What the pox? Review of poxviruses affecting humans.

The re-emergence of human mpox with the multi-country outbreak and a recent report of borealpox (previously Alaskapox) resulting in one death has heightened awareness of the significance of the Poxviridae family and their zoonotic potential. This review examines various poxviruses affecting humans, with discussion of less commonly encountered Poxviridae members, including pathogenesis, epidemiology, and diagnostic methods. Poxvirus treatment is beyond the intended scope of this review and will not be discussed.

Tanapox Virus and Yaba Monkey Tumor Virus K3 Orthologs Inhibit Primate Protein Kinase R in a Species-Specific Fashion.

Yaba monkey tumor virus (YMTV) and Tanapox virus (TPV) are members of the Yatapoxvirus genus and can infect humans and other primates. Despite the threat posed by yatapoxviruses, the factors determining their host range are poorly understood. In this study, we analyzed the ability of YMTV and TPV orthologs of vaccinia virus K3 (called 012 in YMTV and TPV), which share 75% amino acid identity with one another, to inhibit PKR from 15 different primate species. We first used a luciferase-based reporter, and found that YMTV and TPV K3 orthologs inhibited PKR in a species-specific manner and showed distinct PKR inhibition profiles. TPV 012 inhibited PKR from 11 primates, including humans, substantially better than YMTV 012. In contrast, both K3 orthologs inhibited the other four primate PKRs comparably well. Using YMTV 012 and TPV 012 hybrids, we mapped the region responsible for the differential PKR inhibition to the C- terminus of the K3 orthologs. Next, we generated chimeric vaccinia virus strains to investigate whether TPV K3 and YMTV K3 orthologs could rescue the replication of a vaccinia virus strain that lacks PKR inhibitors K3L and E3L. Virus replication in primate-derived cells generally correlated with the patterns observed in the luciferase-based assay. Together, these observations demonstrate that yatapoxvirus K3 orthologs have distinct PKR inhibition profiles and inhibit PKR in a species-specific manner, which may contribute to the differential susceptibility of primate species to yatapoxvirus infections.

Formulation of next-generation polyvalent vaccine candidates against three important poxviruses by targeting DNA-dependent RNA polymerase using an integrated immunoinformatics and molecular modeling approach.

BACKGROUND: Poxviruses comprise a group of large double-stranded DNA viruses and are known to cause diseases in humans, livestock animals, and other animal species. The Mpox virus (MPXV; formerly Monkeypox), variola virus (VARV), and volepox virus (VPXV) are among the prevalent poxviruses of the Orthopoxviridae genera. The ongoing Mpox infectious disease pandemic caused by the Mpox virus has had a major impact on public health across the globe. To date, only limited repurposed antivirals and vaccines are available for the effective treatment of Mpox and other poxviruses that cause contagious diseases. METHODS: The present study was conducted with the primary goal of formulating multi-epitope vaccines against three evolutionary closed poxviruses i.e., MPXV, VARV, and VPXV using an integrated immunoinformatics and molecular modeling approach. DNA-dependent RNA polymerase (DdRp), a potential vaccine target of poxviruses, has been used to determine immunodominant B and T-cell epitopes followed by interactions analysis with Toll-like receptor 2 at the atomic level. RESULTS: Three multi-epitope vaccine constructs, namely DdRp_MPXV (V1), DdRp_VARV (V2), and DdRp_VPXV (V3) were designed. These vaccine constructs were found to be antigenic, non-allergenic, non-toxic, and soluble with desired physicochemical properties. Protein-protein docking and interaction profiling analysis depicts a strong binding pattern between the targeted immune receptor TLR2 and the structural models of the designed vaccine constructs, and manifested a number of biochemical bonds (hydrogen bonds, salt bridges, and non-bonded contacts). State-of-the-art all-atoms molecular dynamics simulations revealed highly stable interactions of vaccine constructs with TLR2 at the atomic level throughout the simulations on 300 nanoseconds. Additionally, the outcome of the immune simulation analysis suggested that designed vaccines have the potential to induce protective immunity against targeted poxviruses. CONCLUSIONS: Taken together, formulated next-generation polyvalent vaccines were found to have good efficacy against closely related poxviruses (MPXV, VARV, and VPXV) as demonstrated by our extensive immunoinformatics and molecular modeling evaluations; however, further experimental investigations are still needed.

Novel strategy for Poxviridae prevention: Thermostable combined subunit vaccine patch with intense immune response.

Poxviruses gained international attention due to the sharp rise in monkeypox cases in recent years, highlighting the urgent need for the development of a secure and reliable vaccine. This study involved the development of an innovative combined subunit vaccine (CSV) targeting poxviruses, with lumpy skin disease virus (LSDV) serving as the model virus. To this end, the potential sites for poxvirus vaccines were fully evaluated to develop and purify four recombinant proteins. These proteins were then successfully delivered to the dermis in a mouse model by utilizing dissolvable microneedle patches (DMPs). This approach simplified the vaccination procedure and significantly mitigated the associated risk. CSV-loaded DMPs contained four recombinant proteins and a novel adjuvant, CpG, which allowed DMPs to elicit the same intensity of humoral and cellular immunity as subcutaneous injection. Following immunization with SC and DMP, the mice exhibited notable levels of neutralizing antibodies, albeit at a low concentration. It is noteworthy that the CSV loaded into DMPs remained stable for at least 4 months at room temperature, effectively addressing the storage and transportation challenges. Based on the study findings, CSV-loaded DMPs are expected to be utilized worldwide as an innovative technique for poxvirus inoculation, especially in underdeveloped regions. This novel strategy is crucial for the development of future poxvirus vaccines.

Role of the Laboratory in the Diagnosis of Poxvirus Infections.

Although WHO-led global efforts led to eradication of smallpox over four decades ago, other poxviruses, especially monkeypox, have re-emerged to occupy the ecological niche vacated by smallpox. Many of these viruses produce similar lesions thus mandating a prompt laboratory confirmation. There has been considerable evolution in the techniques available to diagnose these infections and differentiate between them. With the 2022 multi-country outbreak of monkeypox, significant efforts were made to apprise the laboratory diagnosis of the virus and numerous real-time-PCR-based assays were made commercially available. This chapter discusses the sample collection and biosafety aspects along with the repertoire of diagnostic modalities, both traditional and emerging, for poxviruses which a special focus on monkeypox. The advantages and disadvantages of each technique have been illustrated. We have also reflected upon the newer advances and the existing lacunae.

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.

Natural Immunomodulatory Agents as a Complementary Therapy for Poxviruses.

Poxviruses target innate immunity mediators such as tumor necrosis factors, interleukins, interferons, complement, and chemokines. It also targets adaptive immunity such as CD4+ T cells, CD4+ T cells, and B cells. Emerging of the recent epidemic of monkeypox virus (MPXV), a zoonotic disease native to Central and Western Africa, besides the lack of permitted treatments for poxviruses infections, encouraged researchers to identify effective inhibitors to help in preventing and treating poxviruses infections. Natural bioactive components, particularly polyphenolics, are promising for creating powerful antioxidants, anti-inflammatory, immune-stimulating, and antiviral agents. As a result, they are potentially effective therapies for preventing and treating viral diseases, such as infections caused by poxviruses including the recent pandemic MPXV. Polyphenolics: rosmarinic acid, caffeic acid, resveratrol, quercitrin, myricitrin, gingerol, gallotannin, and propolis-benzofuran A, as well as isoquinoline alkaloids: galanthamine and thalimonine represent prospective antiviral agents against MPXV, they can inhibit MPXV and other poxviruses via targeting different viral elements including DNA Topoisomerase I (TOP1), Thymidine Kinase (TK), serine/threonine protein kinase (Ser/Thr kinase), and protein A48R. The bioactive extracts of different traditional plants including Guiera senegalensis, Larrea tridentata, Sarracenia purpurea, Kalanchoe pinnata (Lam.) Pers., Zingiber officinale Roscoe, Quercus infectoria, Rhus chinensis, Prunella vulgaris L., Salvia rosmarinus, and Origanum vulgare also can inhibit the growth of different poxviruses including MPXV, vaccinia virus (VACV), variola virus, buffalopox virus, fowlpox virus, and cowpox virus. There is an urgent need for additional molecular studies to identify and confirm the anti-poxviruses properties of various natural bioactive components, especially those that showed potent antiviral activity against other viruses.

The Overview of Potential Antiviral Bioactive Compounds in Poxviruses.

Poxviruses belong to the family of double-stranded DNA viruses, and it is pathogenic for humans and spread worldwide. These viruses cause infections and various diseases in human. So, it is required to develop new drugs for the treatment of smallpox or other poxvirus infections. Very few potential compounds for the treatment of poxvirus such as smallpox, chickenpox, and monkeypox have been reported. Most of the compounds has used as vaccines. Cidofovir is most commonly used as a vaccine for the treatment of poxviruses. There are no phytochemicals reported for the treatment of poxviruses. Very few phytochemicals are under investigation for the treatment of poxviruses.

Stressing out-carp edema virus induces stress and modulates immune response in common carp.

Introduction: Carp edema virus (CEV) is a fish poxvirus that primarily infects the gills of common carp. CEV causes koi sleepy disease (KSD), which is highly contagious and can result in mortality of up to 100%. Methods: In the present study, we analyzed the stress and immune responses during KSD in two strains of common carp with different resistance to CEV: susceptible koi and resistant Amur sazan. Experiments were performed at two temperatures: 12°C and 18°C. In the case of koi carp, we also analyzed the effect of supplementation of 0.6% NaCl into tank water, which prevents mortality of the CEV-infected fish (salt rescue model). Results: We found that CEV-infected koi kept at 18°C had the highest viral load, which correlated with the most severe histopathological changes in the gills. CEV infection resulted in the activation of stress response reflected by the upregulated expression of genes involved in stress response in the stress axis organs and increased levels of cortisol and glucose in the blood plasma. These changes were the most pronounced in CEV-infected koi kept at 18°C. At both temperatures, the activation of antiviral immune response was observed in koi kept under freshwater and NaCl conditions upon CEV infection. Interestingly, a clear downregulation of the expression of adaptive immune genes was observed in CEV-infected koi kept under freshwater at 18°C. Conclusion: CEV induces a stress response and modulates adaptive immune response in koi, and this is correlated with the level of viral load and disease development.

Identification of a Potential Entry-Fusion Complex Based on Sequence Homology of African Swine Fever and Vaccinia Virus.

African swine fever virus (ASFV) belongs to the family of Asfarviridae, part of the group of nucleocytoplasmic large DNA viruses (NCLDV). Little is known about the internalization of ASFV in the host cell and the fusion membrane events that take place at early stages of the infection. Poxviruses, also members of the NCLDV and represented by vaccinia virus (VACV), are large, enveloped, double-stranded DNA viruses. Poxviruses were considered unique in having an elaborate entry-fusion complex (EFC) composed of 11 highly conserved proteins integrated into the membrane of mature virions. Recent advances in methodological techniques have again revealed several connections between VACV EFC proteins. In this study, we explored the possibility of an analogous ASFV EFC by identifying ten candidate proteins exhibiting structural similarities with VACV EFC proteins. This could reveal key functions of these ASFV proteins, drawing attention to shared features between the two virus families, suggesting the potential existence of an ASFV entry-fusion complex.

The Global Health Threat of Monkeypox Virus: Understanding Its Biology, Transmission, and Potential Therapeutic Interventions.

Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, shares its genus with Variola virus (VARV), the causative agent of smallpox, and Vaccinia virus (VACV), used for smallpox vaccination. While smallpox has been eradicated, MPXV and related poxviruses continue to pose a global health threat. Monkeypox (Mpox), similar in clinical presentation to smallpox but milder, is endemic in Central and West Africa. Sporadic outbreaks emphasize the potential for wider dissemination. Understanding their biology, transmission, immune evasion, and clinical features informs disease control strategies. The intersection of medical innovation and biotechnology with poxviruses underscores their importance in both disease and scientific advancement. Further research is essential to enhance prevention, management, and therapeutic interventions for these viruses.

Approaches to Evaluating Necroptosis in Virus-Infected Cells.

The immune system functions to protect the host from pathogens. To counter host defense mechanisms, pathogens have developed unique strategies to evade detection or restrict host immune responses. Programmed cell death is a major contributor to the multiple host responses that help to eliminate infected cells for obligate intracellular pathogens like viruses. Initiation of programmed cell death pathways during the early stages of viral infections is critical for organismal survival as it restricts the virus from replicating and serves to drive antiviral inflammation immune recruitment through the release of damage-associated molecular patterns (DAMPs) from the dying cell. Necroptosis has been implicated as a critical programmed cell death pathway in a diverse set of diseases and pathological conditions including acute viral infections. This cell death pathway occurs when certain host sensors are triggered leading to the downstream induction of mixed-lineage kinase domain-like protein (MLKL). MLKL induction leads to cytoplasmic membrane disruption and subsequent cellular destruction with the release of DAMPs. As the role of this cell death pathway in human disease becomes apparent, methods identifying necroptosis patterns and outcomes will need to be further developed. Here, we discuss advances in our understanding of how viruses counteract necroptosis, methods to quantify the pathway, its effects on viral pathogenesis, and its impact on cellular signaling.

Evolution and diversity of nucleotide and dinucleotide composition in poxviruses.

Poxviruses (family Poxviridae) have long dsDNA genomes and infect a wide range of hosts, including insects, birds, reptiles and mammals. These viruses have substantial incidence, prevalence and disease burden in humans and in other animals. Nucleotide and dinucleotide composition, mostly CpG and TpA, have been largely studied in viral genomes because of their evolutionary and functional implications. We analysed here the nucleotide and dinucleotide composition, as well as codon usage bias, of a set of representative poxvirus genomes, with a very diverse host spectrum. After correcting for overall nucleotide composition, entomopoxviruses displayed low overall GC content, no enrichment in TpA and large variation in CpG enrichment, while chordopoxviruses showed large variation in nucleotide composition, no obvious depletion in CpG and a weak trend for TpA depletion in GC-rich genomes. Overall, intergenome variation in dinucleotide composition in poxviruses is largely accounted for by variation in overall genomic GC levels. Nonetheless, using vaccinia virus as a model, we found that genes expressed at the earliest times in infection are more CpG-depleted than genes expressed at later stages. This observation has parallels in betahepesviruses (also large dsDNA viruses) and suggests an antiviral role for the innate immune system (e.g. via the zinc-finger antiviral protein ZAP) in the early phases of poxvirus infection. We also analysed codon usage bias in poxviruses and we observed that it is mostly determined by genomic GC content, and that stratification after host taxonomy does not contribute to explaining codon usage bias diversity. By analysis of within-species diversity, we show that genomic GC content is the result of mutational biases. Poxvirus genomes that encode a DNA ligase are significantly AT-richer than those that do not, suggesting that DNA repair systems shape mutation biases. Our data shed light on the evolution of poxviruses and inform strategies for their genetic manipulation for therapeutic purposes.

Monkeypox Virus: A Comprehensive Overview of Viral Pathology, Immune Response, and Antiviral Strategies.

BACKGROUND: The years 2022-2023 witnessed a monkeypox virus (mpox) outbreak in some countries worldwide, where it exists in an endemic form. However, the number of infectious cases is continuously on the rise, and there has been an unexpected, drastic increase in cases that result from sustained transmission in non-endemic regions of the world. Under this scenario, it is pertinent for the world to be aware of healthcare threats to mpox infection. This review aimed to compile advanced data regarding the different aspects of mpox disease. METHODS: A comprehensive strategy for the compilation of recent data was adopted to add data regarding mpox, biology, viral pathology, immune response, and brief details on the antiviral strategies under trial; the search was limited to 2016-2023. The aim is to make the scientific community aware of diverse aspects of mpox. RESULTS: Consequently, detailed insights have been drawn with regard to the nature, epidemiology, etiology, and biological nature of mpox. Additionally, its host interaction and viral infectious cycle and immune interventions have been briefly elaborated. This comprehensively drawn literature review delivers brief insights into the biological nature, immune responses, and clinical developments in the form of therapeutics against mpox. This study will help scientists understand the biological nature and responses in hosts, which will further help clinicians with therapeutic handling, diagnosis, and treatment options. CONCLUSIONS: This study will provide updated information on mpox's pathology, immune responses, and antiviral strategies. Moreover, it will also help the public to become educated on the healthcare-associated threat and take timely mitigation measures against expected mpox outbreaks in the future.

Highly Attenuated Poxvirus-Based Vaccines Against Emerging Viral Diseases.

Although one member of the poxvirus family, variola virus, has caused one of the most devastating human infections worldwide, smallpox, the knowledge gained over the last 30 years on the molecular, virological and immunological mechanisms of these viruses has allowed the use of members of this family as vectors for the generation of recombinant vaccines against numerous pathogens. In this review, we cover different aspects of the history and biology of poxviruses with emphasis on their application as vaccines, from first- to fourth-generation, against smallpox, monkeypox, emerging viral diseases highlighted by the World Health Organization (COVID-19, Crimean-Congo haemorrhagic fever, Ebola and Marburg virus diseases, Lassa fever, Middle East respiratory syndrome and severe acute respiratory syndrome, Nipah and other henipaviral diseases, Rift Valley fever and Zika), as well as against one of the most concerning prevalent virus, the Human Immunodeficiency Virus, the causative agent of Acquired Immunodeficiency Syndrome. We discuss the implications in human health of the 2022 monkeypox epidemic affecting many countries, and the rapid prophylactic and therapeutic measures adopted to control virus dissemination within the human population. We also describe the preclinical and clinical evaluation of the Modified Vaccinia virus Ankara and New York vaccinia virus poxviral strains expressing heterologous antigens from the viral diseases listed above. Finally, we report different approaches to improve the immunogenicity and efficacy of poxvirus-based vaccine candidates, such as deletion of immunomodulatory genes, insertion of host-range genes and enhanced transcription of foreign genes through modified viral promoters. Some future prospects are also highlighted.

T helper 1 effector memory CD4+ T cells protect the skin from poxvirus infection.

Poxvirus infections of the skin are a recent emerging public health concern, yet the mechanisms that mediate protective immunity against these viral infections remain largely unknown. Here, we show that T helper 1 (Th1) memory CD4+ T cells are necessary and sufficient to provide complete and broad protection against poxvirus skin infections, whereas memory CD8+ T cells are dispensable. Core 2 O-glycan-synthesizing Th1 effector memory CD4+ T cells rapidly infiltrate the poxvirus-infected skin microenvironment and produce interferon γ (IFNγ) in an antigen-dependent manner, causing global changes in gene expression to promote anti-viral immunity. Keratinocytes express IFN-stimulated genes, upregulate both major histocompatibility complex (MHC) class I and MHC class II antigen presentation in an IFNγ-dependent manner, and require IFNγ receptor (IFNγR) signaling and MHC class II expression for memory CD4+ T cells to protect the skin from poxvirus infection. Thus, Th1 effector memory CD4+ T cells exhibit potent anti-viral activity within the skin, and keratinocytes are the key targets of IFNγ necessary for preventing poxvirus infection of the epidermis.

Cowpox Viruses: A Zoo Full of Viral Diversity and Lurking Threats.

Cowpox viruses (CPXVs) exhibit the broadest known host range among the Poxviridae family and have caused lethal outbreaks in various zoo animals and pets across 12 Eurasian countries, as well as an increasing number of human cases. Herein, we review the history of how the cowpox name has evolved since the 1700s up to modern times. Despite early documentation of the different properties of CPXV isolates, only modern genetic analyses and phylogenies have revealed the existence of multiple Orthopoxvirus species that are currently constrained under the CPXV designation. We further chronicle modern outbreaks in zoos, domesticated animals, and humans, and describe animal models of experimental CPXV infections and how these can help shaping CPXV species distinctions. We also describe the pathogenesis of modern CPXV infections in animals and humans, the geographic range of CPXVs, and discuss CPXV-host interactions at the molecular level and their effects on pathogenicity and host range. Finally, we discuss the potential threat of these viruses and the future of CPXV research to provide a comprehensive review of CPXVs.

Arabinofuranosyl Thymine Derivatives-Potential Candidates against Cowpox Virus: A Computational Screening Study.

Cowpox is caused by a DNA virus known as the cowpox virus (CPXV) belonging to the Orthopoxvirus genus in the family Poxviridae. Cowpox is a zoonotic disease with the broadest host range among the known poxviruses. The natural reservoir hosts of CPXV are wild rodents. Recently, the cases of orthopoxviral infections have been increasing worldwide, and cowpox is considered the most common orthopoxviral infection in Europe. Cowpox is often a self-limiting disease, although cidofovir or anti-vaccinia gammaglobulin can be used in severe and disseminated cases of human cowpox. In this computational study, a molecular docking analysis of thymine- and arabinofuranosyl-thymine-related structures (1-21) on two cowpox-encoded proteins was performed with respect to the cidofovir standard and a 3D ligand-based pharmacophore model was generated. Three chemical structures (PubChem IDs: 123370001, 154137224, and 90413364) were identified as potential candidates for anti-cowpox agents. Further studies combining in vitro and in silico molecular dynamics simulations to test the stability of these promising compounds could effectively improve the future design of cowpox virus inhibitors, as molecular docking studies are not sufficient to consider a ligand a potential drug.

Molecular Mechanisms of Poxvirus Evolution.

Poxviruses are often thought to evolve relatively slowly because they are double-stranded DNA pathogens with proofreading polymerases. However, poxviruses have highly adaptable genomes and can undergo relatively rapid genotypic and phenotypic change, as illustrated by the recent increase in human-to-human transmission of monkeypox virus. Advances in deep sequencing technologies have demonstrated standing nucleotide variation in poxvirus populations, which has been underappreciated. There is also an emerging understanding of the role genomic architectural changes play in shaping poxvirus evolution. These mechanisms include homologous and nonhomologous recombination, gene duplications, gene loss, and the acquisition of new genes through horizontal gene transfer. In this review, we discuss these evolutionary mechanisms and their potential roles for adaption to novel host species and modulating virulence.

Is monkeypox a new, emerging sexually transmitted disease? A rapid review of the literature.

Monkeypox, a milder disease compared to smallpox, is caused by a virus initially discovered and described in 1958 by the prominent Danish virologist von Magnus, who was investigating an infectious outbreak affecting monkey colonies. Currently, officially starting from May 2022, an outbreak of monkeypox is ongoing, with 51 000 cases being notified as of September 1, 2022-51 408 confirmed, 28 suspected, and 12 fatalities, for a grand total of 51 448 cases. More than 100 countries and territories are affected, from all the six World Health Organization regions. There are some striking features, that make this outbreak rather unusual when compared with previous outbreaks, including a shift on average age and the most affected age group, affected sex/gender, risk factors, clinical course, presentation, and the transmission route. Initially predominantly zoonotic, with an animal-to-human transmission, throughout the last decades, human-to-human transmission has become more and more sustained and effective. In particular, clusters of monkeypox have been described among men having sex with men, some of which have been epidemiologically linked to international travel to nonendemic countries and participation in mass gathering events/festivals, like the "Maspalomas (Gran Canaria) 2022 pride." This review will specifically focus on the "emerging" transmission route of the monkeypox virus, that is to say, the sexual transmission route, which, although not confirmed yet, seems highly likely in the diffusion of the infectious agent.