Biology of Variola Virus.

Variola virus is an anthroponotic agent that belongs to the orthopoxvirus family. It is an etiological agent of smallpox, an ancient disease that caused massive mortality of human populations. Twentieth century has witnessed the death of about 300 million people due to the unavailability of an effective vaccine. Early detection is the primary strategy to prevent an outbreak of smallpox. Variola virus forms the characteristic pus-filled pustules and centrifugal rash distribution in the infected patients while transmission occurs mainly through respiratory droplets during the early stage of infection. No antiviral drugs are approved for variola virus till date. Generation of first-generation vaccines helped in the eradication of smallpox which was declared by the World Health Organization.

Poxviridae Pneumonia.

Poxviridae family includes several viruses that infecting humans usually causes skin lesions only, but in some cases their clinical course is complicated by viral pneumonia (with or without bacterial superinfections). Historically variola virus has been the poxviridae most frequently associated with the development of pneumonia with many large outbreaks worldwide before its eradication in 1980. It is still considered a biological threat for its potential in biological warfare and bioterrorism. Smallpox pneumonia can be severe with the onset of acute respiratory distress syndrome (ARDS) and death. Vaccinia virus, used for vaccination against smallpox exceptionally, in immunocompromised patients, can induce generalized (with also lung involvement) severe disease after vaccination. MPXV virus occasionally can cause pneumonia particularly in immunocompromised patients. The pathophysiology of poxviridae pneumonia is still an area of active research; however, in animal models these viruses can cause both direct damage to the lower airways epithelium and a hyperinflammatory syndrome, like a cytokine storm. Multiple mechanisms of immune evasion have also been described. The treatment of poxviridae pneumonia is mainly based on careful supportive care. Despite the absence of randomized clinical trials in patients with poxviridae pneumonia there are antiviral drugs, such as tecovirimat, cidofovir and brincidofovir, FDA-approved for use in smallpox and also available under an expanded access protocol for treatment of MPXV. There are 2 (replication-deficient modified vaccinia Ankara and replication-competent vaccinia virus) smallpox vaccines FDA-approved with the first one also approved for prevention of MPXV in adults that are at high risk of infection.

Treatment and Vaccination for Smallpox and Monkeypox.

The smallpox infection with the variola virus was one of the most fatal disorders until a global eradication was initiated in the twentieth century. The last cases were reported in Somalia 1977 and as a laboratory infection in the UK 1978; in 1980, the World Health Organization (WHO) declared smallpox for extinct. The smallpox virus with its very high transmissibility and mortality is still a major biothreat, because the vaccination against smallpox was stopped globally in the 1980s. For this reason, new antivirals (cidofovir, brincidofovir, and tecovirimat) and new vaccines (ACAM2000, LC16m8 and Modified Vaccine Ankara MVA) were developed. For passive immunization, vaccinia immune globulin intravenous (VIGIV) is available. Due to the relationships between orthopox viruses such as vaccinia, variola, mpox (monkeypox), cowpox, and horsepox, the vaccines (LC16m8 and MVA) and antivirals (brincidofovir and tecovirimat) could also be used in the mpox outbreak with positive preliminary data. As mutations can result in drug resistance against cidofovir or tecovirimat, there is need for further research. Further antivirals (NIOCH-14 and ST-357) and vaccines (VACΔ6 and TNX-801) are being developed in Russia and the USA. In conclusion, further research for treatment and prevention of orthopox infections is needed and is already in progress. After a brief introduction, this chapter presents the smallpox and mpox disease and thereafter full overviews on antiviral treatment and vaccination including the passive immunization with vaccinia immunoglobulins.

[Poxvirus-encoded DNA replication proteins: potential targets for antivirals]. / Le complexe de réplication des poxvirus : cible potentielle de molécules antivirales.

In the spring of 2022, an epidemic due to human monkeypox virus (MPXV) of unprecedented magnitude spread across all continents. Although this event was surprising in its suddenness, the resurgence of a virus from the Poxviridae family is not surprising in a world population that has been largely naïve to these viruses since the eradication of the smallpox virus in 1980 and the concomitant cessation of vaccination. Since then, a vaccine and two antiviral compounds have been developed to combat a possible return of smallpox. However, the use of these treatments during the 2022 MPXV epidemic showed certain limitations, indicating the importance of continuing to develop the therapeutic arsenal against these viruses. For several decades, efforts to understand the molecular mechanisms involved in the synthesis of the DNA genome of these viruses have been ongoing. Although many questions remain unanswered up to now, the three-dimensional structures of essential proteins, and in particular of the DNA polymerase holoenzyme in complex with DNA, make it possible to consider the development of a model for poxvirus DNA replication. In addition, these structures are valuable tools for the development of new antivirals targeting viral genome synthesis. This review will first present the molecules approved for the treatment of poxvirus infections, followed by a review of our knowledge of the replication machinery of these viruses. Finally, we will describe how these proteins could be the target of new antiviral compounds.

Bayesian Phylogeography and Pathogenic Characterization of Smallpox Based on HA, ATI, and CrmB Genes.

Variola virus is at risk of re-emergence either through accidental release, bioterrorism, or synthetic biology. The use of phylogenetics and phylogeography to support epidemic field response is expected to grow as sequencing technology becomes miniaturized, cheap, and ubiquitous. In this study, we aimed to explore the use of common VARV diagnostic targets hemagglutinin (HA), cytokine response modifier B (CrmB), and A-type inclusion protein (ATI) for phylogenetic characterization as well as the representativeness of modelling strategies in phylogeography to support epidemic response should smallpox re-emerge. We used Bayesian discrete-trait phylogeography using the most complete data set currently available of whole genome (n = 51) and partially sequenced (n = 20) VARV isolates. We show that multilocus models combining HA, ATI, and CrmB genes may represent a useful heuristic to differentiate between VARV Major and subclades of VARV Minor which have been associated with variable case-fatality rates. Where whole genome sequencing is unavailable, phylogeography models of HA, ATI, and CrmB may provide preliminary but uncertain estimates of transmission, while supplementing whole genome models with additional isolates sequenced only for HA can improve sample representativeness, maintaining similar support for transmission relative to whole genome models. We have also provided empirical evidence delineating historic international VARV transmission using phylogeography. Due to the persistent threat of re-emergence, our results provide important research for smallpox epidemic preparedness in the posteradication era as recommended by the World Health Organisation.

Smallpox Readiness: Modern Strategies Against an Ancient Disease.

This Viewpoint highlights the potential for unintentional or deliberate release of variola virus (smallpox), discusses current medical countermeasures for smallpox, and calls for greater flexibility from the US and its partners in developing safe, reliable, affordable, and equitable countermeasures.

An increasing danger of zoonotic orthopoxvirus infections.

On May 8, 1980, the World Health Assembly at its 33(rd) session solemnly declared that the world and all its peoples had won freedom from smallpox and recommended ceasing the vaccination of the population against smallpox. Currently, a larger part of the world population has no immunity not only against smallpox but also against other zoonotic orthopoxvirus infections. Recently, recorded outbreaks of orthopoxvirus diseases not only of domestic animals but also of humans have become more frequent. All this indicates a new situation in the ecology and evolution of zoonotic orthopoxviruses. Analysis of state-of-the-art data on the phylogenetic relationships, ecology, and host range of orthopoxviruses--etiological agents of smallpox (variola virus, VARV), monkeypox (MPXV), cowpox (CPXV), vaccinia (VACV), and camelpox (CMLV)--as well as the patterns of their evolution suggests that a VARV-like virus could emerge in the course of natural evolution of modern zoonotic orthopoxviruses. Thus, there is an insistent need for organization of the international control over the outbreaks of zoonotic orthopoxvirus infections in various countries to provide a rapid response and prevent them from developing into epidemics.

Poxvirus targeting of E3 ligase ß-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.

The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKß causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase ß-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished ß-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

Variola type IB DNA topoisomerase: DNA binding and supercoil unwinding using engineered DNA minicircles.

Type IB topoisomerases unwind positive and negative DNA supercoils and play a key role in removing supercoils that would otherwise accumulate at replication and transcription forks. An interesting question is whether topoisomerase activity is regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The type IB enzyme from variola virus (vTopo) has proven to be useful in addressing mechanistic questions about topoisomerase function because it forms a reversible 3'-phosphotyrosyl adduct with the DNA backbone at a specific target sequence (5'-CCCTT-3') from which DNA unwinding can proceed. We have synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site that provides highly defined substrates for exploring the effects of supercoil density on DNA binding, strand cleavage and ligation, and unwinding. We observed no topological dependence for binding of vTopo to these supercoiled MC DNAs, indicating that affinity-based targeting to supercoiled DNA regions by vTopo is unlikely. Similarly, the cleavage and religation rates of the MCs were not topologically dependent, but topoisomers with low superhelical densities were found to unwind more slowly than highly supercoiled topoisomers, suggesting that reduced torque at low superhelical densities leads to an increased number of cycles of cleavage and ligation before a successful unwinding event. The K271E charge reversal mutant has an impaired interaction with the rotating DNA segment that leads to an increase in the number of supercoils that were unwound per cleavage event. This result provides evidence that interactions of the enzyme with the rotating DNA segment can restrict the number of supercoils that are unwound. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to be important in regulating the steady-state superhelical density of DNA domains in the cell.

Designing a smallpox B-cell and T-cell multi-epitope subunit vaccine using a comprehensive immunoinformatics approach.

Smallpox is a highly contagious human disease caused by the variola virus. Although the disease was eliminated in 1979 due to its highly contagious nature and historical pathogenicity, with a mortality rate of up to 30%, this virus is an important candidate for biological weapons. Currently, vaccines are the critical measures to prevent this virus infection and spread. In this study, we designed a peptide vaccine using immunoinformatics tools, which have the potential to activate human immunity against variola virus infection efficiently. The design of peptides derives from vaccine-candidate proteins showing protective potential in vaccinia WR strains. Potential non-toxic and nonallergenic T-cell and B-cell binding and cytokine-inducing epitopes were then screened through a priority prediction using special linkers to connect B-cell epitopes and T-cell epitopes, and an appropriate adjuvant was added to the vaccine construction to enhance the immunogenicity of the peptide vaccine. The 3D structure display, docking, and free energy calculation analysis indicate that the binding affinity between the vaccine peptide and Toll-like receptor 3 is high, and the vaccine receptor complex is highly stable. Notably, the vaccine we designed is obtained from the protective protein of the vaccinia and combined with preventive measures to avoid side effects. This vaccine is highly likely to produce an effective and safe immune response against the variola virus infection in the body. IMPORTANCE: In this work, we designed a vaccine with a cluster of multiple T-cell/B-cell epitopes, which should be effective in inducing systematic immune responses against variola virus infection. Besides, this work also provides a reference in vaccine design for preventing monkeypox virus infection, which is currently prevalent.

Activity, specificity, and probe design for the smallpox virus protease K7L.

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

Structural basis of chemokine sequestration by CrmD, a poxvirus-encoded tumor necrosis factor receptor.

Pathogens have evolved sophisticated mechanisms to evade detection and destruction by the host immune system. Large DNA viruses encode homologues of chemokines and their receptors, as well as chemokine-binding proteins (CKBPs) to modulate the chemokine network in host response. The SECRET domain (smallpox virus-encoded chemokine receptor) represents a new family of viral CKBPs that binds a subset of chemokines from different classes to inhibit their activities, either independently or fused with viral tumor necrosis factor receptors (vTNFRs). Here we present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a ß-sandwich fold and utilizes its ß-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs. Structure-based mutagenesis and biochemical analysis identified important basic residues in the 40s loop of CX3CL1 for the interaction. Mutation of corresponding acidic residues in the SECRET domain also affected the binding for other chemokines, indicating that the SECRET domain binds different chemokines in a similar manner. We further showed that heparin inhibited the binding of CX3CL1 by the SECRET domain and the SECRET domain inhibited RAW264.7 cell migration induced by CX3CL1. These results together shed light on the structural basis for the SECRET domain to inhibit chemokine activities by interfering with both chemokine-GAG and chemokine-receptor interactions.

Smallpox: can we still learn from the journey to eradication?

One of the most celebrated achievements of immunology and modern medicine is the eradication of the dreaded plague smallpox. From the introduction of smallpox vaccination by Edward Jenner, to its popularization by Louis Pasteur, to the eradication effort led by Donald Henderson, this story has many lessons for us today, including the characteristics of the disease and vaccine that permitted its eradication, and the obviousness of the vaccine as a vector for other intractable Infectious diseases. The disease itself, interpreted in the light of modern molecular immunology, is an obvious immunopathological disease, which occurs after a latent interval of 1-2 weeks, and manifests as a systemic cell-mediated delayed type hypersensitivity (DTH) syndrome. The vaccine that slayed this dragon was given the name vaccinia, and was thought to have evolved from cowpox virus, but is now known to be most closely related to a poxvirus isolated from a horse. Of interest is the fact that of the various isolates of orthopox viruses, only variola, vaccinia and monkeypox viruses can infect humans. In contrast to the systemic disease of variola, vaccinia only replicates locally at the site of inoculation, and causes a localized DTH response that usually peaks after 7-10 days. This difference in the pathogenicity of variola vs. vaccinia is thought to be due to the capacity of variola to circumvent innate immunity, which allows it to disseminate widely before the adaptive immune response occurs. Thus, the fact that vaccinia virus is attenuated compared to variola, but is still replication competent, makes for its remarkable efficacy as a vaccine, as the localized infection activates all of the cells and molecules of both innate and adaptive immunity. Accordingly vaccinia itself, and not modified replication incompetent vaccina, is the hope for use as a vector in the eradication of additional pathogenic microbes from the globe.

Extraction-free LAMP assays for generic detection of Old World Orthopoxviruses and specific detection of Mpox virus.

Mpox is a neglected zoonotic disease endemic in West and Central Africa. The Mpox outbreak with more than 90,000 cases worldwide since 2022 generated great concern about future outbreaks and highlighted the need for a simple and rapid diagnostic test. The Mpox virus, MPV, is a member of the Orthopoxvirus (OPV) genus that also contains other pathogenic viruses including variola virus, vaccinia virus, camelpox virus, and cowpox virus. Phylogenomic analysis of 200 OPV genomes identified 10 distinct phylogroups with the New World OPVs placed on a very long branch distant from the Old World OPVs. Isolates derived from infected humans were found to be distributed across multiple phylogroups interspersed with isolates from animal sources, indicating the zoonotic potential of these viruses. In this study, we developed a simple and sensitive colorimetric LAMP assay for generic detection of Old World OPVs. We also developed an MPV-specific probe that differentiates MPV from other OPVs in the N1R LAMP assay. In addition, we described an extraction-free protocol for use directly with swab eluates in LAMP assays, thereby eliminating the time and resources needed to extract DNA from the sample. Our direct LAMP assays are well-suited for low-resource settings and provide a valuable tool for rapid and scalable diagnosis and surveillance of OPVs and MPV.

[The rapid ELISA method for detection of orthopoxviruses].

INTRODUCTION: Following the successful eradication of smallpox, mass vaccination against this disease was discontinued in 1980. The unvaccinated population continues to be at risk of infection due to military use of variola virus or exposure to monkeypox virus in Africa and non-endemic areas. In cases of these diseases, rapid diagnosis is of great importance, since the promptness and effectiveness of therapeutic and quarantine measures depend on it. The aim of work is to develop a kit of reagents for enzyme-linked immunosorbent assay (ELISA) for fast and highly sensitive detection of orthopoxviruses (OPV) in clinical samples. MATERIALS AND METHODS: The efficiency of virus detection was evaluated by single-stage ELISA in the cryolisate of CV-1 cell culture samples infected with vaccinia, cowpox, rabbitpox, and ectromelia viruses, as well as in clinical samples of infected rabbits and mice. RESULTS: The method of rapid ELISA was shown to allow the detection of OPV in crude viral samples in the range of 5.0 1025.0 103 PFU/ml, and in clinical samples with a viral load exceeding 5 103 PFU/ml. CONCLUSIONS: The assay involves a minimum number of operations and can be performed within 45 minutes, which makes it possible to use it in conditions of a high level of biosecurity. Rapid ELISA method was developed using polyclonal antibodies, which significantly simplifies and reduces the cost of manufacturing a diagnostic system.