Development of a multiplex real-time PCR assay for the simultaneous detection of mpox virus and orthopoxvirus infections.

Since May 2022, the multi-country outbreak of monkeypox (mpox) has raised a great concern worldwide. Early detection of mpox virus infection is recognized as an efficient way to prevent mpox transmission. Mpox specific detection methods reported up to now are based on the SNPs among mpox virus and other orthopoxviruses. We have therefore developed a real-time PCR based mpox detection method targeting mpox virus specific sequences (N3R and B18Rplus). We have also optimized an orthopoxvirus detection system which targets the highly conserved E9L and D6R genes. The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction) and specificity. Mpox viral DNA and clinical samples from mpox patients are detected with the mpox detection system. Furthermore, we have established a multiplex real-time PCR detection system allowing simultaneous and efficient detection of mpox and orthopoxvirus infections.

Molecular Virology of Orthopoxviruses with Special Reference to Monkeypox Virus.

Poxviruses are large (200-450 nm) and enveloped viruses carrying double-stranded DNA genome with an epidermal cell-specific adaptation. The genus Orthopoxvirus within Poxviridae family constitutes several medically and veterinary important viruses including variola (smallpox), vaccinia, monkeypox virus (MPXV), and cowpox. The monkeypox disease (mpox) has recently emerged as a public health emergency caused by MPXV. An increasing number of human cases of MPXV have been documented in non-endemic nations without any known history of contact with animals brought in from endemic and enzootic regions, nor have they involved travel to an area where the virus was typically prevalent. Here, we review the MPXV replication, virus pathobiology, mechanism of viral infection transmission, virus evasion the host innate immunity and antiviral therapies against Mpox. Moreover, preventive measures including vaccination were discussed and concluded that cross-protection against MPXV may be possible using antibodies that are directed against an Orthopoxvirus. Despite the lack of a specialised antiviral medication, several compounds such as Cidofovir and Ribavirin warrant consideration against mpox.

Safety and Efficacy of the Modified Vaccinia Ankara-Bavaria Nordic Vaccine Against Mpox in the Real World: Systematic Review and Meta-Analysis.

In May 2022, mpox began to spread worldwide, posing a serious threat to human public health. Modified Vaccinia Ankara-Bavaria Nordic (MVA-BN) is a live attenuated orthopoxvirus vaccine that has been authorized by the U.S. Food and Drug Administration as the vaccine of choice for the prevention of mpox. In this study, we conducted a meta-analysis of all currently published literature on the efficacy and safety of the MVA-BN vaccine in the real world, showing that the MVA-BN vaccine is effective and safe, with efficacy of up to 75% with a single dose and up to 80% with a two-dose vaccine. Meanwhile, we found that subcutaneous injection has lower local and systemic adverse events than intradermal injection, regardless of single- or two-dose vaccination, and subcutaneous injection is better tolerated in children, the elderly, or people with underlying medical conditions. These results have important reference value for clinical practice.

Exploring Viral Genome Profile in Mpox Patients during the 2022 Outbreak, in a North-Eastern Centre of Italy.

In 2022, an unprecedented outbreak of mpox raged in several nations. Sequences from the 2022 outbreak reveal a higher nucleotide substitution if compared with the estimated rate for orthopoxviruses. Recently, intra-lesion SNVs (single nucleotide variants) have been described, and these have been suggested as possible sources of genetic variation. Until now, it has not been clear if the presence of several SNVs could represents the result of local mutagenesis or a possible co-infection. We investigated the significance of SNVs through whole-genome sequencing analysis of four unrelated mpox cases. In addition to the known mutations harboured by the circulating strains of virus (MPXV), 7 novel mutations were identified, including SNVs located in genes that are involved in immune evasion mechanisms and/or viral fitness, six of these appeared to be APOBEC3-driven. Interestingly, three patients exhibited the coexistence of mutated and wild-type alleles for five non-synonymous variants. In addition, two patients, apparently unrelated, showed an analogous pattern for two novel mutations, albeit with divergent frequencies. The coexistence of mixed viral populations, harbouring non-synonymous mutations in patients, supports the hypothesis of possible co-infection. Additional investigations of larger clinical cohorts are essential to validating intra-patient viral genome heterogeneity and determining the possibility of co-presence events of slightly divergent MPXV strains.

Breakthrough Mpox Outbreak Investigation, the Delicate Balance Between Host Immune Response and Viral Immune Escape.

BACKGROUND: Limited data are available on Mpox breakthrough infections. PURPOSE: The purpose of this study is to investigate a Mpox breakthrough outbreak in 3 vaccinated individuals. METHODS: Study participants provided informed consent. Serology testing was performed in one involved individual (ID-1) using an in-house assay detecting anti-orthopoxvirus IgG. Whole genome sequencing (WGS) was carried out and compared with the reference sequence ON563414.3 ( https://www.ncbi.nlm.nih.gov/nuccore/ON563414.3/ ). RESULTS: Three individuals vaccinated with modified vaccinia Ankara-Bavaria Nordic contracted Mpox following one sexual intercourse event. One of them (ID-1) had received only one vaccine dose, while the other two were fully vaccinated. ID-1 presented to the sexual health clinic of the Universitair Ziekenhuis Brussel with proctitis related to Mpox. Despite one vaccination, serology testing Three months post vaccine showed absence of Mpox virus (MPXV) specific antibodies in ID-1. In contrast, 2 weeks after the sexual intercourse, seroconversion occurred. Whole genome sequencing of the isolated MPXV showed, compared with the reference sequence, a total of seven single nucleotide variants with four of them indicating protein amino-acid changes. CONCLUSION: Incomplete MPXV vaccination as well as MPXV variants might result in breakthrough infections. Preventive measures, such as MPVX vaccination, could maintain immunity in individuals with higher risk of MPXV infection, and might lower disease severity.

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.

Molecular investigations of camelpox virus circulating in dromedary camel population in Rajasthan, India.

Camelpox is an important viral disease of dromedary camel in Rajasthan, India. In the present study, partial C18L gene sequences (n = 6) of camelpox virus (CMLV) obtained in an outbreak in Bikaner, Rajasthan, India in year 2022 were compared with other similar sequences obtained in the past in similar geographical location. Clinical and epidemiological features of the disease were also compared. Genomic study suggested variations in C18L gene sequences obtained in the present outbreak from those obtained during the past outbreaks. CMLV were genetically different from cowpox viruses, but appeared identical to CMLV causing disease in Israel, Egypt and Kazakhstan. Genomes of CMLV virus circulating in dromedary camel population of Rajasthan, India appeared diverse and changing, hence complete genome sequencing and identification of genomic changes altering infectivity and pathogenicity is warranted for designing control strategies.

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.

Rendezvous with Vaccinia Virus in the Post-smallpox Era: R&D Advances.

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.

Evaluation of a Zoonotic Orthopoxvirus PCR Assay for the Detection of Mpox Virus Infection.

An epidemic caused by an outbreak of mpox (formerly monkeypox) in May 2022 rapidly spread internationally, requiring an urgent response from the clinical diagnostics community. A detailed description of the clinical validation and implementation of a laboratory-developed real-time PCR test for detecting nonvariola Orthopoxvirus-specific DNA based on the newly designed RealStar Zoonotic Orthopoxvirus assay is presented. The validation was performed using an accuracy panel (n = 97) comprising skin lesion swabs in universal transport media and from mpox virus genomic DNA spiked into pooled mpox virus-negative remnant universal transport media of lesion specimens submitted for routine clinical testing in the NewYork-Presbyterian Hospital clinical laboratory system. Accuracy testing demonstrated excellent assay agreement between expected and observed results and comparable diagnostic performance to three different reference tests. Analytical sensitivity with 95% detection probability was 126 copies/mL, and analytical specificity, clinical sensitivity, and clinical specificity were 100%. In summary, the RealStar Zoonotic Orthopoxvirus assay provides a sensitive and reliable method for routine diagnosis of mpox infections.

CRISPR-Cas12a assisted specific detection of mpox virus.

Mpox virus, a member of genus Orthopoxvirus, causes rash and flu-like symptoms in humans. In the recent global outbreak, it was reported from several geographical areas that have not historically reported mpox. Point of care, sensitive and specific mpox diagnostic assays are critical in checking the spread of the disease. We have developed a clustered regularly interspaced short palindromic repeats associated Cas12a nuclease-based assay for detecting mpox virus. Mpox specific conserved sequences were identified in polA (E9L) gene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in the genus Orthopoxvirus. This SNP was exploited in our assay to specifically distinguish mpox virus from other related orthopox viruses with a limit of detection of 1 copy/µl in 30 min. The assay exhibits a sensitive and specific detection of mpox virus which can prove to be of practical value for its surveillance in areas infected with multiple orthopox viruses, especially in hotspots of mpox virus infections.

[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.

Systems biology of the genomes' microsatellite signature of Orthopoxvirus including the Monkeypox virus.

This study is an attempt to extract and analyse the microsatellites or simple sequence repeats (SSRs) from the genomes of eight species of the genus Orthopoxvirus. The average size of genomes included in the study was 205 kb while the GC% was 33% for all but one. A total of 10,584 SSRs and 854 cSSRs were observed. POX2 with the largest genome of 224.499 kb had maximum of 1493 SSRs and 121 cSSRs (compound SSR) while POX7 with the smallest genome of 185.578 kb had minimum incident SSRs and cSSRs at 1181 and 96, respectively. There was significant correlation between genome size and SSR incidence. Di-nucleotide repeats were the most prevalent (57.47%) followed by mono- at 33% and tri- at 8.6%. Mono-nucleotide SSRs were predominantly T (51%) and A (48.4%). A majority of 80.32% SSRs were in the coding region. The three most similar genomes as per heat map POX1, POX7 and POX5 (93% similarity) are adjacent to one another in the phylogenetic tree. Ankyrin/Ankyrin like protein and Kelch protein which are associated with host determination and divergence have the highest SSR density in almost all studied viruses. Thus, SSRs are involved in genome evolution and host determination of viruses.

Three neutralizing mAbs induced by MPXV A29L protein recognizing different epitopes act synergistically against orthopoxvirus.

The worldwide outbreak of the monkeypox virus (MPXV) has become a "Public Health Emergency of International Concern" (PHEIC). Severe monkeypox virus infection can be fatal, however, effective therapeutic methods are yet to be developed. Mice were immunized with A35R protein and A29L protein of MPXV, and the binding and neutralizing activities of the immune sera against poxvirus-associated antigens and viruses were identified. A29L protein and A35R protein-specific monoclonal antibodies (mAbs) were generated and their antiviral activities of these mAbs were characterized in vitro and in vivo. Immunization with the MPXV A29L protein and A35R protein induced neutralizing antibodies against the orthopoxvirus in mice. None of the mAbs screened in this study against A35R could effectively neutralize the vaccinia virus (VACV), while three mAbs against A29L protein, 9F8, 3A1 and 2D1 were confirmed to have strong broad binding and neutralizing activities against orthopoxvirus, among which 9F8 showed the best neutralizing activity. 9F8, 3A1, and 2D1 recognized different epitopes on MPXV A29L protein, showing synergistic antiviral activity in vitro against the VACV Tian Tan and WR strains; the best activity was observed when the three antibodies were combined. In the vivo antiviral prophylactic and therapeutic experiments, 9F8 showed complete protective activity, whereas 3A1 and 2D1 showed partial protective activity. Similarly, the three antibodies showed synergistic antiviral protective activity against the two VACVs. In conclusion, three mAbs recognized different epitopes on MPXV A29L protein were developed and showed synergistic effects against orthopoxvirus.

EPIPOX: A Resource Facilitating Epitope-Vaccine Design Against Human Pathogenic Orthopoxviruses.

EPIPOX is a specialized online resource intended to facilitate the design of epitope-based vaccines against orthopoxviruses. EPIPOX is built upon a collection of T cell epitopes that are shared by eight pathogenic orthopoxviruses, including variola minor and major strains, monkeypox, cowpox, and vaccinia viruses. In EPIPOX, users can select T cell epitopes attending to the predicted binding to distinct major histocompatibility molecules (MHC) and according to various features that may have an impact on epitope immunogenicity. Among others, EPIPOX allows to discern epitopes by their structural location in the virion and the temporal expression of the counterpart antigens. Overall, the annotations in EPIPOX are optimized to facilitate the rational design of T cell epitope-based vaccines. In this chapter, we describe the main features of EPIPOX and exemplify its use, retrieving orthopoxvirus-specific T cell epitopes with features set to enhance their immunogenicity. EPIPOX is available for free public use at http://bio.med.ucm.es/epipox/ .

Orthopoxvirus Circulation in an Endemic Area in Brazil: Investigation of Infections in Small Mammals during an Absence of Outbreaks.

Vaccinia virus (VACV) is the causative agent of an emerging viral zoonosis called bovine vaccinia (BV). Several studies have documented characteristics of VACV infections in Brazil; however, the manner in which this virus is maintained in wildlife remains unknown. This work investigated the presence of viral DNA and anti-orthopoxvirus (OPXV) antibodies in samples collected from small mammals in a VACV-endemic area in Minas Gerais, Brazil, in the absence of current outbreaks. Samples did not show amplification of OPXV DNA in molecular tests. However, 5/142 serum samples demonstrated the presence of anti-OPXV neutralizing antibodies in serological tests. These data reinforce the involvement of small mammals in the natural cycle of VACV, highlighting the need for further ecological studies to better understand how this virus is maintained in nature and to develop measures to prevent BV outbreaks.

Mpox Virus: Its Molecular Evolution and Potential Impact on Viral Epidemiology.

Mpox (previously known as monkeypox) is an infectious viral illness caused by the mpox virus (MPXV), an orthopoxvirus that belongs to the family Poxviridae. The symptoms of mpox in humans are similar to those of smallpox, although the mortality rate is lower. In recent years, the concern over a potential global pandemic has increased due to reports of mpox spreading across Africa and other parts of the world. Prior to this discovery, mpox was a rare zoonotic disease restricted to endemic regions of Western and Central Africa. The sudden emergence of MPXV cases in multiple regions has raised concerns about its natural evolution. This review aims to provide an overview of previously available information about MPXV, including its genome, morphology, hosts and reservoirs, and virus-host interaction and immunology, as well as to perform phylogenetic analysis on available MPXV genomes, with an emphasis on the evolution of the genome in humans as new cases emerge.

Exaptation of Inactivated Host Enzymes for Structural Roles in Orthopoxviruses and Novel Folds of Virus Proteins Revealed by Protein Structure Modeling.

Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds. IMPORTANCE Protein structures are more strongly conserved in evolution than are amino acid sequences. Comparative structural analysis is particularly important for inferring the origins of viral proteins that typically evolve at high rates. We used a powerful protein structure modeling method, namely, AlphaFold2, to model the structures of all orthopoxvirus proteins and compared them to all available protein structures. Multiple cases of recruitment of host enzymes for structural roles in viruses, accompanied by the disruption of catalytic sites, were discovered. However, many viral proteins appear to have evolved unique structural folds.