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.

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.

HDAC5 enhances IRF3 activation and is targeted for degradation by protein C6 from orthopoxviruses including Monkeypox virus and Variola virus.

Histone deacetylases (HDACs) regulate gene expression and innate immunity. Previously, we showed that HDAC5 is degraded during Vaccinia virus (VACV) infection and is a restriction factor for VACV and herpes simplex virus type 1. Here, we report that HDAC5 promotes interferon regulatory factor 3 (IRF3) activation downstream of Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 or Sendai virus-mediated stimulation without requiring HDAC activity. Loss of HDAC5-mediated IRF3 activation is restored by re-introduction of HDAC5 but not HDAC1 or HDAC4. The antiviral activity of HDAC5 is antagonized by VACV protein C6 and orthologs from the orthopoxviruses cowpox, rabbitpox, camelpox, monkeypox, and variola. Infection by many of these viruses induces proteasomal degradation of HDAC5, and expression of C6 alone can induce HDAC5 degradation. Mechanistically, C6 binds to the dimerization domain of HDAC5 and prevents homodimerization and heterodimerization with HDAC4. Overall, this study describes HDAC5 as a positive regulator of IRF3 activation and provides mechanistic insight into how the poxviral protein C6 binds to HDAC5 to antagonize its function.

Variola Virus and Clade I Mpox Virus Differentially Modulate Cellular Responses Longitudinally in Monocytes During Infection.

Variola virus (VARV), the etiological agent of smallpox, had enormous impacts on global health prior to its eradication. In the absence of global vaccination programs, mpox virus (MPXV) has become a growing public health threat that includes endemic and nonendemic regions across the globe. While human mpox resembles smallpox in clinical presentation, there are considerable knowledge gaps regarding conserved molecular pathogenesis between these 2 orthopoxviruses. Thus, we sought to compare MPXV and VARV infections in human monocytes through kinome analysis. We performed a longitudinal analysis of host cellular responses to VARV infection in human monocytes as well as a comparative analysis to clade I MPXV-mediated responses. While both viruses elicited strong activation of cell responses early during infection as compared to later time points, several key differences in cell signaling events were identified and validated. These observations will help in the design and development of panorthopoxvirus therapeutics.

Guidance for the identification of bony lesions related to smallpox.

OBJECTIVE: This research aimed to address the underrepresentation of smallpox (osteomyelitis variolosa) in palaeopathology, providing a synthesis of published literature and presenting guidance for the identification of osteomyelitis variolosa in non-adult and adult skeletal remains. MATERIALS AND METHODS: Literature regarding smallpox and published reports of individuals with osteomyelitis variolosa were synthesised and critiqued to produce clear diagnostic criteria for the identification of smallpox osteologically. RESULTS: Associated osteological changes begin in non-adults, where skeletal morphology is rapidly changing. Characteristic lesions associated with non-adult osteomyelitis variolosa include inflammation and destructive remodelling of long-bone joints and metaphyses. Where childhood infection was survived, residual osteomyelitis variolosa lesions should also be visible in adults in the osteoarchaeological record. CONCLUSIONS: Despite long-term clinical recognition, only limited osteological and archaeological evidence of osteomyelitis variolosa has yet emerged. With improved diagnostic criteria, osteomyelitis variolosa may be more frequently identified. SIGNIFICANCE: This is the first synthesis of osteomyelitis variolosa encompassing both clinical and palaeopathological literature, providing detailed guidance for the identification of osteomyelitis variolosa in skeletal remains. It will lead to the increased identification of smallpox osteologically. LIMITATIONS: Differential diagnoses should always be considered. The archaeological longevity of smallpox, and the potential for archaeological VARV to cause clinically recognised smallpox, is currently unknown. Characteristic bone changes in the archaeological record may be other, extinct human-infecting-orthopoxviruses. SUGGESTIONS FOR FURTHER RESEARCH: Further consideration of the implications of age of smallpox contraction on bony pathology: whether epiphyses are affected differently due to state of fusion. Reassessment of individuals previously identified with smallpox-consistent lesions, but otherwise diagnosed.

Functional epitopes and neutralizing antibodies of vaccinia virus.

Smallpox is an infectious disease caused by the variola virus, and it has a high mortality rate. Historically it has broken out in many countries and it was a great threat to human health. Smallpox was declared eradicated in 1980, and Many countries stopped nation-wide smallpox vaccinations at that time. In recent years the potential threat of bioterrorism using smallpox has led to resumed research on the treatment and prevention of smallpox. Effective ways of preventing and treating smallpox infection have been reported, including vaccination, chemical drugs, neutralizing antibodies, and clinical symptomatic therapies. Antibody treatments include anti-sera, murine monoclonal antibodies, and engineered humanized or human antibodies. Engineered antibodies are homologous, safe, and effective. The development of humanized and genetically engineered antibodies against variola virus via molecular biology and bioinformatics is therefore a potentially fruitful prospect with respect to field application. Natural smallpox virus is inaccessible, therefore most research about prevention and/or treatment of smallpox were done using vaccinia virus, which is much safer and highly homologous to smallpox. Herein we summarize vaccinia virus epitope information reported to date, and discuss neutralizing antibodies with potential value for field application.

Gene duplication, gene loss, and recombination events with variola virus shaped the complex evolutionary path of historical American horsepox-based smallpox 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.

Comparison of human monkeypox, chickenpox and smallpox: a comprehensive review of pathology and dermatological manifestations.

Variola virus, the causing agent of smallpox, was eradicated in 1980s and today no new cases are reported. The first human infectious illness to be eliminated globally is variola. On the contrary to Variola, monkeypox, which is a zoonotic and variola-like disease, has nowadays turned to be a major health problem worldwide. VZV is a neurotropic virus and the cause of varicella (chickenpox) and herpes zoster (shingles), which is also a highly infectious disease, especially prevalent in children. These three skin diseases-monkeypox, smallpox, and chickenpox-are frequently mistaken with one another due to similar manifestations including fever, rash, myalgia, chills and headache, but they can all be distinguished by their distinctive symptoms. Although these rash-causing disorders might present different skin lesions; diagnostic tests can be extremely useful in their differentiation. We searched for these concepts on a search engine like Google Scholar, scanning the results for alternative words and phrases, and examined relevant abstracts or articles for alternative words. The clinical diagnosis of monkeypox infection is commonly made based on the occurrence pattern of its skin rash. It is possible in varicella to concurrently identify lesions in their various stages including macular, papular, vesicular, pustular, and crusts; however, monkeypox lesions are all in the same stage and evolve with the same rate. In this review, we have tried to provide a holistic and comprehensive comparison between these three skin infections with a focus on the newly epidemic monkeypox, bringing about the most recent knowledge about its features and its diagnosis.

Identification of novel potential inhibitor of thymidylate kinase from Variola virus.

A hit compound was designed using Fragment Based Drug Designing (FBDD) approach, density functional theory (DFT) calculations were performed to find the structural and electronic properties. Additionally, pharmacokinetic properties were studied to understand the biological response of the compound. Docking studies were carried out with the protein structure of VrTMPK and HssTMPK with the reported hit compound. The favored docked complex was further carried to perform MD simulations; the RMSD plot and H-bond analysis was done for 200 ns. Also, MM-PBSA was done to understand the binding energy constituents and stability of the complex. A comparative study of the designed hit compound was done with FDA approved Tecovirimat. As a result, it was found that the reported compound (POX-A)is a potential selective inhibitor for Variola virus. Hence, it can be used to study further in vivo and in vitro behavior of the compound.Communicated by Ramaswamy H. Sarma.

An APOBEC3 Mutational Signature in the Genomes of Human-Infecting Orthopoxviruses.

The ongoing worldwide monkeypox outbreak is caused by viral lineages (globally referred to as hMPXV1) that are related to but distinct from clade IIb MPXV viruses transmitted within Nigeria. Analysis of the genetic differences has indicated that APOBEC-mediated editing might be responsible for the unexpectedly high number of mutations observed in hMPXV1 genomes. Here, using 1,624 publicly available hMPXV1 sequences, we analyzed the mutations that accrued between 2017 and the emergence of the current predominant variant (B.1), as well as those that that have been accumulating during the 2022 outbreak. We confirmed an overwhelming prevalence of C-to-T and G-to-A mutations, with a sequence context (5'-TC-3') consistent with the preferences of several human APOBEC3 enzymes. We also found that mutations preferentially occur in highly expressed viral genes, although no transcriptional asymmetry was observed. A comparison of the mutation spectrum and context was also performed against the human-specific variola virus (VARV) and the zoonotic cowpox virus (CPXV), as well as fowlpox virus (FWPV). The results indicated that in VARV genomes, C-to-T and G-to-A changes were more common than the opposite substitutions, although the effect was less marked than for hMPXV1. Conversely, no preference toward C-to-T and G-to-A changes was observed in CPXV and FWPV. Consistently, the sequence context of C-to-T changes confirmed a preference for a T in the -1 position for VARV, but not for CPXV or FWPV. Overall, our results strongly support the view that, irrespective of the transmission route, orthopoxviruses infecting humans are edited by the host APOBEC3 enzymes. IMPORTANCE Analysis of the viral lineages responsible for the 2022 monkeypox outbreak suggested that APOBEC enzymes are driving hMPXV1 evolution. Using 1,624 public sequences, we analyzed the mutations that accumulated between 2017 and the emergence of the predominant variant and those that characterize the last outbreak. We found that the mutation spectrum of hMPXV1 has been dominated by TC-to-TT and GA-to-AA changes, consistent with the editing activity of human APOBEC3 proteins. We also found that mutations preferentially affect highly expressed viral genes, possibly because transcription exposes single-stranded DNA (ssDNA), a target of APOBEC3 editing. Notably, analysis of the human-specific variola virus (VARV) and the zoonotic cowpox virus (CPXV) indicated that in VARV genomes, TC-to-TT and GA-to-AA changes are likewise extremely frequent. Conversely, no preference toward TC-to-TT and GA-to-AA changes is observed in CPXV. These results suggest that APOBEC3 proteins have an impact on the evolution of different human-infecting orthopoxviruses.

Analysis of variola virus molecular evolution suggests an old origin of the virus consistent with historical records.

Archaeovirology efforts provided a rich portrait of the evolutionary history of variola virus (VARV, the cause of smallpox), which was characterized by lineage extinctions and a relatively recent origin of the virus as a human pathogen (~1700 years ago, ya). This contrasts with historical records suggesting the presence of smallpox as early as 3500 ya. By performing an analysis of ancestry components in modern, historic, and ancient genomes, we unveil the progressive drifting of VARV lineages from a common ancestral population and we show that a small proportion of Viking Age ancestry persisted until the 18th century. After the split of the P-I and P-II lineages, the former experienced a severe bottleneck. With respect to the emergence of VARV as a human pathogen, we revise time estimates by accounting for the time-dependent rate phenomenon. We thus estimate that VARV emerged earlier than 3800 ya, supporting its presence in ancient societies, as pockmarked Egyptian mummies suggest.

Pathogen genomics study of an early medieval community in Germany reveals extensive co-infections.

BACKGROUND: The pathogen landscape in the Early European Middle Ages remains largely unexplored. Here, we perform a systematic pathogen screening of the rural community Lauchheim "Mittelhofen," in present-day Germany, dated to the Merovingian period, between fifth and eighth century CE. Skeletal remains of individuals were subjected to an ancient DNA metagenomic analysis. Genomes of the detected pathogens were reconstructed and analyzed phylogenetically. RESULTS: Over 30% of the individuals exhibit molecular signs of infection with hepatitis B virus (HBV), parvovirus B19, variola virus (VARV), and Mycobacterium leprae. Seven double and one triple infection were detected. We reconstructed four HBV genomes and one genome each of B19, VARV, and M. leprae. All HBV genomes are of genotype D4 which is rare in Europe today. The VARV strain exhibits a unique pattern of gene loss indicating that viruses with different gene compositions were circulating in the Early Middle Ages. The M. leprae strain clustered in branch 3 together with the oldest to-date genome from the UK. CONCLUSIONS: The high burden of infectious disease, together with osteological markers of physiological stress, reflect a poor health status of the community. This could have been an indirect result of the climate decline in Europe at the time, caused by the Late Antique Little Ice Age (LALIA). Our findings suggest that LALIA may have created an ecological context in which persistent outbreaks set the stage for major epidemics of severe diseases such as leprosy and smallpox hundreds of years later.

Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens.

As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and the population's immunity to other OPV-related diseases such as MPX. In parallel, there was a need for differential diagnosis between the different OPVs' clinical manifestations and diseases with similar symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a rapid genetic-based diagnostic tool for accurate and specific identification of MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic regions that harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5'-phosphonate were simultaneously added, thus making the PCR product ligation ready for nanopore sequencing. Following rapid sequencing (a few minutes), the reads were compared to a reference database and the nearest strain was identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced during the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we obtained definite results, identifying other vesicle-forming viruses: Human herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was a proof-of-concept study, demonstrating the potential of the RANS approach for rapid and discriminatory identification of a panel of closely related pathogens. The simplicity and affordability of our approach makes it straightforward to implement in any genetics lab. Moreover, other differential diagnostics panels might benefit from the implementation of the RANS approach into their diagnostics pipelines.

Phylogeographical and evolutionary history of variola major virus; a question of timescales?

Aim of this study was to reconstruct the phylogeography of variola virus (VARV) in the XX century, using 47 VARV whole genome sequences available in public databases, through two different methods for ancestral character reconstruction: a frequently used Bayesian framework and a fast maximum-likelihood (ML) based method. The substitution rate of the whole VARV genome was estimated to be between 6.7×10-6 and 1.1×10-5 substitutions/site/year. Both ML and Bayesian methods gave similar trees topology, showing two distinct monophyletic groups: one (known as P1) including the great part of variola major and the second (P2) including West African and American (variola minor) isolates and close evolutionary rate estimations, between 6.73×10-6 and 1.1×10-5 for the whole genome. The phylogeographical reconstruction of P1 suggested that the common ancestor of the variola major circulating in the Old World between the 1940s and the 1970s most probably originated in the Far East in the first decades of the XX century, and then spread to Indian subcontinent in the 1920s. India represented a center of further spread of VARV to eastern Africa in the 1940s and to the Middle East in the 1960s. The phylogeographic scenario obtained by the maximum-likelihood based method was congruent with that obtained by Bayesian framework, but the analysis was faster indicating the usefulness of this method in the analyses of large viral genomes. Our results may help to explain the controversial reconstructions of the history of VARV obtained using long or short timescale for calibration.

An Update of Orthopoxvirus Molecular Evolution.

Although variola virus (VARV) has been eradicated through widespread vaccination, other orthopoxviruses pathogenic for humans circulate in nature. Recently, new orthopoxviruses, including some able to infect humans, have been found and their complete genomes have been sequenced. Questions about the orthopoxvirus mutation rate and the emergence of new threats to humankind as a result of the evolution of circulating orthopoxviruses remain open. Based on contemporary data on ancient VARV DNA and DNA of new orthopoxvirus species, an analysis of the molecular evolution of orthopoxviruses was carried out and the timescale of their emergence was estimated. It was calculated that the orthopoxviruses of the Old and New Worlds separated approximately 40,000 years ago; the recently discovered Akhmeta virus and Alaskapox virus separated from other orthopoxviruses approximately 10,000-20,000 years ago; the rest of modern orthopoxvirus species originated from 1700 to 6000 years ago, with the exception of VARV, which emerged in approximately 300 AD. Later, there was a separation of genetic variants of some orthopoxvirus species, so the monkeypox virus West African subtype originated approximately 600 years ago, and the VARV minor alastrim subtype emerged approximately 300 years ago.

Smallpox, Monkeypox and Other Human Orthopoxvirus Infections.

Considering that vaccination against smallpox with live vaccinia virus led to serious adverse effects in some cases, the WHO, after declaration of the global eradication of smallpox in 1980, strongly recommended to discontinue the vaccination in all countries. This led to the loss of immunity against not only smallpox but also other zoonotic orthopoxvirus infections in humans over the past years. An increasing number of human infections with zoonotic orthopoxviruses and, first of all, monkeypox, force us to reconsider a possible re-emergence of smallpox or a similar disease as a result of natural evolution of these viruses. The review contains a brief analysis of the results of studies on genomic organization and evolution of human pathogenic orthopoxviruses, development of modern methods for diagnosis, vaccination, and chemotherapy of smallpox, monkeypox, and other zoonotic human orthopoxvirus infections.

Comment on the article "With regard to the bicentennial of the independence of Colombia: Reading practices of Antonio Nariño and the development of a presumably effective vaccine against smallpox / [Comentario en torno al artículo "A propósito del bicentenario de la independencia de Colombia: las prácticas de lectura de Antonio Nariño y el desarrollo de una vacuna presuntamente efectiva contra la viruela"].

For the American colonies, smallpox implied a process that dramatically destabilized their sociodemographic dynamics, which explains why scientific development took place around the causative virus. Each book about smallpox in Nariño's library was a tool in the fight against smallpox undertaken by the founding father. After reviewing the article "About the bicentennial of the independence of Colombia: The reading practices of Antonio Nariño and the development of a vaccine that is presumably effective against smallpox" (1), I set myself to study Antonio Nariño's medical knowledge further. Through the approach to the works that Nariño used to educate himself on smallpox and the development of a biographical sketch of each of them, I analyzed the process of variolization in the Kingdom of Nueva Granada and the need to manufacture a vaccine locally.

La viruela significó para las colonias americanas un proceso que desestabilizaba de forma dramática las dinámicas sociodemográficas de las colonias, lo que incentivó el desarrollo de estudios científicos sobre el virus causante. Cada libro acerca de la viruela en la biblioteca de Nariño constituyó una herramienta en la lucha contra el virus emprendida por el prócer. Tras la revisión del artículo "A propósito del bicentenario de la independencia de Colombia: las prácticas de lectura de Antonio Nariño y el desarrollo de una vacuna presuntamente efectiva contra la viruela" (1) quise comentar y profundizar en torno al saber médico de Nariño mediante el acercamiento a las obras a las que recurrió para instruirse sobre la enfermedad. A partir de la semblanza de cada una de ellas, analicé el proceso de variolización en el Reino de la Nueva Granada y la necesidad de fabricar una vacuna propia.

In Silico Studies of Potential Selective Inhibitors of Thymidylate Kinase from Variola virus.

Continuing the work developed by our research group, in the present manuscript, we performed a theoretical study of 10 new structures derived from the antivirals cidofovir and ribavirin, as inhibitor prototypes for the enzyme thymidylate kinase from Variola virus (VarTMPK). The proposed structures were subjected to docking calculations, molecular dynamics simulations, and free energy calculations, using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, inside the active sites of VarTMPK and human TMPK (HssTMPK). The docking and molecular dynamic studies pointed to structures 2, 3, 4, 6, and 9 as more selective towards VarTMPK. In addition, the free energy data calculated through the MM-PBSA method, corroborated these results. This suggests that these compounds are potential selective inhibitors of VarTMPK and, thus, can be considered as template molecules to be synthesized and experimentally evaluated against smallpox.