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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.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

The emergence of monkeypox has become a global health threat after the COVID‐19 pandemic. Due to the lack of available specifically treatment against MPV, developing an available vaccine is thus the most prospective and urgent strategy. Herein, a programmable macrophage vesicle based bionic self‐adjuvanting vaccine (AM@AEvs‐PB) is first developed for defending against monkeypox virus (MPV). Based on MPV‐related antigen‐stimulated macrophage‐derived vesicles, the nanovaccine is constructed by loading the mature virion (MV)‐related intracellular protein (A29L/M1R) and simultaneously modifying with the enveloped virion (EV) antigen (B6R), enabling them to effectively promote antigen presentation and enhance adaptive immune through self‐adjuvant strategy. Owing to the synergistic properties of bionic vaccine coloaded MV and EV protein in defensing MPV, the activation ratio of antigen‐presenting cells is nearly four times than that of single antigen in the same dose, resulting in stronger immunity in host. Notably, intramuscular injection uptake of AM@AEvs‐PB demonstrated vigorous immune‐protective effects in the mouse challenge attempt, offering a promising strategy for pre‐clinical monkeypox vaccine development. The monkeypox‐specific bionic vaccine (AM@AEvs‐PB) is consists of IMV antigens (A29L, M1R), the EEV antigen (B6R), and MPV‐preactivated macrophagederived vesicles. AM@AEvs‐PB can induce enhanced innate immune responses, promote cross‐presentation of antigens to dendritic cells (DCs), and elicit robust adaptive immune responses, realizing immunization protection against Monkeypox Virus.

Viruses are infectious agents that hijack the host cell machinery in order to replicate and generate progeny. Viral infection is initiated by attachment to host cell receptors, and typical viral receptors are cell-surface-borne molecules such as proteins or glycan structures. Sialylated glycans (glycans bearing sialic acids) and glycosaminoglycans (GAGs) represent major classes of carbohydrate receptors and have been implicated in facilitating viral entry for many viruses. As interactions between viruses and sialic acids have been extensively reviewed in the past, this review provides an overview of the current state of structural knowledge about interactions between non-enveloped human viruses and GAGs. We focus here on adeno-associated viruses, human papilloma viruses (HPVs), and polyomaviruses, as at least some structural information about the interactions of these viruses with GAGs is available. We also discuss the multivalent potential for GAG binding, highlighting the importance of charged interactions and positively charged amino acids at the binding sites, and point out challenges that remain in the field.

Vesicle-encapsulated nonenveloped viruses are a recently recognized alternate form of nonenveloped viruses that can avoid immune detection and potentially increase systemic transmission. Avian orthoreoviruses (ARVs) are the leading cause of various disease conditions among birds and poultry. However, whether ARVs use cellular vesicle trafficking routes for egress and cell-to-cell transmission is still poorly understood. We demonstrated that fusogenic ARV-infected quail cells generated small (~100 nm diameter) extracellular vesicles (EVs) that contained electron-dense material when observed by transmission electron microscope. Cryo-EM tomography indicated that these vesicles did not contain ARV virions or core particles, but the EV fractions of OptiPrep gradients did contain a small percent of the ARV virions released from cells. Western blotting of detergent-treated EVs revealed that soluble virus proteins and the fusogenic p10 FAST protein were contained within the EVs. Notably, virus particles mixed with the EVs were up to 50 times more infectious than virions alone. These results suggest that EVs and perhaps fusogenic FAST-EVs could contribute to ARV virulence.

In 2022, the global outbreak of monkeypox virus (MPXV) with increased human-to-human transmission triggered urgent public health interventions. Plant-derived monoclonal antibodies (mAbs) are being explored as potential therapeutic strategies due to their diverse mechanisms of antiviral activity. MPXV produces two key infectious particles: the mature virion (MV) and the extracellular enveloped virion (EV), both essential for infection and spread. Effective therapies must target both to halt replication and transmission. Our prior research demonstrated the development of a potent neutralizing mAb against MPXV MV. This study focuses on developing a plant-derived mAb targeting MPXV EV, which is critical for viral dissemination within the host and generally resistant to antibody neutralization. Our findings reveal that the mAb (H2) can be robustly produced in Nicotiana benthamiana plants via transient expression. The plant-made H2 mAb effectively targets MPXV EV by binding specifically to the A35 MPXV antigen. Importantly, H2 mAb shows notable neutralizing activity against the infectious MPXV EV particle. This investigation is the first to report the development of a plant-derived anti-EV mAb for MPXV prevention and treatment, as well as the first demonstration of anti-MPXV EV activity by an mAb across any production platform. It highlights the potential of plant-produced mAbs as therapeutics for emerging infectious diseases, including the MPXV outbreak.

Delivering protein therapeutics specifically into target cells and tissues is a promising avenue in medicine. Advancing this process will significantly enhance the efficiency of the designed drugs. In this regard, natural membrane-based systems are of particular interest. Extracellular vesicles (EVs), being the bilayer lipid particles secreted by almost all types of cells, have several principal advantages: biocompatibility, carrier stability, and blood–brain barrier penetrability, which make them a perspective tool for protein therapeutic delivery. Here, we evaluate the engineered genetically encoded EVs produced by a human cell line, which allow efficient cargo loading. In the devised system, the protein of interest is captured by self-assembling structures, i.e., “enveloped protein nanocages” (EPN). In their turn, EPNs are encapsulated in fusogenic EVs by the overexpression of vesicular stomatitis virus G protein (VSV-G). The proteomic profiles of different engineered EVs were determined for a comprehensive evaluation of their therapeutic potential. EVs loading mediated by bio-safe Fos–Jun heterodimerization demonstrates an increased efficacy of active cargo loading and delivery into target cells. Our results emphasize the outstanding technological and biomedical potential of the engineered EV systems, including their application in adoptive cell transfer and targeted cell reprogramming.

Extracellular vesicles (EVs) are mediators of intercellular communication through the transfer of nucleic acids, lipids and proteins between cells. This property makes bioengineered EVs promising therapeutic vectors. However, it remains challenging to isolate EVs with a therapeutic payload due to the heterogeneous nature of cargo loading into EVs. In this study, enrichment of EVs with a desired cargo was possible through engineering of the hallmark CD63 transmembrane protein. E‐NoMi refers to engineered CD63 with mCherry on the inside of the EV membrane and a tag (3xFLAG) exposed on the outside of the EV membrane. To facilitate EV loading during biogenesis, cargo proteins, such as EGFP, Cre recombinase and the CRISPR‐Cas nuclease (SaCas9), were fused to a nanobody (Nb) protein with a high affinity for mCherry. FLAG‐tag‐based immunocapture from cell conditioned media allowed selection of cargo‐loaded E‐NoMi‐EVs, and tobacco etch virus (TEV) protease cleavage sites were used to remove the 3xFLAG‐tag from the surface of E‐NoMi‐EVs after capture. For functional payload delivery to recipient cells, the vesicular stomatitis virus G (VSV‐G) fusogenic protein was incorporated into E‐NoMi‐EVs to form fusogenic EV‐based vectors (EVVs) and proved to be 10‐fold more effective at cargo delivery than EVs generated by size‐exclusion chromatography. Functional delivery of cargo with E‐NoMi‐EVVs was validated in two mouse brain models in vivo.

ABSTRACT Lumpy skin disease virus (LSDV) is a highly transmissible bovine disease caused by a virus belongs to Poxviridae family (genus Capripox). The disease was originally isolated from cattle in Egypt in 1988 and it later became widespread in the most of governorates. Because of the appearance of vaccine-associated disease recently, it is critical to differentiate infected from vaccinated animals (DIVA) strategies. Therefore, the aim of this study was to detect LSDV in suspected clinically diseased cows from 6 herds in Al-Sharqia governorate, Egypt, between May 2021-April 2022. Moreover, to detect whether this infection is due to a field strain or vaccine strain based on partial sequence of the EEV Glycoprotein gene that firstly used in Egypt, for LSDV detection from 2 infected cows and three types of live attenuated vaccines used in Egypt. In all, 42 of the 145 cows displayed characteristic LSD clinical signs in form of spontaneous eruption of many intradermal nodules varied in size and numbers. Conventional PCR was employed for LSDV confirmation as LSDV DNA was identified in 11 out of 12 (91.6%) samples [6/6 (100%) skin nodule biopsies and 5/6 (83.3%) nasal swabs] using EEV Glycoprotein gene. The nucleotide sequences of the EEV Glycoprotein gene of LSDV from 2 diseased cows aligned with those received from Gene Bank demonstrating that, the two detected LSDV were 100% similar and shared high sequence homology with the virulent strain from Egypt 1988; South Africa; Cameron; Kenya and Ein-Zurim/Israel with identities ranging from 99.7 % to 99.8%. Moreover, the nucleotide sequence alignment for LSDVs from 2 diseased cows and Al-Abbasya LSDV vaccine revealed the presence of 27 nucleotides that were absent in Romanian MEVAC-SPV and MEVAC-LSDV. So the conventional PCR targeting the partial EEV Glycoprotein gene is a quick and precise method for identifying LSDV. Also, the Partial sequence of EEV Glycoprotein gene has the ability to perform DIVA approach when use MEVAC LSD vaccine. In contrast it's not capable to do the same on Al-Abbasya LSDV vaccine due to unlikely presence of 27 nucleotides that specific for field strain in this type of vaccine.

Viral vectors and viral vaccines are invaluable tools in prevention and treatment of diseases. Many infectious diseases are controlled using vaccines designed from subunits or whole viral structures, whereas other genetic diseases and cancers are being treated by viruses used as vehicles for delivering genetic material in gene therapy or as therapeutic agents in virotherapy protocols. Viral vectors and vaccines are produced in different platforms, from traditional embryonated chicken eggs to more advanced cell cultures. All these expression systems, like most cells and cellular tissues, are known to spontaneously release extracellular vesicles (EVs). EVs share similar sizes, biophysical characteristics and even biogenesis pathways with enveloped viruses, which are currently used as key ingredients in a number of viral vectors and licensed vaccine products. Herein, we review distinctive features and similarities between EVs and enveloped viruses as we revisit the downstream processing steps and analytical technologies currently implemented to produce and document viral vector and vaccine products. Within a context of well-established viral vector and vaccine safety profiles, this review provides insights on the likely presence of EVs in the final formulation of enveloped virus products and discusses the potential to further resolve and document these components.