthcare sector, around the globe. Since its first identification, monkeypox disease (mpox) remained enzootic in Central and West African countries where reports of human cases are sporadically described. Recent trends in mpox spread outside the Africa have highlighted increased incidence of spillover of the MPXV from animal to humans. While nature of established animal reservoirs remained undefined, several small mammals including rodents, carnivores, lagomorphs, insectivores, non-human primates, domestic/farm animals, and several species of wildlife are proposed to be carrier of the MPXV infection. There are established records of animal-to-human (zoonotic) spread of MPXV through close interaction of humans with animals by eating bushmeat, contracting bodily fluids or trading possibly infected animals. In contrast, there are reports and increasing possibilities of human-to-animal (zooanthroponotic) spread of the MPXV through petting and close interaction with pet owners and animal care workers. We describe here the rationales and molecular factors which predispose the spread of MPXV not only amongst humans but also from animals to humans. A range of continuing opportunities for the spread and evolution of MPXV are discussed to consider risks beyond the currently identified groups. With the possibility of MPXV establishing itself in animal reservoirs, continued and broad surveillance, investigation into unconventional transmissions, and exploration of spillover events are warranted.

Biological Characteristics and Pathogenesis of Monkeypox Virus: An Overview.

Although the smallpox virus has been eradicated worldwide, the World Health Organization (WHO) has issued a warning about the virus's potential to propagate globally. The WHO labeled monkeypox a world public health emergency in July 2022, requiring urgent prevention and treatment. The monkeypox virus is a part of the Poxviridae family, Orthopoxvirus genus, and is accountable for smallpox, which has killed over a million people in the past. Natural hosts of the virus include squirrels, Gambian rodents, chimpanzees, and other monkeys. The monkeypox virus has transmitted to humans through primary vectors (various animal species) and secondary vectors, including direct touch with lesions, breathing particles from body fluids, and infected bedding. The viral particles are ovoid or brick-shaped, 200-250 nm in diameter, contain a single double-stranded DNA molecule, and reproduce only in the cytoplasm of infected cells. Monkeypox causes fever, cold, muscle pains, headache, fatigue, and backache. The phylogenetic investigation distinguished between two genetic clades of monkeypox: the more pathogenic Congo Basin clade and the West Africa clade. In recent years, the geographical spread of the human monkeypox virus has accelerated despite a paucity of information regarding the disease's emergence, ecology, and epidemiology. Using lesion samples and polymerase chain reaction (PCR), the monkeypox virus was diagnosed. In the USA, the improved Ankara vaccine can now be used to protect people who are at a higher risk of getting monkeypox. Antivirals that we have now work well against smallpox and may stop the spread of monkeypox, but there is no particular therapy for monkeypox.

Type I Interferon: Monkeypox/Mpox Viruses Achilles Heel?

Poxviruses are notorious for having acquired/evolved numerous genes to counteract host innate immunity. Chordopoxviruses have acquired/evolved at least three different inhibitors of host necroptotic death: E3, which blocks ZBP1-dependent necroptotic cell death, and vIRD and vMLKL that inhibit necroptosis downstream of initial cell death signaling. While this suggests the importance of the necroptotic cell death pathway in inhibiting chordopoxvirus replication, several chordopoxviruses have lost one or more of these inhibitory functions. Monkeypox/mpox virus (MPXV) has lost a portion of the N-terminus of its E3 homologue. The N-terminus of the vaccinia virus E3 homologue serves to inhibit activation of the interferon-inducible antiviral protein, ZBP1. This likely makes MPXV unique among the orthopoxviruses in being sensitive to interferon (IFN) treatment in many mammals, including humans, which encode a complete necroptotic cell death pathway. Thus, IFN sensitivity may be the Achille's Heel for viruses like MPXV that cannot fully inhibit IFN-inducible, ZBP1-dependent antiviral pathways.

Molecular Virology of Orthopoxviruses with Special Reference to Monkeypox Virus.

Poxviruses are large (200-450 nm) and enveloped viruses carrying double-stranded DNA genome with an epidermal cell-specific adaptation. The genus Orthopoxvirus within Poxviridae family constitutes several medically and veterinary important viruses including variola (smallpox), vaccinia, monkeypox virus (MPXV), and cowpox. The monkeypox disease (mpox) has recently emerged as a public health emergency caused by MPXV. An increasing number of human cases of MPXV have been documented in non-endemic nations without any known history of contact with animals brought in from endemic and enzootic regions, nor have they involved travel to an area where the virus was typically prevalent. Here, we review the MPXV replication, virus pathobiology, mechanism of viral infection transmission, virus evasion the host innate immunity and antiviral therapies against Mpox. Moreover, preventive measures including vaccination were discussed and concluded that cross-protection against MPXV may be possible using antibodies that are directed against an Orthopoxvirus. Despite the lack of a specialised antiviral medication, several compounds such as Cidofovir and Ribavirin warrant consideration against mpox.

Human Monkeypox Virus and Host Immunity: New Challenges in Diagnostics and Treatment Strategies.

The monkeypox virus (MPXV), responsible for human disease, has historically been limited to the African countries, with only a few isolated instances reported elsewhere in the world. Nevertheless, in recent years, there have been occurrences of monkeypox in regions where the disease is typically absent, which has garnered global interest. Within a period of less than four months, the incidence of MPXV infections has surged to over 48,000 cases, resulting in a total of 13 deaths. This chapter has addressed the genetics of the pox virus, specifically the human monkeypox virus, and its interaction with the immune systems of host organisms. The present chapter is skillfully constructed, encompassing diagnostic methodologies that span from traditional to developing molecular techniques. Furthermore, the chapter provides a succinct analysis of the therapeutic methods employed, potential future developments, and the various emerging difficulties encountered in illness management.

Poxvirus Vaccines: Past, Present, and Future.

Smallpox was a significant cause of mortality for over three thousand years, amounting to 10% of deaths yearly. Edward Jenner discovered smallpox vaccination in 1796, which rapidly became a smallpox infection preventive practice throughout the world and eradicated smallpox infection by 1980. After smallpox eradication, monkeypox vaccines have been used primarily in research and in outbreaks in Africa, where the disease is endemic. In the present, the vaccines are being used for people who work with animals or in high-risk areas, as well as for healthcare workers treating patients with monkeypox. Among all orthopoxviruses (OPXV), monkeypox viral (MPXV) infection occurs mainly in cynomolgus monkeys, natural reservoirs, and occasionally causes severe multi-organ infection in humans, who were the incidental hosts. The first case of the present epidemic of MXPV was identified on May 7, 2022, and rapidly increased the number of cases. In this regard, the WHO declared the outbreak, an international public health emergency on July 23, 2022. The first monkeypox vaccine was developed in the 1960s by the US Army and was based on the vaccinia virus, which is also used in smallpox vaccines. In recent years, newer monkeypox vaccines have been developed based on other viruses such as Modified Vaccinia Ankara (MVA). These newer vaccines are safer and can provide longer-lasting immunity with fewer side effects. For the future, there is ongoing research to improve the current vaccines and to develop new ones. One notable advance has been the development of a recombinant vaccine that uses a genetically modified vaccinia virus to express monkeypox antigens. This vaccine has shown promising results in pre-clinical trials and is currently undergoing further testing in clinical trials. Another recent development has been the use of a DNA vaccine, which delivers genetic material encoding monkeypox antigens directly into cells. This type of vaccine has shown effectiveness in animal studies and is also undergoing clinical testing in humans. Overall, these recent advances in monkeypox vaccine development hold promise for protecting individuals against this potentially serious disease.

Monkeypox Diagnosis in Clinical Settings: A Comprehensive Review of Best Laboratory Practices.

An outbreak of monkeypox (Mpox) was reported in more than 40 countries in early 2022. Accurate diagnosis of Mpox can be challenging, but history, clinical findings, and laboratory diagnosis can establish the diagnosis. The pre-analytic phase of testing includes collecting, storing, and transporting specimens. It is advised to swab the lesion site with virus transport medium (VTM) containing Dacron or polyester flock swabs from two different sites. Blood, urine, and semen samples may also be used. Timely sampling is necessary to obtain a sufficient amount of virus or antibodies. The analytical phase of infectious disease control involves diagnostic tools to determine the presence of the virus. While polymerase chain reaction (PCR) is the gold standard for detecting Mpox, genome sequencing is for identifying new or modified viruses. As a complement to these methods, isothermal amplification methods have been designed. ELISA assays are also available for the determination of antibodies. Electron microscopy is another effective diagnostic method for tissue identification of the virus. Wastewater fingerprinting provides some of the most effective diagnostic methods for virus identification at the community level. The advantages and disadvantages of these methods are further discussed. Post-analytic phase requires proper interpretation of test results and the preparation of accurate patient reports that include relevant medical history, clinical guidelines, and recommendations for follow-up testing or treatment.

Treatment and Vaccination for Smallpox and Monkeypox.

The smallpox infection with the variola virus was one of the most fatal disorders until a global eradication was initiated in the twentieth century. The last cases were reported in Somalia 1977 and as a laboratory infection in the UK 1978; in 1980, the World Health Organization (WHO) declared smallpox for extinct. The smallpox virus with its very high transmissibility and mortality is still a major biothreat, because the vaccination against smallpox was stopped globally in the 1980s. For this reason, new antivirals (cidofovir, brincidofovir, and tecovirimat) and new vaccines (ACAM2000, LC16m8 and Modified Vaccine Ankara MVA) were developed. For passive immunization, vaccinia immune globulin intravenous (VIGIV) is available. Due to the relationships between orthopox viruses such as vaccinia, variola, mpox (monkeypox), cowpox, and horsepox, the vaccines (LC16m8 and MVA) and antivirals (brincidofovir and tecovirimat) could also be used in the mpox outbreak with positive preliminary data. As mutations can result in drug resistance against cidofovir or tecovirimat, there is need for further research. Further antivirals (NIOCH-14 and ST-357) and vaccines (VACΔ6 and TNX-801) are being developed in Russia and the USA. In conclusion, further research for treatment and prevention of orthopox infections is needed and is already in progress. After a brief introduction, this chapter presents the smallpox and mpox disease and thereafter full overviews on antiviral treatment and vaccination including the passive immunization with vaccinia immunoglobulins.

Current Status of Vaccine Development for Monkeypox Virus.

Monkeypox virus (MPXV) of poxviridae family causes a zoonotic disease called monkeypox (Mpox). MPXV cases have a fatality ratio ranging from 0 to 11% globally and have been more prevalent in children. There are three generations of smallpox vaccines that protect against MPXV. First and second generation of the vaccinia virus (VACV) vaccine protects MPXV. However, various adverse side effects were associated with the first and second generations of vaccines. In contrast, the Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN) replication-incompetent vaccine shows fewer adverse effects and a significant amount of neutralizing antibodies in mammalian cells. A third-generation Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN) was approved to prevent Mpox in 2019. Recently, MVA-BN-based Imvanex, Imvamune, and JYNNEOS vaccines have also been administered against MPXV. Globally, the World Health Organization (WHO) declared a global health emergency in May 2022 due to increased MPXV cases. Various computational studies have also designed a multi-epitope-based vaccine against the MPXV. In the multi-epitope-based vaccine, different epitopes like B-cell, Cytotoxic T Lymphocyte (CTL), CD8+, and CD4+ epitopes were derived from MPXV proteins. Further, these epitopes were linked with the help of various linkers to design a multi-epitope vaccine against MPXV. In summary, we have provided an overview of the current status of the vaccine against MPXV.