Bioinformatic identification of monkeypox virus phylogenetic gene trees that are representative of its whole-genome phylogenetic tree.

Phylogenetic analysis based on whole-genome sequences is the gold standard for monkeypox virus (MPXV) phylogeny. However, genomic epidemiology capability and capacity are lacking or limited in resource poor countries of sub-Saharan Africa. Therefore, these make real-time genome surveillance of MPXV virtually impossible. We hypothesized that phylogenetic analysis based on single, conserved genes will produce phylogenetic tree topology consistent with MPXV whole-genome phylogeny, thus serving as a reliable proxy to phylogenomic analysis. In this study, we analyzed 62 conserved MPXV genes and showed that Bayesian phylogenetic analysis based on five genes (OPG 066/E4L, OPG068/E6R, OPG079/I3L, OPG145/A18R, and OPG150/A23R) generated phylogenetic trees with 72.2-96.3% topology similarity index to the reference phylogenomic tree topology. Our results showed that phylogenetic analysis of the identified five genes singly or in combination can serve as surrogate for whole-genome phylogenetic analysis, and thus obviates the need for whole-genome sequencing and phylogenomic analysis in regions where genomic epidemiology competence and capacity are lacking or unavailable. This study is relevant to evolution and genome surveillance of MPXV in resource limited countries.

First report of a chemokine from camelids: Dromedary CXCL8 is induced by poxvirus and heavy metal toxicity.

Low molecular weight proteins, known as chemokines, facilitate the migration and localization of immune cells to the site of infection and injury. One of the first chemokines identified, CXCL8 functions as a key neutrophil activator, recruiting neutrophils to sites of inflammation. Several viral infections, including zoonotic coronaviruses and poxviruses, have been reported to induce the expression of CXCL8. Dromedary camels are known to harbor several potentially zoonotic pathogens, but critical immune molecules such as chemokines remain unidentified. We report here the identification of CXCL8 from the dromedary camel - the first chemokine identified from camelids. The complete dromedary CXCL8 cDNA sequence as well as the corresponding gene sequence from dromedary and two New World camelids - alpaca and llama were cloned. CXCL8 mRNA expression was relatively higher in PBMC, spleen, lung, intestine, and liver. Poly(I:C) and lipopolysaccharide stimulated CXCL8 expression in vitro, while interferon treatment inhibited it. In vitro infection with potentially zoonotic camelpox virus induced the expression of CXCL8 in camel kidney cells. Toxicological studies on camelids have been limited, and no biomarkers have been identified. Hence, we also evaluated CXCL8 mRNA expression as a potential biomarker to assess heavy metal toxicity in camel kidney cells in vitro. CXCL8 expression was increased after in vitro exposure to heavy metal compounds of cobalt and cadmium, suggesting potential utility as a biomarker for renal toxicity in camels. The results of our study demonstrate that camel CXCL8 plays a significant role in immunomodulatory and induced toxicity responses in dromedary camels.

Systematic analysis of traditional Chinese medicine prescriptions provides new insights into drug combination therapy for pox.

ETHNOPHARMACOLOGICAL RELEVANCE: The decline in cross-protection provided by the smallpox vaccine increases the risk of infection from other poxviruses. While drug combinations are a promising management, they remain underdeveloped for poxviruses. Prior to the development of the smallpox vaccine, China had long relied on herbal medicine to combat pox and accumulated a wealth of knowledge regarding different herb combinations and symptoms related to pox. The information was documented in the form of prescriptions. AIM OF THE STUDY: The extensive data of prescriptions offer the potential for uncovering commonalities underlying these prescriptions, thereby providing valuable insights into the development of drug combinations against pox. MATERIALS AND METHODS: The 2344 prescriptions were collected from the LTM-TCM database and 12 traditional Chinese medicine books. Firstly, the relative frequency of citation was utilized to identify the most used herbs among these prescriptions. TCMSP and LTM-TCM databases were employed to gather information about active compounds and their targets. GeneCards and DisGeNET databases were utilized to determine the associated targets for smallpox, cowpox, chickenpox, and mpox. Subsequently, network pharmacology analysis was conducted to investigate potential pathway information related to the most used herbs. A comparison of active compounds from these herbs resulted in the identification of 29 high-frequency compounds. The functions of these compounds were elucidated through gene overlap analysis, docking, and literature review. Finally, we summarized pox-related symptoms and used fidelity levels to distinguish specific herbs for corresponding symptoms. RESULTS: Based on 2344 traditional pox-related prescriptions, we identified 19 most used herbs and 64 associated bio-functional modules for poxvirus treatment, with the most significant one being immunoregulation primarily involving CD4+ regulation. We also identified 29 leads that possess anti-inflammatory, antimicrobial, and antiviral properties. These herbs and leads hold the potential for pox treatment. Additionally, docking analysis suggested that these leads could inhibit poxvirus DNA synthesis, RNA capping machinery processes, and mature poxvirus particle formation, as well as immunosuppressors. The clinical features of mpox in 2022 were found to align well with our description of symptoms related to the pox. CONCLUSION: Through the analysis of 2344 prescriptions for pox treatment, we obtained a comprehensive library of the most used herbs and high-frequency compounds, along with their potential functional spectrum. These libraries served as raw resources for drug combination development, while the identified symptom patterns and specific herbs greatly enhanced our insight into diverse treatments for pox patients.

What the pox? Review of poxviruses affecting humans.

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

Exploring the transcriptomic profile of human monkeypox virus via CAGE and native RNA sequencing approaches.

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.

The roles of CD4+ T cell help, sex, and dose in the induction of protective CD8+ T cells against a lethal poxvirus by mRNA-LNP vaccines.

The role of CD4+ T cells in the induction of protective CD8+ T cells by mRNA lipid nanoparticle (LNP) vaccines is unknown. We used B6 or Tlr9 -/- mice depleted or not of CD4+ T cells and LNP vaccines loaded with mRNAs encoding the ectromelia virus (ECTV) MHC class I H-2 Kb-restricted immunodominant CD8+ T cell epitope TSYKFESV (TSYKFESV mRNA-LNPs) or the ECTV EVM158 protein, which contains TSYKFESV (EVM-158 mRNA-LNPs). Following prime and boost with 10 µg of either vaccine, Kb-TSYKFESV-specific CD8+ T cells fully protected male and female mice from ECTV at 29 (both mRNA-LNPs) or 90 days (EVM158 mRNA-LNPs) post boost (dpb) independently of CD4+ T cells. However, at 29 dpb with 1 µg mRNA-LNPs, males had lower frequencies of Kb-TSYKFESV-specific CD8+ T cells and were much less well protected than females from ECTV, also independently of CD4+ T cells. At 90 dpb with 1 µg EVM158 mRNA-LNPs, the frequencies of Kb-TSYKFESV-specific CD8+ T cells in males and females were similar, and both were similarly partially protected from ECTV, independently of CD4+ T cells. Therefore, at optimal or suboptimal doses of mRNA-LNP vaccines, CD4+ T cell help is unnecessary to induce protective anti-poxvirus CD8+ T cells specific to a dominant epitope. At suboptimal doses, protection of males requires more time to develop.

Animal and bacterial viruses share conserved mechanisms of immune evasion.

Animal and bacterial cells sense and defend against viral infections using evolutionarily conserved antiviral signaling pathways. Here, we show that viruses overcome host signaling using mechanisms of immune evasion that are directly shared across the eukaryotic and prokaryotic kingdoms of life. Structures of animal poxvirus proteins that inhibit host cGAS-STING signaling demonstrate architectural and catalytic active-site homology shared with bacteriophage Acb1 proteins, which inactivate CBASS anti-phage defense. In bacteria, phage Acb1 proteins are viral enzymes that degrade host cyclic nucleotide immune signals. Structural comparisons of poxvirus protein-2'3'-cGAMP and phage Acb1-3'3'-cGAMP complexes reveal a universal mechanism of host nucleotide immune signal degradation and explain kingdom-specific additions that enable viral adaptation. Chimeric bacteriophages confirm that animal poxvirus proteins are sufficient to evade immune signaling in bacteria. Our findings identify a mechanism of immune evasion conserved between animal and bacterial viruses and define shared rules that explain host-virus interactions across multiple kingdoms of life.

Comparative analysis of two novel complete genomes of myxoma virus vaccine strains.

Myxoma virus (MYXV) is a double-stranded DNA-containing virus of the family Poxviridae, genus Leporipoxvirus. MYXV is an important model virus for evolutionary and immunological research and a promising oncolytic. In this study, we sequenced and analyzed two complete genomes of MYXV virus vaccine strains B-82 and Rabbivac-B, which are widely used for vaccine production in Russia. Here, we first show that MYXV vaccine strains B-82 and Rabbivac-B share a common origin with the American recombinant MYXV MAV vaccine strain. In addition, our data suggest that the MYXV B-82 and Rabbivac-B strains contain a number of genes at the 5' and 3' ends that are identical to the virulent MYXV Lausanne strain. Several unique genetic signatures were identified in the M013L, M017L, M023, and M121R genes, helping to achieve high genetic resolution between vaccine strains. Overall, these findings highlight the evolutionary flexibility of certain genes in the MYXV genome and provide insights into the molecular epidemiology of the virus and subsequent vaccine development.

Host range of a parasitoid wasp is linked to host susceptibility to its mutualistic viral symbiont.

Parasitoid wasps are one of the most species-rich groups of animals on Earth, due to their ability to successfully develop as parasites of nearly all types of insects. Unlike most known parasitoid wasps that specialize towards one or a few host species, Diachasmimorpha longicaudata is a generalist that can survive within multiple genera of tephritid fruit fly hosts, including many globally important pest species. Diachasmimorpha longicaudata has therefore been widely released to suppress pest populations as part of biological control efforts in tropical and subtropical agricultural ecosystems. In this study, we investigated the role of a mutualistic poxvirus in shaping the host range of D. longicaudata across three genera of agricultural pest species: two of which are permissive hosts for D. longicaudata parasitism and one that is a nonpermissive host. We found that permissive hosts Ceratitis capitata and Bactrocera dorsalis were highly susceptible to manual virus injection, displaying rapid virus replication and abundant fly mortality. However, the nonpermissive host Zeugodacus cucurbitae largely overcame virus infection, exhibiting substantially lower mortality and no virus replication. Investigation of transcriptional dynamics during virus infection demonstrated hindered viral gene expression and limited changes in fly gene expression within the nonpermissive host compared with the permissive species, indicating that the host range of the viral symbiont may influence the host range of D. longicaudata wasps. These findings also reveal that viral symbiont activity may be a major contributor to the success of D. longicaudata as a generalist parasitoid species and a globally successful biological control agent.

High Rates of Miscarriage and Stillbirth among Pregnant Women with Clade I Mpox (Monkeypox) Are Confirmed during 2023-2024 DR Congo Outbreak in South Kivu Province.

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.

Swinepox virus: an unusual outbreak in free-range pig farms in Sicily (Italy).

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.

Unique ankyrin repeat proteins in the genome of poxviruses-Boon or Wane, a critical review.

Ankyrin repeat is a 33-amino acid motif commonly observed in eukaryotes and, to a lesser extent, in prokaryotes and archaea and rarely in viruses. This motif plays a crucial role in regulating various cellular processes like the cell cycle, transcription, cell signaling, and inflammatory responses through interactions between proteins. Poxviruses exhibit a distinctive feature of containing multiple ankyrin repeat proteins within their genomes. All the genera of poxviruses possess these proteins except molluscipox virus, crocodylidpox virus, and red squirrel poxvirus. An intriguing characteristic has generated notable interest in studying the functions of these proteins within poxvirus biology. Within poxviruses, ankyrin repeat proteins exhibit a distinct configuration, featuring ankyrin repeats in the N-terminal region and a cellular F-box homolog in the C-terminal region, which enables interactions with the cellular Skp, Cullin, F-box containing ubiquitin ligase complex. Through the examination of experimental evidences and discussions from current literature, this review elucidates the organization and role of ankyrin repeat proteins in poxviruses. Various research studies have highlighted the significant importance of these proteins in poxviral pathogenesis and, acting as factors that enhance virulence. Consequently, they represent viable targets for developing genetically altered viruses with decreased virulence, thus displaying potential as candidates for vaccines and antiviral therapeutic development contributing to safer and more effective strategies against poxviral infections.

Poxvirus A51R Proteins Negatively Regulate Microtubule-Dependent Transport by Kinesin-1.

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.

Poxvirus Immune Evasion.

Poxviruses have evolved a wide array of mechanisms to evade the immune response, and we provide an overview of the different immunomodulatory strategies. Poxviruses prevent the recognition of viral DNA that triggers the immune responses and inhibit signaling pathways within the infected cell. A unique feature of poxviruses is the production of secreted proteins that mimic cytokines and cytokine receptors, acting as decoy receptors to neutralize the activity of cytokines and chemokines. The capacity of these proteins to evade cellular immune responses by inhibiting cytokine activation is complemented by poxviruses' strategies to block natural killer cells and cytotoxic T cells, often through interfering with antigen presentation pathways. Mechanisms that target complement activation are also encoded by poxviruses. Virus-encoded proteins that target immune molecules and pathways play a major role in immune modulation, and their contribution to viral pathogenesis, facilitating virus replication or preventing immunopathology, is discussed.

Current Status of Poultry Recombinant Virus Vector Vaccine Development.

Inactivated and live attenuated vaccines are the mainstays of preventing viral poultry diseases. However, the development of recombinant DNA technology in recent years has enabled the generation of recombinant virus vector vaccines, which have the advantages of preventing multiple diseases simultaneously and simplifying the vaccination schedule. More importantly, some can induce a protective immune response in the presence of maternal antibodies and offer long-term immune protection. These advantages compensate for the shortcomings of traditional vaccines. This review describes the construction and characterization of primarily poultry vaccine vectors, including fowl poxvirus (FPV), fowl adenovirus (FAdV), Newcastle disease virus (NDV), Marek's disease virus (MDV), and herpesvirus of turkey (HVT). In addition, the pathogens targeted and the immunoprotective effect of different poultry recombinant virus vector vaccines are also presented. Finally, this review discusses the challenges in developing vector vaccines and proposes strategies for improving immune efficacy.

Differential Diagnosis, Prevention Measures, and Therapeutic Interventions for Enhanced Monkeypox (Mpox) Care.

Monkeypox (Mpox) is a rare viral disease that presents considerable challenges in healthcare settings, necessitating enhanced nursing care for effective management. This review thoroughly explores key aspects related to improving nursing care for Mpox. It commences by examining the background information on Mpox, encompassing its etiology, epidemiology, and modes of transmission. The differential diagnosis of Mpox is investigated, elucidating its clinical presentation, symptoms, and diagnostic methods to differentiate it from similar conditions. Prevention and control measures at both the public health and healthcare levels are scrutinized, including surveillance and reporting, contact tracing, isolation, and vaccination programs. In healthcare settings, infection prevention and control strategies, such as proper utilization of personal protective equipment, hand hygiene, and environmental management, are discussed. Furthermore, therapeutic interventions for Mpox, including symptomatic management, antiviral therapy, and supportive care, are outlined, with a specific emphasis on pain management, fever control, and psychosocial support. Nursing care strategies encompass patient assessment and monitoring, infection prevention strategies, psychosocial support, and patient education. The challenges encountered in enhancing nursing care for Mpox are acknowledged, along with research gaps and areas for further investigation. Finally, innovations in nursing practice for improved care, such as technology integration and simulation-based training, are explored. Enhancing nursing care in Mpox is crucial for positive patient outcomes, reducing transmission risks, and promoting overall well-being. By addressing the unique challenges, conducting further research, and embracing innovative practices, healthcare professionals, particularly nurses, can provide optimal care and contribute to better management of Mpox cases.

Widespread Horizontal Gene Transfer Among Animal Viruses.

The initial objective of this study was to shed light on the evolution of small DNA tumor viruses by analyzing de novo assemblies of publicly available deep sequencing datasets. The survey generated a searchable database of contig snapshots representing more than 100,000 Sequence Read Archive records. Using modern structure-aware search tools, we iteratively broadened the search to include an increasingly wide range of other virus families. The analysis revealed a surprisingly diverse range of chimeras involving different virus groups. In some instances, genes resembling known DNA-replication modules or known virion protein operons were paired with unrecognizable sequences that structural predictions suggest may represent previously unknown replicases and novel virion architectures. Discrete clades of an emerging group called adintoviruses were discovered in datasets representing humans and other primates. As a proof of concept, we show that the contig database is also useful for discovering RNA viruses and candidate archaeal phages. The ancillary searches revealed additional examples of chimerization between different virus groups. The observations support a gene-centric taxonomic framework that should be useful for future virus-hunting efforts.

T Cell Surveillance during Cutaneous Viral Infections.

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.

Poxviruses from the Concept of One Health.

In the last 4 years, the world has experienced two pandemics of bat-borne viruses. Firstly, in 2019 the SARS-CoV-2 pandemic started and has been causing millions of deaths around the world. In 2022, a Monkeypox pandemic rose in various countries of the world. Those pandemics have witnessed movements and initiatives from healthcare and research institutions to establish a worldwide understanding to battle any future pandemics and biological threats. One Health concept is a modern, comprehensive, unifying ways to improve humans, animals, and ecosystems' health. This concept shows how much they are intertwined and related to one another, whether it is an environmental, or a pathological relation. This review aims to describe Poxviridae and its impact on the One Health concept, by studying the underlying causes of how poxviruses can affect the health of animals, humans, and environments. Reviewing the effect of disease transmission between animal to human, human to human, and animal to animal with pox viruses as a third party to achieve a total understanding of infection and viral transmission. Thus, contributing to enhance detection, diagnosis, research, and treatments regarding the application of One Health.

Assembly and Evolution of Poxviruses.

Poxvirus assembly has been an intriguing area of research for several decades. While advancements in experimental techniques continue to yield fresh insights, many questions are still unresolved. Large genome sizes of up to 380 kbp, asymmetrical structure, an exterior lipid bilayer, and a cytoplasmic life cycle are some notable characteristics of these viruses. Inside the particle are two lateral bodies and a protein wall-bound-biconcave core containing the viral nucleocapsid. The assembly progresses through five major stages-endoplasmic reticulum (ER) membrane alteration and rupture, crescent formation, immature virion formation, genome encapsidation, virion maturation and in a subset of viruses, additional envelopment of the virion prior to its dissemination. Several large dsDNA viruses have been shown to follow a comparable sequence of events. In this chapter, we recapitulate our understanding of the poxvirus morphogenesis process while reviewing the most recent advances in the field. We also briefly discuss how virion assembly aids in our knowledge of the evolutionary links between poxviruses and other Nucleocytoplasmic Large DNA Viruses (NCLDVs).