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The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2α, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix αG) determined sensitivity to K3. The expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix αG was resistant to wild-type VACV infection and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA.IMPORTANCEThe molecular mechanisms that govern the host range of viruses are incompletely understood. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. The current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters double-stranded (ds) RNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete and that dsRNA sequestration is not the primary mechanism for E3 activity.

The Africa Centers for Disease Control and Prevention declared mpox a Public Health Emergency of Continental Security (PHECS) in Africa. African public health systems have moved to mobilize a response against a backdrop of inherent significant challenges. With this commentary, we discuss how lessons from past public health emergencies, particularly COVID-19 and Ebola outbreaks, have prepared the region for improved disease surveillance, rapid response strategies, and effective public health communication and how these lessons can be applied to the mpox response, emphasizing the importance of strong healthcare infrastructure, effective data sharing, community engagement, targeted interventions, and robust contact tracing. Additionally, addressing misinformation and building public trust are crucial for controlling the spread of any disease. By leveraging these strategies, African countries can enhance their response to mpox. This includes improving diagnostic capabilities, strengthening cross-border collaborations, and prioritizing vaccination campaigns where needed. Ultimately, by applying the hard-earned lessons from the COVID-19 pandemic and Ebola outbreak, the East Africa region can better address the challenges posed by mpox and safeguard public health. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, the endoplasmic reticulum is particularly important in post-translational modification of proteins before they are released extracellularly or sent to another endomembrane system. The correct three-dimensional folding of most proteins occurs in the ER lumen, which has an oxidative environment that is essential for the formation of disulfide bridges, which are important in maintaining protein structure. The ER is a versatile organelle that ensures the correct structure of proteins and is essential in the synthesis of lipids and sterols, in addition to offering support in the maintenance of intracellular calcium. Consequently, the cells needed to respond to demands caused by physiological conditions and pathological disturbances in the organelle homeostasis, leading to proper functioning of the cell or even programmed cell death. Disturbances to the ER function trigger a response to the accumulation of unfolded or misfolded proteins, known as the unfolded protein response. Such disturbances include abiotic stress, pharmacological agents, and intracellular pathogens, such as viruses. When misfolded proteins accumulate in the ER, they can undergo ubiquitination and proteasomal degradation through components of the ER-associated degradation system. Once a prolonged activity of the UPR pathway occurs, indicating that homeostasis cannot be reestablished, components of this pathway induce cell death by apoptosis. Here, we discuss how viruses have evolved ways to counteract UPR responses to maximize replication. This evolutionary viral ability is important to understand cell pathology and should be taken into account when designing therapeutic interventions and vaccines. [ABSTRACT FROM AUTHOR]

Members of the family Poxviridae cause diseases such as smallpox and monkeypox, which are pathogenic to humans and negatively affect animal husbandry and development. The deadly smallpox virus was eradicated by large-scale intensive vaccination with live Vaccinia virus (VACV). However, in recent years, other VACVs have emerged as threats to human and animal health in developing countries. Vaccination is the most effective and widely used means of prevention and control of viral diseases. Different types of vaccines have been used to control poxvirus infection, including traditional attenuated, nucleic acids, recombinant subunits, virus vectors, polypeptides, gene deletions, and inactived vaccines. This review summarizes recent advances in the field of poxvirus vaccine research. It also discusses existing gaps and forecasts potential directions for future research.

Abstract Background Two outbreaks of swinepox were investigated in free-range domestic pig farms located in the northeastern side of Sicily, Italy. The disease is generally self-limiting with a low mortality rate, but morbidity can reach high rates in case of poor sanitary conditions, improper husbandry practices and ectoparasitic infestation. The presented cases are the first ever reported on the island and part of the few cases reported in domestic pigs. Case presentation Carcasses condemned at the slaughterhouse and deceased pigs from Farm A and Farm B respectively, were referred for post-mortem examination and further investigations, with a strong suspect of SwinePox virus (SWPV) infection. Twelve deceased pigs were examined in total, showing poor body condition and pustular lesions scattered all over the cutaneous surfaces. Moreover, pigs from Farm B showed ocular lesions classified from Grade I to IV (from mild conjunctivitis to severe keratoconjunctivitis with corneal oedema, opacity, and ulcers). Final diagnosis was pursued by the microscopic assessment of skin lesions in both farms, which revealed the typical SWPV-lesion appearance, such as severe and disseminated ulcerative dermatitis and suspected inclusion bodies multifocally observed in the epidermis. Moreover, negative staining Electron Microscopy (nsEM) was performed on skin lesions and ocular swabs from Farm B, revealing in two samples the presence of brick-shaped viral particles, 220 nm long and 160 nm wide, with irregularly arranged surface tubules, identified as SWPV. The gene encoding the 482-bp fragment of the virus late transcription factor–3 was detected by PCR and sequencing revealed 99.79% identity and 100% query-cover with a strain previously isolated in Germany. Field clinical assessment was then performed in Farm B, revealing high overcrowding, poor sanitary conditions and improper husbandry practices, which are relevant risk factors for SWPV transmission. Conclusions The present is the first case report of SWPV in free-range pigs raised in Sicily, an island of the Southern coast of Italy, and wants to raise awareness on a neglected disease, and cause of animal health and welfare issues.

Background: Two outbreaks of swinepox were investigated in free-range domestic pig farms located in the northeastern side of Sicily, Italy. The disease is generally self-limiting with a low mortality rate, but morbidity can reach high rates in case of poor sanitary conditions, improper husbandry practices and ectoparasitic infestation. The presented cases are the first ever reported on the island and part of the few cases reported in domestic pigs. Case presentation: Carcasses condemned at the slaughterhouse and deceased pigs from Farm A and Farm B respectively, were referred for post-mortem examination and further investigations, with a strong suspect of SwinePox virus (SWPV) infection. Twelve deceased pigs were examined in total, showing poor body condition and pustular lesions scattered all over the cutaneous surfaces. Moreover, pigs from Farm B showed ocular lesions classified from Grade I to IV (from mild conjunctivitis to severe keratoconjunctivitis with corneal oedema, opacity, and ulcers). Final diagnosis was pursued by the microscopic assessment of skin lesions in both farms, which revealed the typical SWPV-lesion appearance, such as severe and disseminated ulcerative dermatitis and suspected inclusion bodies multifocally observed in the epidermis. Moreover, negative staining Electron Microscopy (nsEM) was performed on skin lesions and ocular swabs from Farm B, revealing in two samples the presence of brick-shaped viral particles, 220 nm long and 160 nm wide, with irregularly arranged surface tubules, identified as SWPV. The gene encoding the 482-bp fragment of the virus late transcription factor–3 was detected by PCR and sequencing revealed 99.79% identity and 100% query-cover with a strain previously isolated in Germany. Field clinical assessment was then performed in Farm B, revealing high overcrowding, poor sanitary conditions and improper husbandry practices, which are relevant risk factors for SWPV transmission. Conclusions: The present is the first case report of SWPV in free-range pigs raised in Sicily, an island of the Southern coast of Italy, and wants to raise awareness on a neglected disease, and cause of animal health and welfare issues. [ABSTRACT FROM AUTHOR]

Microtubule (MT)-dependent transport is a critical means of intracellular movement of cellular cargo by kinesin and dynein motors. MT-dependent transport is tightly regulated by cellular MT-associated proteins (MAPs) that directly bind to MTs and either promote or impede motor protein function. Viruses have been widely shown to usurp MT-dependent transport to facilitate their virion movement to sites of replication and/or for exit from the cell. However, it is unclear if viruses also negatively regulate MT-dependent transport. Using single-molecule motility and cellular transport assays, we show that the vaccinia virus (VV)-encoded MAP, A51R, inhibits kinesin-1-dependent transport along MTs in vitro and in cells. This inhibition is selective as the function of kinesin-3 is largely unaffected by VV A51R. Interestingly, we show that A51R promotes the perinuclear accumulation of cellular cargo transported by kinesin-1 such as lysosomes and mitochondria during infection. Moreover, A51R also regulates the release of specialized VV virions that exit the cell using kinesin-1-dependent movement. Using a fluorescently tagged rigor mutant of kinesin-1, we show that these motors accumulate on A51R-stabilized MTs, suggesting these stabilized MTs may form a "kinesin-1 sink" to regulate MT-dependent transport in the cell. Collectively, our findings uncover a new mechanism by which viruses regulate host cytoskeletal processes. [ABSTRACT FROM AUTHOR]

Mpox (monkeypox) is a neglected tropical disease that has received increased attention since the multi-nation outbreak that began in 2022. The virus is endemic in West and Central Africa, where the Democratic Republic of the Congo (DRC) is the most affected country. Clade I monkeypox virus (MPXV) infection is endemic in the DRC and has an overall case fatality rate of 10.6% among children and adults. A study conducted in Sankuru Province, DRC, from 2007 to 2011 demonstrated that 75% of pregnant women with mpox had miscarriages or stillbirth. Further analysis of a stillborn fetus showed that MPXV could infect both the placenta and fetus, causing congenital infection. No additional cases of Clade I MPXV in pregnant women were reported until a new outbreak occurred in South Kivu Province during 2023 and 2024. Eight pregnant women having Clade I MPXV infection were identified, of whom four had either miscarriages or stillbirth, representing a 50% fetal mortality rate. These reports confirm previous data from the DRC that indicate the capability of Clade I MPXV to affect the fetus, causing congenital infection and fetal loss in a high percentage of cases. In this article, we review both past and new data from the DRC on the effects of Clade I MPXV during pregnancy and discuss the association of mpox with fetal loss. [ABSTRACT FROM AUTHOR]

Background and Aims: A double‐stranded DNA virus called monkeypox virus (MPV) belonging to the Poxviridae family and Orthopoxvirus genus causes monkeypox (mpox) infection. This virus used to infect only Central, East, and West Africa. However, it has spread to an extent outside Africa recently. The range of MPV outbreaks was so high that on July 23, 2022, the World Health Organization (WHO) declared it a Public Health Emergency of International Concern (PHEIC). About a year later, the WHO notified the end of a global public health emergency for mpox on May 11, 2023. Here, we aimed to assess the current pathogenicity and potential risk of MPV causing public health emergencies. Methods: We searched information from published articles available in PubMed, Scopus, and ScienceDirect. We used monkeypox, mpox, monkeypox outbreak, and monkeypox virus as keywords during the literature search. Results: Many new variants of MPV have emerged throughout the world that created PHEIC for mpox. Considering the low lethality and transmission rate, mpox is no longer a global public health threat. In addition, the availability of therapeutic and preventive measures helped the healthcare authorities fight the mpox infection in an efficient manner. In this review, we have portrayed the history and evolution of mpox from past to present and an idea of its future outcomes. Also, we have discussed the symptoms related to mpox and approved antiviral treatment strategies to fight off the infection in this piece. This review also emphasized the preventive guidelines set by the WHO for patients, caregivers, and healthcare providers to control the outbreak of mpox infection. Conclusion: We believe this article would give an idea about the potential public health threats of the recent multi‐country monkeypox outbreak to the healthcare authorities for taking measures accordingly. [ABSTRACT FROM AUTHOR]

The skin is a complex tissue that provides a strong physical barrier against invading pathogens. Despite this, many viruses can access the skin and successfully replicate in either the epidermal keratinocytes or dermal immune cells. In this review, we provide an overview of the antiviral T cell biology responding to cutaneous viral infections and how these responses differ depending on the cellular targets of infection. Much of our mechanistic understanding of T cell surveillance of cutaneous infection has been gained from murine models of poxvirus and herpesvirus infection. However, we also discuss other viral infections, including flaviviruses and papillomaviruses, in which the cutaneous T cell response has been less extensively studied. In addition to the mechanisms of successful T cell control of cutaneous viral infection, we highlight knowledge gaps and future directions with possible impact on human health. [ABSTRACT FROM AUTHOR]

Viral vector vaccines represent a substantial advancement in immunization technology, offering numerous benefits over traditional vaccine modalities. The Orf virus (ORFV) strain D1701-VrV is a particularly promising candidate for vaccine development due to its distinctive attributes, such as a good safety profile, the ability to elicit both humoral and cellular immunity, and its favorable genetic and thermal stability. Despite ORFV's theoretical safety advantages, such as its narrow host range and limited systemic spread post-inoculation, a critical gap persists between these theoretical benefits and the empirical evidence regarding its in vivo safety profile. This discrepancy underscores the need for comprehensive preclinical validations to bridge this knowledge gap, especially considering ORFV's use in humans. Our research introduces Prime-2-CoV, an innovative ORFV-based vaccine candidate against COVID-19, designed to elicit a robust immune response by expressing SARS-CoV-2 Nucleocapsid and Spike proteins. Currently under clinical trials, Prime-2-CoV marks the inaugural application of ORFV in human subjects. Addressing the aforementioned safety concerns, our extensive preclinical evaluation, including an environmental risk assessment (ERA) and detailed pharmacokinetic studies in rats and immunocompromised NOG mice, demonstrates Prime-2-CoV's favorable pharmacokinetic profile, negligible environmental impact, and minimal ERA risks. These findings not only affirm the vaccine's safety and efficacy but also pioneer the use of ORFV-based therapeutics, highlighting its potential for wider therapeutic applications. [ABSTRACT FROM AUTHOR]

ABSTRACT In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.

Monkeypox (mpox) outbreak in 2022 has caused more than 91,000 cases, has spread to 115 countries, regions, and territories, and has thus attracted much attention. The stability of poxvirus particles in the environment is recognized as an important factor in determining their transmission. However, few studies have investigated the persistence of poxviruses on material surfaces under various environmental conditions, and their sensitivity to biocides. Here, we systematically measured the stability of vaccinia virus (VACV) under different environmental conditions and sensitivity to inactivation methods via plaque assay, quantitative real-time polymerase chain reaction (qPCR), and Gaussia luciferase (G-luciferase) reporter system. The results show that VACV is stable on the surface of stainless steel, glass, clothing, plastic, towel, A4 paper, and tissue and persists much longer at 4 °C and −20 °C, but is effectively inactivated by ultraviolet (UV) irradiation, heat treatment, and chemical reagents. Our study raises the awareness of long persistence of poxviruses in the environment and provides a simple solution to inactivate poxviruses using common disinfectants, which is expected to help the control and prevention of mpox virus and future poxvirus outbreaks.

Modified vaccinia virus Ankara (MVA) has been widely tested in clinical trials as recombinant vector vaccine against infectious diseases and cancers in humans and animals. However, one biosafety concern about the use of MVA vectored vaccine is the potential forMVA to recombinewith naturally occurring orthopoxviruses in cells and hosts in which itmultiplies poorly and, therefore, producing viruses with mosaic genomes with altered genetic and phenotypic properties. We previously conducted co-infection and superinfection experiments with MVA vectored influenza vaccine (MVA-HANP) and a feline Cowpox virus (CPXV-No-F1) in Vero cells (that were semipermissive to MVA infection) and showed that recombination occurred in both coinfected and superinfected cells. In this study, we selected the putative recombinant viruses and performed genomic characterization of these viruses. Some putative recombinant viruses displayed plaque morphology distinct of that of the parental viruses. Our analysis demonstrated that they had mosaic genomes of different lengths. The recombinant viruses, with a genome more similar to MVA-HANP (>50%), rescued deleted and/or fragmented genes in MVA and gained new host ranges genes. Our analysis also revealed that someMVA-HANP contained a partially deleted transgene expression cassette and one recombinant virus contained part of the transgene expression cassette similar to that incomplete MVA-HANP. The recombination in co-infected and superinfected Vero cells resulted in recombinant viruses with unpredictable biological and genetic properties as well as recovery of delete/fragmented genes in MVA and transfer of the transgene into replication competent CPXV. These results are relevant to hazard characterization and risk assessment of MVA vectored biologicals. [ABSTRACT FROM AUTHOR]

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies. [ABSTRACT FROM AUTHOR]

The persistent epidemic of human mpox, caused by mpox virus (MPXV), raises concerns about the future spread of MPXV and other poxviruses. MPXV is a typical zoonotic virus which can infect human and cause smallpox-like symptoms. MPXV belongs to the Poxviridae family, which has a relatively broad host range from arthropods to vertebrates. Cross-species transmission of poxviruses among different hosts has been frequently reported and resulted in numerous epidemics. Poxviruses have a complex linear double-strand DNA genome that encodes hundreds of proteins. Genes related to the host range of poxvirus are called host range genes (HRGs). This review briefly introduces the taxonomy, phylogeny and hosts of poxviruses, and then comprehensively summarizes the current knowledge about the cross-species transmission of poxviruses. In particular, the HRGs of poxvirus are described and their impacts on viral host range are discussed in depth. We hope that this review will provide a comprehensive perspective about the current progress of researches on cross-species transmission and HRG variation of poxviruses, serving as a valuable reference for academic studies and disease control in the future. [ABSTRACT FROM AUTHOR]

Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection. Synopsis: Poxvirus membranes are organized into distinct binding and fusion domains. To facilitate fusion, the virus binding protein D8 actively curves host membranes to promote their contact with the virus fusion machinery. Interactions between vaccinia D8 and host chondroitin sulfates drive low pH-dependent host membrane curvature. VACV proteins A26, D8 and H3 show hierarchical and compensatory contributions to virus binding affinity and orientation. VACV adapts cellular attachment factor usage depending on which viral binding proteins and cell factors are available. Membrane organization into functionally distinct domains maximizes virion binding and fusion efficiency for productive infection. Poxvirus membranes are organized into distinct binding and fusion domains. To facilitate fusion, the virus binding protein D8 actively curves host membranes to promote their contact with the virus fusion machinery. [ABSTRACT FROM AUTHOR]

Myxoma virus (MYXV) is a Leporipoxvirus (genus) belonging to the family Poxviridae; it is characterised by a genome of approximately 161 kb dsDNA encoding for several proteins that play an essential role in both host spectrum determination and immunomodulation. The healthy reservoir of the virus is Sylvilagus spp. At the same time, in wild and domestic European rabbits (Oryctolagus cuniculus), MYXV is the etiologic agent of myxomatosis, a disease with an extremely high mortality rate. In 2014, an interspecies jump of MYXV was reported in Lepus europaeus in the UK. In 2018, myxomatosis induced by a new recombinant strain called MYXV-To was identified during a large outbreak in Iberian hares (Lepus granatensis) in Spain. Here, we describe the case of myxomatosis in another hare species: an adult male Italian hare (Lepus corsicanus) found dead in 2018 in Sicily with lesions suggestive of myxomatosis and treponema infection. Laboratory tests, e.g., end-point PCR and negative staining electron microscopy, confirmed the presence of both pathogens. MYXV was then isolated from tissue samples in permissive cells and sequenced using NGS technology. Main genomic differences concerning known MYXV strains are discussed. [ABSTRACT FROM AUTHOR]

Infection at barrier sites, e.g., skin, activates local immune defenses that limit pathogen spread, while preserving tissue integrity. Phenotypically distinct γδ T cell populations reside in skin, where they shape immunity to cutaneous infection prior to onset of an adaptive immune response by conventional αβ CD4+ (TCD4+) and CD8+ (TCD8+) T cells. To examine the mechanisms used by γδ T cells to control cutaneous virus replication and tissue pathology, we examined γδ T cells after infection with vaccinia virus (VACV). Resident γδ T cells expanded and combined with recruited γδ T cells to control pathology after VACV infection. However, γδ T cells did not play a role in control of local virus replication or blockade of systemic virus spread. We identified a unique wound healing signature that has features common to, but also features that antagonize, the sterile cutaneous wound healing response. Tissue repair generally occurs after clearance of a pathogen, but viral wound healing started prior to the peak of virus replication in the skin. γδ T cells contributed to wound healing through induction of multiple cytokines/growth factors required for efficient wound closure. Therefore, γδ T cells modulate the wound healing response following cutaneous virus infection, maintaining skin barrier function to prevent secondary bacterial infection. [ABSTRACT FROM AUTHOR]

The sudden emergence of SARS-CoV-2 demonstrates the need for new vaccines that rapidly protect in the case of an emergency. In this study, we developed a recombinant MVA vaccine co-expressing SARS-CoV-2 prefusion-stabilized spike protein (ST) and SARS-CoV-2 nucleoprotein (N, MVA-SARS-2-ST/N) as an approach to further improve vaccine-induced immunogenicity and efficacy. Single MVA-SARS-2-ST/N vaccination in K18-hACE2 mice induced robust protection against lethal respiratory SARS-CoV-2 challenge infection 28 days later. The protective outcome of MVA-SARS-2-ST/N vaccination correlated with the activation of SARS-CoV-2-neutralizing antibodies (nABs) and substantial amounts of SARS-CoV-2-specific T cells especially in the lung of MVA-SARS-2-ST/N-vaccinated mice. Emergency vaccination with MVA-SARS-2-ST/N just 2 days before lethal SARS-CoV-2 challenge infection resulted in a delayed onset of clinical disease outcome in these mice and increased titers of nAB or SARS-CoV-2-specific T cells in the spleen and lung. These data highlight the potential of a multivalent COVID-19 vaccine co-expressing S- and N-protein, which further contributes to the development of rapidly protective vaccination strategies against emerging pathogens. [ABSTRACT FROM AUTHOR]

Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17− particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17− particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17− particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17− particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17− particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17− particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17− particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17− particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein. [ABSTRACT FROM AUTHOR]

Myxoma virus (MYXV) causes localized cutaneous fibromas in its natural hosts, tapeti and brush rabbits; however, in the European rabbit, MYXV causes the lethal disease myxomatosis. Currently, the molecular mechanisms underlying this increased virulence after cross-species transmission are poorly understood. In this study, we investigated the interaction between MYXV M156 and the host protein kinase R (PKR) to determine their crosstalk with the proinflammatory nuclear factor kappa B (NF-κB) pathway. Our results demonstrated that MYXV M156 inhibits brush rabbit PKR (bPKR) more strongly than European rabbit PKR (ePKR). This moderate ePKR inhibition could be improved by hyperactive M156 mutants. We hypothesized that the moderate inhibition of ePKR by M156 might incompletely suppress the signal transduction pathways modulated by PKR, such as the NF-κB pathway. Therefore, we analyzed NF-κB pathway activation with a luciferase-based promoter assay. The moderate inhibition of ePKR resulted in significantly higher NF-κB–dependent reporter activity than complete inhibition of bPKR. We also found a stronger induction of the NF-κB target genes TNFα and IL-6 in ePKR-expressing cells than in bPKR-expressing cells in response to M156 in both transfection and infections assays. Furthermore, a hyperactive M156 mutant did not cause ePKR-dependent NF-κB activation. These observations indicate that M156 is maladapted for ePKR inhibition, only incompletely blocking translation in these hosts, resulting in preferential depletion of short–half-life proteins, such as the NF-κB inhibitor IκBα. We speculate that this functional activation of NF-κB induced by the intermediate inhibition of ePKR by M156 may contribute to the increased virulence of MYXV in European rabbits.

Horizontal gene transfer (HGT) provides a major source of genetic variation. Many viruses, including poxviruses, encode genes with crucial functions directly gained by gene transfer from hosts. The mechanism of transfer to poxvirus genomes is unknown. Using genome analysis and experimental screens of infected cells, we discovered a central role for Long Interspersed Nuclear Element-1 retrotransposition in HGT to virus genomes. The process recapitulates processed pseudogene generation, but with host messenger RNA directed into virus genomes. Intriguingly, hallmark features of retrotransposition appear to favor virus adaption through rapid duplication of captured host genes on arrival. Our study reveals a previously unrecognized conduit of genetic traffic with fundamental implications for the evolution of many virus classes and their hosts.

There is ample phylogenetic evidence that many critical virus functions, like immune evasion, evolved by the acquisition of genes from their hosts through horizontal gene transfer (HGT). However, the lack of an experimental system has prevented a mechanistic understanding of this process. We developed a model to elucidate the mechanisms of HGT into vaccinia virus, the prototypic poxvirus. All identified gene capture events showed signatures of long interspersed nuclear element-1 (LINE-1)-mediated retrotransposition, including spliced-out introns, polyadenylated tails, and target site duplications. In one case, the acquired gene integrated together with a polyadenylated host U2 small nuclear RNA. Integrations occurred across the genome, in some cases knocking out essential viral genes. These essential gene knockouts were rescued through a process of complementation by the parent virus followed by nonhomologous recombination during serial passaging to generate a single, replication-competent virus. This work links multiple evolutionary mechanisms into one adaptive cascade and identifies host retrotransposons as major drivers for virus evolution.

Modified vaccinia virus Ankara (MVA) has been widely tested in clinical trials as recombinant vector vaccine against infectious diseases and cancers in humans and animals. However, one biosafety concern about the use of MVA vectored vaccine is the potential for MVA to recombine with naturally occurring orthopoxviruses in cells and hosts in which it multiplies poorly and, therefore, producing viruses with mosaic genomes with altered genetic and phenotypic properties. We previously conducted co-infection and superinfection experiments with MVA vectored influenza vaccine (MVA-HANP) and a feline Cowpox virus (CPXV-No-F1) in Vero cells (that were semi-permissive to MVA infection) and showed that recombination occurred in both co-infected and superinfected cells. In this study, we selected the putative recombinant viruses and performed genomic characterization of these viruses. Some putative recombinant viruses displayed plaque morphology distinct of that of the parental viruses. Our analysis demonstrated that they had mosaic genomes of different lengths. The recombinant viruses, with a genome more similar to MVA-HANP (>50%), rescued deleted and/or fragmented genes in MVA and gained new host ranges genes. Our analysis also revealed that some MVA-HANP contained a partially deleted transgene expression cassette and one recombinant virus contained part of the transgene expression cassette similar to that incomplete MVA-HANP. The recombination in co-infected and superinfected Vero cells resulted in recombinant viruses with unpredictable biological and genetic properties as well as recovery of delete/fragmented genes in MVA and transfer of the transgene into replication competent CPXV. These results are relevant to hazard characterization and risk assessment of MVA vectored biologicals.

ABSTRACT Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a pseudohexagonal palisade layer, composed of trimers projecting from an inner wall. To understand the assembly of this complex core architecture, we obtained a subnanometer structure of the palisade trimer by cryo-electron tomography and subtomogram averaging of purified intact virions. Using AlphaFold2 structure predictions, we determined that the palisade is formed from trimers of the proteolytically processed form of the viral protein A10. In addition, we found that each A10 protomer associates with an α-helix (residues 24–66) of A4. Cellular localization assays outside the context of infection demonstrate that the A4 N-terminus is necessary and sufficient to interact with A10. The interaction between A4 and A10 provides insights into how the palisade layer might become tightly associated with the viral membrane during virion maturation. Reconstruction of the palisade layer reveals that, despite local hexagonal ordering, the A10/A4 trimers are widely spaced, suggesting that additional components organize the lattice. This spacing would, however, allow the adoption of the characteristic biconcave shape of the viral core. Finally, we also found that the palisade incorporates multiple copies of a hexameric portal structure. We suggest that these portals are formed by E6, a viral protein that is essential for virion assembly and required to release viral mRNA from the core early in infection.IMPORTANCEPoxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.

Aging is associated with a decline in immune system functionality. So-called immunosenescence may impair the successful vaccination of elderly people. Thus, improved vaccination strategies also suitable for an aged immune system are required. Modified Vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus that has been established as a multipurpose viral vector for vaccine development against various infections. We characterized a recombinant MVA expressing a prefusion-stabilized version of SARS-CoV-2 S protein (MVA-ST) in an aged-hamster model for COVID-19. Intramuscular MVA-ST immunization resulted in protection from disease and severe lung pathology. Importantly, this protection was correlated with a potent activation of SARS-CoV-2 specific T-cells and neutralizing antibodies. Our results suggest that MVA vector vaccines merit further evaluation in preclinical models to contribute to future clinical development as candidate vaccines in elderly people to overcome the limitations of age-dependent immunosenescence. [ABSTRACT FROM AUTHOR]

Smallpox was a highly contagious disease caused by the variola virus. The disease affected millions of people over thousands of years and variola virus ranked as one of the deadliest viruses in human history. The complete eradication of smallpox in 1980, a major triumph in medicine, was achieved through a global vaccination campaign using a less virulent poxvirus, vaccinia virus. Despite this success, the herd immunity established by this campaign has significantly waned, and concerns are rising about the potential reintroduction of variola virus as a biological weapon or the emergence of zoonotic poxviruses. These fears were further fueled in 2022 by a global outbreak of monkeypox virus (mpox), which spread to over 100 countries, thereby boosting interest in developing new vaccines using molecular approaches. However, poxviruses are complex and creating modern vaccines against them is challenging. This review focuses on the structural biology of the six major neutralization determinants on poxviruses (D8, H3, A27, L1, B5, and A33), the localization of epitopes targeted by neutralizing antibodies, and their application in the development of subunit vaccines. [ABSTRACT FROM AUTHOR]

The 2022 global Mpox outbreak swiftly introduced unforeseen diversity in the monkeypox virus (MPXV) population, resulting in numerous Clade IIb sublineages. This propagation of new MPXV mutations warrants the thorough re-investigation of previously recommended or validated primers designed to target MPXV genomes. In this study, we explored 18 PCR primer sets and examined their binding specificity against 5210 MPXV genomes, representing all the established MPXV lineages. Our results indicated that only five primer sets resulted in almost all perfect matches against the targeted MPXV lineages, and the remaining primer sets all contained 1–2 mismatches against almost all the MPXV lineages. We further investigated the mismatched primer-genome pairs and discovered that some of the primers overlapped with poorly sequenced and assembled regions of the MPXV genomes, which are consistent across multiple lineages. However, we identified 173 99% genome-wide conserved regions across all 5210 MPXV genomes, representing 30 lineages/clades with at least 80% lineage-specific consensus for future primer development and primer binding evaluation. This exercise is crucial to ensure that the current detection schemes are robust and serve as a framework for primer evaluation in clinical testing development for other infectious diseases. [ABSTRACT FROM AUTHOR]

The aim of personalized cancer vaccines is to elicit potent and tumor-specific immune responses against neoantigens specific to each patient and to establish durable immunity, while minimizing the adverse events. Over recent years, there has been a renewed interest in personalized cancer vaccines, primarily due to the advancement of innovative technologies for the identification of neoantigens and novel vaccine delivery platforms. Here, we review the emerging field of personalized cancer vaccination, with a focus on the use of viral vectors as a vaccine platform. The recent advancements in viral vector technology have led to the development of efficient production processes, positioning personalized viral vaccines as one of the preferred technologies. Many clinical trials have shown the feasibility, safety, immunogenicity and, more recently, preliminary evidence of the anti-tumor activity of personalized vaccination, fostering active research in the field, including further clinical trials for different tumor types and in different clinical settings. [ABSTRACT FROM AUTHOR]

Vaccinia virus (VACV) infection induces prominent changes in host cell metabolism. Little is known about the global metabolic reprogramming that takes place in whole tissue during viral infection. Here, we performed a longitudinal metabolomics study in VACV-infected mouse skin to investigate metabolic changes in the tissue during infection. We assessed metabolites in homogenized skin of the ear pinnae over time in the presence or absence of antigen-specific T cells using untargeted mass spectrometry. VACV infection induced several significant metabolic changes in the tissue, including in the levels of nucleic acid metabolites (reflecting the impact of viral replication on the skin metabolome). Furthermore, monocyte- and antiviral T cell-produced metabolites, such as itaconic acid and glutamine, were significantly increased following infection, highlighting the immune response's contribution to the global skin metabolome. Additional RNA-Seq of infected skin tissue recapitulated metabolic changes identified by metabolomics analysis. Overall, our study reveals the metabolic balance of viral replication and the antiviral immune response in the skin, elucidating metabolic pathways that could contribute to cutaneous poxvirus control in vivo. [ABSTRACT FROM AUTHOR]

Oncolytic viruses (OVs) have emerged as one of the most promising cancer immunotherapy agents that selectively target and kill cancer cells while sparing normal cells. OVs are from diverse families of viruses and can possess either a DNA or an RNA genome. These viruses also have either a natural or engineered tropism for cancer cells. Oncolytic DNA viruses have the additional advantage of a stable genome and multiple-transgene insertion capability without compromising infection or replication. Herpes simplex virus 1 (HSV-1), a member of the oncolytic DNA viruses, has been approved for the treatment of cancers. This success with HSV-1 was achievable by introducing multiple genetic modifications within the virus to enhance cancer selectivity and reduce the toxicity to healthy cells. Here, we review the natural characteristics of and genetically engineered changes in selected DNA viruses that enhance the tumor tropism of these oncolytic viruses. [ABSTRACT FROM AUTHOR]

We detected a novel poxvirus from a gray seal (Halichoerus grypus) from the North Sea, Germany. The juvenile animal showed pox-like lesions and deteriorating overall health condition and was finally euthanized. Histology, electron microscopy, sequencing, and PCR confirmed a previously undescribed poxvirus of the Chordopoxvirinae subfamily, tentatively named Wadden Sea poxvirus.

Viral vector vaccines represent a substantial advancement in immunization technology, offering numerous benefits over traditional vaccine modalities. The Orf virus (ORFV) strain D1701-VrV is a particularly promising candidate for vaccine development due to its distinctive attributes, such as a good safety profile, the ability to elicit both humoral and cellular immunity, and its favorable genetic and thermal stability. Despite ORFV’s theoretical safety advantages, such as its narrow host range and limited systemic spread post-inoculation, a critical gap persists between these theoretical benefits and the empirical evidence regarding its in vivo safety profile. This discrepancy underscores the need for comprehensive preclinical validations to bridge this knowledge gap, especially considering ORFV’s use in humans. Our research introduces Prime-2-CoV, an innovative ORFV-based vaccine candidate against COVID-19, designed to elicit a robust immune response by expressing SARS-CoV-2 Nucleocapsid and Spike proteins. Currently under clinical trials, Prime-2-CoV marks the inaugural application of ORFV in human subjects. Addressing the aforementioned safety concerns, our extensive preclinical evaluation, including an environmental risk assessment (ERA) and detailed pharmacokinetic studies in rats and immunocompromised NOG mice, demonstrates Prime-2-CoV’s favorable pharmacokinetic profile, negligible environmental impact, and minimal ERA risks. These findings not only affirm the vaccine’s safety and efficacy but also pioneer the use of ORFV-based therapeutics, highlighting its potential for wider therapeutic applications.

Objetivou-se com este trabalho relatar um surto de mixomatose em uma criação de coelhos na região rural do município de Cachoeiras de Macacu no estado do Rio de Janeiro. De um total de 103 coelhos, 8 óbitos ocorreram pela doença e 51 indivíduos foram eutanasiados com sinais clínicos típicos. Os animais apresentaram blefaroconjuntivite bilateral com secreção purulenta, progredindo para rinite com secreção sanguinolenta, edema de face e surgimento de pequenos nódulos na face, orelha e escroto. À necropsia, foram observados linfonodomegalia, esplenomegalia, pulmões congestos e edema escrotal. Nesta, foram colhidos fragmentos de fígado, pulmão, rins e tecido cutâneo dos nódulos da face e orelhas para exame histopatológico. Na microscopia das amostras cutâneas foi observada na epiderme: vacuolização dos queratinócitos com vesículas, bolhas e pústulas, além de áreas de acantose, ulceração e presença de crosta serocelular. Na derme foi observado edema pronunciado, infiltrado marcante, difuso e misto de células heterófilas e mononucleares, além de degeneração mucinosa pronunciada. Adicionalmente, constatou-se a presença de grandes células “estreladas” com cariomegalia e cromatina salpicada. As carcaças foram incineradas e os 44 animais restantes que não apresentaram sintomatologia foram submetidos à quarentena por seis meses em galpão com telas, repelentes eletrônicos e monitorados através de exames clínicos e testes complementares para confirmar a sanidade do plantel. Além disso, foi administrada selamectina tópica a cada 30 dias para prevenir e controlar vetores.

After recognition of cognate antigen (Ag), effector CD8+ T cells secrete serine proteases called granzymes in conjunction with perforin, allowing granzymes to enter and kill target cells. While the roles for some granzymes during antiviral immune responses are well characterized, the function of others, such as granzyme C and its human ortholog granzyme H, is still unclear. Granzyme C is constitutively expressed by mature, cytolytic innate lymphoid 1 cells (ILC1s). Whether other antiviral effector cells also produce granzyme C and whether it is continually expressed or responsive to the environment is unknown. To explore this, we analyzed granzyme C expression in different murine skin-resident antiviral lymphocytes. At steady-state, dendritic epidermal T cells (DETCs) expressed granzyme C while dermal γδ T cells did not. CD8+ tissue-resident memory T cells (TRM) generated in response to cutaneous viral infection with the poxvirus vaccinia virus (VACV) also expressed granzyme C. Both DETCs and virus-specific CD8+ TRM upregulated granzyme C upon local VACV infection. Continual Ag exposure was not required for maintained TRM expression of granzyme C, although re-encounter with cognate Ag boosted expression. Additionally, IL-15 treatment increased granzyme C expression in both DETCs and TRM. Together, our data demonstrate that granzyme C is widely expressed by antiviral T cells in the skin and that expression is responsive to both environmental stimuli and TCR engagement. These data suggest that granzyme C may have functions other than killing in tissue-resident lymphocytes. [ABSTRACT FROM AUTHOR]

Poxviruses belong to the Poxviridae family, a group of pathogens known for their high infectivity in humans, posing significant health threats. One of the most well‐known representatives of poxvirus infections is smallpox, which has been successfully eradicated. However, in recent years, there has been a resurgence in cases of mpox, another member of the Poxviridae family, raising concerns about the potential for a global pandemic or a worldwide health crisis. While the typical clinical presentation of mpox and other poxvirus infections often involves cutaneous lesions, there have been reports of various atypical and non‐classic clinical manifestations. Dermoscopy has emerged as a crucial diagnostic tool, aiding dermatologists in clinical practice to make informed decisions. In this summary, we provide an overview of the clinical and dermoscopic features of representative cutaneous lesions associated with human poxvirus infections, including mpox, orf, milker's nodule, and molluscum contagiosum. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate serine proteases in the central coagulation and immune pathways, representing 2–10% of circulating proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian proteolytic pathways. Through interacting with extracellular and intracellular serine and cysteine proteases, viral serpins provide a new class of highly active virus-derived coagulation-, immune-, and apoptosis-modulating drug candidates. Viral serpins have unique characteristics: (1) function at micrograms per kilogram doses; (2) selectivity in targeting sites of protease activation; (3) minimal side effects at active concentrations; and (4) the demonstrated capacity to be modified, or fine-tuned, for altered protease targeting. To date, the virus-derived serpin class of biologics has proven effective in a wide range of animal models and in one clinical trial in patients with unstable coronary disease. Here, we outline the known viral serpins and review prior studies with viral serpins, considering their potential for application as new sources for immune-, coagulation-, and apoptosis-modulating therapeutics. [ABSTRACT FROM AUTHOR]

Zoonotic poxviruses such as mpox virus (MPXV) continue to threaten public health safety since the eradication of smallpox. Vaccinia virus (VACV), the prototypic poxvirus used as the vaccine strain for smallpox eradication, is the best-characterized member of the poxvirus family. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified VACV Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A (family with sequence similarity 111 member A), a nuclear protein that regulates host DNA replication, was shown to restrict the replication of a VACV SPI-1 deletion mutant (VACV-ΔSPI-1) in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-ΔSPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA-binding domain but not the PCNA-interacting motif. We further identified that FAM111A translocated into the cytoplasm upon VACV infection by degrading the nuclear pore complex via its protease activity, interacted with VACV DNA-binding protein I3, and promoted I3 degradation through autophagy. Moreover, SPI-1 from VACV, MPXV, or lumpy skin disease virus was able to antagonize FAM111A by prohibiting its nuclear export. Our findings reveal the detailed mechanism by which FAM111A inhibits VACV and provide explanations for the immune evasive function of VACV SPI-1. [ABSTRACT FROM AUTHOR]

Smallpox was eradicated in less than 200 years after Edward Jenner's practice of cowpox variolation in 1796. The forty-three years of us living free of smallpox, beginning in 1979, never truly separated us from poxviruses. The recent outbreak of monkeypox in May 2022 might well warn us of the necessity of keeping up both the scientific research and public awareness of poxviruses. One of them in particular, the vaccinia virus (VACV), has been extensively studied as a vector given its broad host range, extraordinary thermal stability, and exceptional immunogenicity. Unceasing fundamental biological research on VACV provides us with a better understanding of its genetic elements, involvement in cellular signaling pathways, and modulation of host immune responses. This enables the rational design of safer and more efficacious next-generation vectors. To address the new technological advancement within the past decade in VACV research, this review covers the studies of viral immunomodulatory genes, modifications in commonly used vectors, novel mechanisms for rapid generation and purification of recombinant virus, and several other innovative approaches to studying its biology. [ABSTRACT FROM AUTHOR]

In African countries where mpox (monkeypox) is endemic, infection is caused by two genetically related clades—Clade I (formerly Congo Basin), and Clade IIa (formerly West Africa), both of which are potentially life-threatening infections. Prior to the 2022–2023 global outbreak, mpox infections among pregnant women caused by Clade I were reported to have a 75% perinatal case fatality rate in the Democratic Republic of Congo, including the only documented case of placental infection and stillbirth from the Congenital Mpox Syndrome, and the Clade IIa mpox infection was associated with stillbirths in Nigeria. The 2022–2023 global mpox outbreak, caused by a genetically distinct strain, Clade IIb, has focused attention on the effects of mpox on pregnant women and fetal outcomes. There have been at least 58 cases of mpox infection occurring in pregnant women during the 2022–2023 outbreak. No confirmed cases of adverse perinatal outcome, including stillbirth, have been reported. The absence of perinatal morbidity and mortality from Clade IIb corresponds to the overall case fatality rate among non-pregnant women of <0.1%, as this clade has been demonstrated to produce a less-severe disease than the mpox Clade I or IIa variants. Thus, there are apparently important differences between mpox clades affecting pregnant women and perinatal outcomes. [ABSTRACT FROM AUTHOR]

Monkeypox virus (MPXV) is closely related to the infamous variola (smallpox) virus, causing a febrile rash illness in humans similar to but milder than smallpox. In the twentieth century, human monkeypox had been mostly a rare zoonotic disease confined to forested areas in West and Central Africa. However, the case number and geographic range have increased significantly in this century, coincided with the waning of the smallpox vaccine-induced immunity in the global population. The outbreak of human monkeypox in multiple countries since May 2022 has been unusual in its large case number and the absence of direct links to endemic countries, raising concerns for a possible change in monkeypox transmission pattern that could pose a greater global threat. Here, we review aspects of MPXV biology that are relevant for risk assessment and preparedness for a monkeypox epidemic, with an emphasis on recent progress in understanding of the virus host range, evolutionary potential, and neutralization targets.

Abstract The famed variola (smallpox) virus and the monkeypox virus (MPXV) are closely related, and MPXV causes a milder form of febrile rash disease in people. Human monkeypox was mostly an uncommon zoonotic illness that was restricted to West and Central African wooded areas in the twentieth century. The global population’s smallpox vaccine-induced immunity, however, has started to decrease as the number of cases and the geographic breadth have both increased significantly in this century. Several countries have seen human monkeypox outbreaks since May 2022. A possible shift in the monkeypox transmission pattern that might represent a bigger worldwide danger is raised by the atypical nature of these outbreaks, which are characterized by their high-case numbers and absence of ties to endemic countries. Here, we explore a wide range of MPXV biology topics.

Abstract Zoonotic poxvirus infections pose significant threat to human health as we have witnessed recent spread of monkeypox. Therefore, insights into molecular mechanism behind poxvirus replication cycle are needed for the development of efficient antiviral strategies. Virion assembly is one of the key steps that determine the fate of replicating poxviruses. However, in-depth understanding of poxvirus assembly is challenging due to the complex nature of multi-step morphogenesis and heterogeneous virion structures. Despite these challenges, decades of research have revealed virion morphologies at various maturation stages, critical protein components and interactions with host cell compartments. Transmission electron microscopy has been employed as an indispensable tool for the examination of virion morphology, and more recently for the structure determination of protein complexes. In this review, we describe some of the major findings in poxvirus morphogenesis and the contributions of continuously advancing electron microscopy techniques.

The Orf virus (ORFV) is a promising candidate for vector vaccines as well as for immunomodulatory and oncolytic therapies. However, few publications are available on its infectivity degradation or on suitable additives for prolonging its viral stability. In this study, the non-supplemented ORFV itself showed a very high stability at storage temperatures up to 28 °C, with a linear titer loss of 0.10 log infectious particles per day at 4 °C over a period of five weeks. To prolong this inherent stability, thirty additives, i.e., detergents, sugars, proteins, salts, and buffers as well as amino acids, were tested for their time- and temperature-dependent influence on the ORFV infectivity. A stabilizing effect on the infectivity was identified for the addition of all tested proteins, i.e., gelatine, bovine serum albumin, and recombinant human serum albumin (rHSA), of several sugars, i.e., mannitol, galactose, sucrose, and trehalose, of amino acids, i.e., arginine and proline, of the detergent Pluronic F68, and of the salt Na2SO4. The infectivity preservation was especially pronounced for proteins in liquid and frozen formulations, sugars in frozen state, and arginine und Pluronic in liquid formulations at high storage temperatures (37 °C). The addition of 1% rHSA with and without 5% sucrose was evaluated as a very stable formulation with a high safety profile and economic validity at storage temperatures up to 28 °C. At increased temperatures, the supplementation with 200 mM arginine performed better than with rHSA. In summary, this comprehensive data provides different options for a stable ORFV formulation, considering temperature, storage time, economic aspects, and downstream processing integrity.

ABSTRACT In addition to the 3′-poly(A) tail, vaccinia virus mRNAs synthesized after viral DNA replication (post-replicative mRNAs) possess a 5′-poly(A) leader that confers a translational advantage in virally infected cells. These mRNAs are synthesized in viral factories, the cytoplasmic compartment where vaccinia virus DNA replication, mRNA synthesis, and translation occur. However, a previous study indicates that the poly(A)-binding protein (PABPC1)—which has a well-established role in RNA stability and translation—is absent in the viral factories. This prompts the question of whether other poly(A)-binding proteins engage vaccinia virus post-replicative mRNA in viral factories. Here, in this study, we found that La-related protein 4 (LARP4), a poly(A) binding protein, was enriched in viral factories in multiple types of cells during vaccinia virus infection. Further studies showed that LARP4 enrichment in the viral factories required viral post-replicative gene expression and functional decapping enzymes encoded by vaccinia virus. We further showed that knockdown of LARP4 expression in human foreskin fibroblasts (HFFs) reduced vaccinia virus DNA replication, post-replicative protein levels, and viral production. Interestingly, the knockdown of LARP4 expression also reduced protein levels from transfected mRNA containing a 5′-poly(A) leader in vaccinia virus-infected and uninfected HFFs. Taken together, our results identified a poly(A)-binding protein, LARP4, being enriched in the vaccinia virus viral factories and facilitating viral replication in HFFs. IMPORTANCE Vaccinia virus, the prototype poxvirus, encodes over 200 open reading frames (ORFs). Over 90 of vaccinia virus ORFs are transcribed post-viral DNA replication. All these mRNAs contain a 5′-poly(A) leader, as well as a 3′-poly(A) tail. They are synthesized in viral factories, where vaccinia virus DNA replication, mRNA synthesis, and translation occur. However, surprisingly, the poly(A) binding protein, PABPC1, that is important for mRNA metabolism and translation is not present in the viral factories, suggesting other poly(A) binding protein(s) may be present in viral factories. Here, we found another poly(A)-binding protein, La-related protein 4 (LARP4), enriched in viral factories during vaccinia virus infection. We also showed that LARP4 enrichment in the viral factories depends on viral post-replicative gene expression and functional viral decapping enzymes. The knockdown of LARP4 expression in human foreskin fibroblasts reduced vaccinia virus DNA replication, post-replicative gene expression, and viral production.

ABSTRACT Vaccinia virus is the active component of all modern smallpox vaccines after the mid-20th century, but it is uncertain to what extent cowpox, vaccinia, and horsepox viruses were used to produce vaccines before then. Genome sequences of six smallpox vaccines used in the United States between 1850 and 1902, namely VK01, VK02, VK05, VK08, VK12, and Mulford_1902 vaccines, revealed >99.5% similarity with a 1976 strain of horsepox in the genome core. However, how these historical vaccines relate to horsepox and vaccinia viruses is still unknown. Here, we present a detailed investigation of the gene content and genomic structure of these historical smallpox vaccines. Except for VK05, all historical vaccines differ from horsepox in the genomic architecture of the flanking variable regions showing complex patterns of gene duplication/transposition, gene fragmentation, and gene loss. The Mulford_1902 vaccine is the closest virus to contemporary vaccinia viruses and the VK02 vaccine is the most different, with several stretches of variola virus genes recombined in its genome. Our data suggest that in the late 19th and early 20th centuries, different horsepox-based vaccines and probably related unsampled progenitors of modern vaccinia virus coexisted. A better understanding of the evolutionary path of the now extinct horsepox-based vaccines will increase our knowledge of the origins of contemporary vaccinia viruses and the pathways that led to the consolidation of current smallpox vaccines. This is particularly important now that the resumption of production of smallpox vaccines for use against mpox is widely discussed, as is the improvement of available vaccines. IMPORTANCE Modern smallpox vaccines, such as those used against mpox, are made from vaccinia viruses, but it is still unknown whether cowpox, horsepox, or vaccinia viruses were used in the early 20th century or earlier. The mystery began to be solved when the genomes of six historical smallpox vaccines used in the United States from 1850 to 1902 were determined. Our work analyzed in detail the genomes of these six historical vaccines, revealing a complex genomic structure. Historical vaccines are highly similar to horsepox in the core of their genomes, but some are closer to the structure of vaccinia virus at the ends of the genome. One of the vaccines is a recombinant virus with parts of variola virus recombined into its genome. Our data add valuable information for understanding the evolutionary path of current smallpox vaccines and the genetic makeup of the potentially extinct group of horsepox viruses.

ABSTRACT Vaccinia virus, the prototypical poxvirus and smallpox/monkeypox vaccine, has proven a challenging entity for structural biology, defying many of the approaches leading to molecular and atomic models for other viruses. Via a combination of deep learning and cross-linking mass spectrometry, we have developed an atomic-level model and an integrated processing/assembly pathway for a structural component of the vaccinia virion, protein P4a. Within the pathway, proteolytic separation of the C-terminal P4a-3 segment of P4a triggers a massive conformational rotation within the N-terminal P4a-1 segment that becomes fixed by disulfide-locking while removing a steric block to trimerization of the processing intermediate P4a-1+2. These events trigger the proteolytic separation of P4a-2, allowing the assembly of P4a-1 into a hexagonal lattice that encloses the nascent virion core. IMPORTANCE An outstanding problem in the understanding of poxvirus biology is the molecular structure of the mature virion. Via deep learning methods combined with chemical cross-linking mass spectrometry, we have addressed the structure and assembly pathway of P4a, a key poxvirus virion core component.

The SARS-CoV-2 pandemic demonstrated the need for potent and broad-spectrum vaccines. This study reports the development and testing of a lumpy skin disease virus (LSDV)-vectored vaccine against SARS-CoV-2, utilizing stabilized spike and conserved nucleocapsid proteins as antigens to develop robust immunogenicity. Construction of the vaccine (LSDV-SARS2-S,N) was confirmed by polymerase chain reaction (PCR) amplification and sequencing. In vitro characterization confirmed that cells infected with LSDV-SARS2-S,N expressed SARS-CoV-2 spike and nucleocapsid protein. In BALB/c mice, the vaccine elicited high magnitude IFN-γ ELISpot responses (spike: 2808 SFU/106 splenocytes) and neutralizing antibodies (ID50 = 6552). Testing in hamsters, which emulate human COVID-19 disease progression, showed the development of high titers of neutralizing antibodies against the Wuhan and Delta SARS-CoV-2 variants (Wuhan ID50 = 2905; Delta ID50 = 4648). Additionally, hamsters vaccinated with LSDV-SARS2-S,N displayed significantly less weight loss, lung damage, and reduced viral RNA copies following SARS-CoV-2 infection with the Delta variant as compared to controls, demonstrating protection against disease. These data demonstrate that LSDV-vectored vaccines display promise as an effective SARS-CoV-2 vaccine and as a potential vaccine platform for communicable diseases in humans and animals. Further efficacy testing and immune response analysis, particularly in non-human primates, are warranted. [ABSTRACT FROM AUTHOR]