Background/Objectives: Monkeypox (mpox) is currently the most common orthopoxvirus (OPXV) zoonotic disease, and, since 2022, there has been atypical person-to-person transmission observed in non-endemic countries. The present study aimed to investigate the frequency of monkeypox virus (MPXV) and OPXV DNA detection in recommended and alternative clinical materials taken during the acute and convalescent phases of infection in Bulgarian patients. Methods: The study included laboratory investigation by real time PCR of 181 clinical samples from 42 Bulgarian patients with possible mpox infections. Results: MPXV DNA was detected in 23/181 (12.71%), and OPXV DNA in 20/181 (11.05%) clinical samples. There were six mpox-confirmed patients aged 23 to 44. At the highest frequency, MPXV and OPXV DNA were detected in samples of vesicular contents (6/6) and nasal/oropharyngeal secretions (5/6 and 4/6) during the first three days from the appearance of clinical symptoms. We demonstrated MPXV and OPXV DNA in alternative samples (urine, feces, ejaculate, and saliva), and in follow-up patient samples, taken two weeks after mpox confirmation in the convalescent phase (vesicular contentsand urine). Conclusions: Our findings suggested that MPXV may be detected in a larger set of clinical materials, including alternatives, where the virus can persist for more than two weeks.

The vaccinia virus (VACV) has been used for prophylactic immunization against smallpox for many decades. However, the VACV-based vaccine had been highly reactogenic. Therefore, after the eradication of smallpox, the World Health Organization in 1980 recommended that vaccination against this infection be discontinued. As a result, there has been a rise in the occurrence of orthopoxvirus infections in humans in recent years, with the most severe being the 2022 monkeypox epidemic that reached all continents. Thus, it is crucial to address the pressing matter of developing safe and highly immunogenic vaccines for new generations to combat orthopoxvirus infections. In a previous study, we created a LAD strain by modifying the LIVP (L) VACV strain, which is used as a first-generation smallpox vaccine in Russia. This modification involved introducing mutations in the A34R gene to enhance extracellular virion production and deleting the A35R gene to counteract the antibody response to the viral infection. In this study, a strain LADA was created with an additional deletion in the DNA of the LAD strain ati gene. This ati gene directs the production of a major non-virion immunogen. The findings indicate that the LADA VACV variant exhibits lower levels of reactogenicity in BALB/c mice during intranasal infection, as compared to the original L strain. Following intradermal immunization with a 105 PFU dose, both the LAD and LADA strains were found to induce a significantly enhanced cellular immune response in mice when compared to the L strain. At the same time, the highest level of virus-specific IFN-γ producing cells for the LAD variant was detected on the 7th day post-immunization (dpi), whereas for LADA, it was observed on 14 dpi. The LAD and LADA strains induced significantly elevated levels of VACV-specific IgG compared to the original L strain, particularly between 28 and 56 dpi. The vaccinated mice were intranasally infected with the cowpox virus at a dose of 460 LD50 to assess the protective immunity at 62 dpi. The LADA virus conferred complete protection to mice, with the LAD strain providing 70% protection and the parent strain L offering protection to only 60% of the animals.

The evolutionary pressures exerted by viral infections have led to the development of various cellular proteins with potent antiviral activities, some of which are known as antiviral restriction factors. TRIpartite Motif-containing protein 5 alpha (TRIM5α) is a well-studied restriction factor of retroviruses that exhibits virus- and host-species-specific functions in protecting against cross-primate transmission of specific lentiviruses. This specificity is achieved at the level of the host gene through positive selection predominantly within its C-terminal B30.2/PRYSPRY domain, which is responsible for the highly specific recognition of retroviral capsids. However, more recent work has challenged this paradigm, demonstrating TRIM5α as a restriction factor for retroelements as well as phylogenetically distinct viral families, acting similarly through the recognition of viral gene products via B30.2/PRYSPRY. This spectrum of antiviral activity raises questions regarding the genetic and structural plasticity of this protein as a mediator of the recognition of a potentially diverse array of viral molecular patterns. This review highlights the dynamic evolutionary footprint of the B30.2/PRYSPRY domain in response to retroviruses while exploring the guided \'specificity\' conferred by the totality of TRIM5α\'s additional domains that may account for its recently identified promiscuity.

The basis for criteria of the taxonomic classification of DNA and RNA viruses based on data of the genomic sequencing are viewed in this review. The genomic sequences of viruses, which have genome represented by double-stranded DNA (orthopoxviruses as example), positive-sense single-stranded RNA (alphaviruses and flaviviruses as example), non-segmented negative-sense single-stranded RNA (filoviruses as example), segmented negative-sense single-stranded RNA (arenaviruses and phleboviruses as example) are analyzed. The levels of genetic variability that determine the assignment of compared viruses to taxa of various orders are established for each group of viruses.
В обзоре рассмотрено обоснование критериев идентификации таксономической принадлежности некоторых групп патогенных ДНК- и РНК-содержащих вирусов на основе результатов секвенирования генома. Проанализированы данные секвенирования геномной нуклеиновой кислоты вирусов, геном которых представлен двухцепочечной ДНК (на примере ортопоксвирусов), одноцепочечной «плюс» РНК (на примере альфавирусов и флавивирусов), одноцепочечной несегментированной «минус» РНК (на примере филовирусов), одноцепочечной сегментированной «минус» РНК (на примере аренавирусов и флебовирусов). Для каждой группы вирусов установлены уровни генетической изменчивости, определяющие отнесение сравниваемых вирусов к таксонам разных порядков.

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.

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.

Monkeypox (now Mpox), a zoonotic disease caused by the monkeypox virus (MPXV) is an emerging threat to global health. In the time span of only six months, from May to October 2022, the number of MPXV cases breached 80,000 and many of the outbreaks occurred in locations that had never previously reported MPXV. Currently there are no FDA-approved MPXV-specific vaccines or treatments, therefore, finding drugs to combat MPXV is of utmost importance. The A42R profilin-like protein of the MPXV is involved in cell development and motility making it a critical drug target. A42R protein is highly conserved across orthopoxviruses, thus A42R inhibitors may work for other family members. This study sought to identify potential A42R inhibitors for MPXV treatment using computational approaches. The energy minimized 3D structure of the A42R profilin-like protein (PDB ID: 4QWO) underwent virtual screening using a library of 36,366 compounds from Traditional Chinese Medicine (TCM), AfroDb, and PubChem databases as well as known inhibitor tecovirimat via AutoDock Vina. A total of seven compounds comprising PubChem CID: 11371962, ZINC000000899909, ZINC000001632866, ZINC000015151344, ZINC000013378519, ZINC000000086470, and ZINC000095486204, predicted to have favorable binding were shortlisted. Molecular docking suggested that all seven proposed compounds have higher binding affinities to A42R (-7.2 to -8.3 kcal/mol) than tecovirimat (-6.7 kcal/mol). This was corroborated by MM/PBSA calculations, with tecovirimat demonstrating the highest binding free energy of -68.694 kJ/mol (lowest binding affinity) compared to the seven shortlisted compounds that ranged from -73.252 to -97.140 kJ/mol. Furthermore, the 7 compounds in complex with A42R demonstrated higher stability than the A42R-tecovirimat complex when subjected to 100 ns molecular dynamics simulations. The protein-ligand interaction maps generated using LigPlot+ suggested that residues Met1, Glu3, Trp4, Ile7, Arg127, Val128, Thr131, and Asn133 are important for binding. These seven compounds were adequately profiled to be potential antivirals via PASS predictions and structural similarity searches. All seven potential lead compounds were scored Pa > Pi for antiviral activity while ZINC000001632866 and ZINC000015151344 were predicted as poxvirus inhibitors with Pa values of 0.315 and 0.215, and Pi values of 0.052 and 0.136, respectively. Further experimental validations of the identified lead compounds are required to corroborate their predicted activity. These seven identified compounds represent solid footing for development of antivirals against MPXV and other orthopoxviruses.

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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).

Orthopoxviruses (OPVs), including the causative agents of smallpox and mpox have led to devastating outbreaks in human populations worldwide. However, the discontinuation of smallpox vaccination, which also provides cross-protection against related OPVs, has diminished global immunity to OPVs more broadly. We apply machine learning models incorporating both host ecological and viral genomic features to predict likely reservoirs of OPVs. We demonstrate that incorporating viral genomic features in addition to host ecological traits enhanced the accuracy of potential OPV host predictions, highlighting the importance of host-virus molecular interactions in predicting potential host species. We identify hotspots for geographic regions rich with potential OPV hosts in parts of southeast Asia, equatorial Africa, and the Amazon, revealing high overlap between regions predicted to have a high number of potential OPV host species and those with the lowest smallpox vaccination coverage, indicating a heightened risk for the emergence or establishment of zoonotic OPVs. Our findings can be used to target wildlife surveillance, particularly related to concerns about mpox establishment beyond its historical range.