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Background Human monkeypox is a zoonotic disease endemic to parts of Africa. Similar to other orthopoxviruses, virus and host have considerable interactions through immunomodulation. These interactions likely drive the establishment of a productive infection and disease progression, resulting in the range of disease presentations and case fatality rates observed for members of the Orthopoxvirus genus. Objectives Much of our understanding about the immune response to orthopoxvirus infection comes from either in vitro or in vivo studies performed in small animals or non-human primates. Here, we conducted a detailed assessment of cytokine responses to monkeypox virus using serum from acutely ill humans collected during monkeypox active disease surveillance (2005–2007) in the Democratic Republic of the Congo. Study design Nineteen serum samples that were from patients with confirmed monkeypox virus infections were selected for cytokine profiling. Cytokine profiling was performed on the Bio-Rad Bioplex 100 system using a 30-plex human cytokine panel. Results Cytokine profiling revealed elevated cytokine concentrations in all samples. Overproduction of certain cytokines (interleukin [IL]-2R, IL-10, and granulocyte macrophage-colony stimulating factor were observed in patients with serious disease (defined as >250 lesions based on the World Health Organization scoring system). Conclusions The data suggest that cytokine modulation affects monkeypox disease severity in humans. [ABSTRACT FROM AUTHOR]

Monkeypox virus belongs to the orthopoxvirus genus, infects rodents and monkeys in Africa, produces a smallpox-like zoonotic disease in humans, and has the potential for global spread and exploitation for bioterrorism. Several small animal models for studying monkeypox virus pathogenesis have been investigated. The African dormouse is a candidate natural host but is outbred and no immunological reagents exist. Although not a natural host, the CAST/EiJ mouse is inbred and animals and reagents are commercially available. We compared the dissemination of monkeypox virus by bioluminescence imaging in CAST/EiJ mice and dormice. In CAST/EiJ mice, intense replication occurred at the intranasal site of inoculation and virus spread rapidly to lungs and abdominal organs, which had a lower virus burden. Compared to CAST/EiJ mice, dormice exhibited a greater variation of virus spread, a slower time course, less replication in the head and chest, and more replication in abdominal organs prior to death. [ABSTRACT FROM AUTHOR]

The species cowpox virus (CPXV), genus Orthopoxvirus (OPV), consists of isolates highly variable in their biological properties and their genotypes. A TaqMan PCR assay for the specific detection of CPXV DNA based on sequences of the ORF D11L has been developed recently. ( Gavrilova et al . , 2010; Shchelkunov et al . , 2011 ); however, a rather limited panel of CPXV stains has been used. When a much larger panel of 47 CPXV DNAs has been tested, three strains could not be amplified at all because of large deletions in their respective ORF D11L. In addition, a deletion of 23 bp led to low-efficiency detection of five other CPXV strains. To solve this problem a new primer/probe combinations was selected based on sequences of ORF D8L, and a new real-time PCR method for (i) a genus-specific detection of OPVs and (ii) a simultaneous CPXV-specific differentiation is described in this study. The specificity and sensitivity were assessed by analyzing DNA of 67 strains belonging to human-pathogenic OPV species, including variola virus, as well as specimens of CPXV-infected mice. [ABSTRACT FROM AUTHOR]

Poxviruses are composed of large double-stranded DNA (dsDNA) genomes coding for several hundred genes whose variation has supported virus adaptation to a wide variety of hosts over their long evolutionary history. Comparative genomics has suggested that the Orthopoxvirus genus in particular has undergone reductive evolution, with the most recent common ancestor likely possessing a gene complement consisting of all genes present in any existing modern-day orthopoxvirus species, similar to the current Cowpox virus species. As orthopoxviruses adapt to new environments, the selection pressure on individual genes may be altered, driving sequence divergence and possible loss of function. This is evidenced by accumulation of mutations and loss of protein-coding open reading frames (ORFs) that progress from individual missense mutations to gene truncation through the introduction of early stop mutations (ESMs), gene fragmentation, and in some cases, a total loss of the ORF. In this study, we have constructed a whole-genome alignment for representative isolates from each Orthopoxvirus species and used it to identify the nucleotide-level changes that have led to gene content variation. By identifying the changes that have led to ESMs, we were able to determine that short indels were the major cause of gene truncations and that the genome length is inversely proportional to the number of ESMs present. We also identified the number and types of protein functional motifs still present in truncated genes to assess their functional significance. [ABSTRACT FROM AUTHOR]

Cantagalo virus is a strain of vaccinia virus (genus Orthopoxvirus) and the etiological agent of an important vesicopustular disease that affects dairy cows and milkers in Brazil. The reservoirs involved in the maintenance of this virus in nature are unknown. In the present work, the detection of neutralizing antibodies to Orthopoxvirus in capybaras collected in São Paulo state is reported. Capybaras are the largest rodent species native to South America and have already been reported as putative reservoirs of other pathogenic microorganisms. Thirteen out of thirty-three serum samples were found positive in plaque-reduction neutralization tests, some of them showing high titers compared to positive controls. These results suggest that capybaras may play a role in the infection cycle of vaccinia virus in Brazil. [ABSTRACT FROM AUTHOR]

Natural orthopoxvirus outbreaks such as vaccinia, cowpox, cattlepox and buffalopox continue to cause morbidity in the human population. Monkeypox virus remains a significant agent of morbidity and mortality in Africa. Furthermore, monkeypox virus’s broad host-range and expanding environs make it of particular concern as an emerging human pathogen. Monkeypox virus and variola virus (the etiological agent of smallpox) are both potential agents of bioterrorism. The first line response to orthopoxvirus disease is through vaccination with first-generation and second-generation vaccines, such as Dryvax and ACAM2000. Although these vaccines provide excellent protection, their widespread use is impeded by the high level of adverse events associated with vaccination using live, attenuated virus. It is possible that vaccines could be used in combination with antiviral drugs to reduce the incidence and severity of vaccine-associated adverse events, or as a preventive in individuals with uncertain exposure status or contraindication to vaccination. We have used the intranasal mousepox (ectromelia) model to evaluate the efficacy of vaccination with Dryvax or ACAM2000 in conjunction with treatment using the broad spectrum antiviral, brincidofovir (BCV, CMX001). We found that co-treatment with BCV reduced the severity of vaccination-associated lesion development. Although the immune response to vaccination was quantifiably attenuated, vaccination combined with BCV treatment did not alter the development of full protective immunity, even when administered two days following ectromelia challenge. Studies with a non-replicating vaccine, ACAM3000 (MVA), confirmed that BCV’s mechanism of attenuating the immune response following vaccination with live virus was, as expected, by limiting viral replication and not through inhibition of the immune system. These studies suggest that, in the setting of post-exposure prophylaxis, co-administration of BCV with vaccination should be considered a first response to a smallpox emergency in subjects of uncertain exposure status or as a means of reduction of the incidence and severity of vaccine-associated adverse events. [ABSTRACT FROM AUTHOR]

Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (MAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 MAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins, such as H3, D8, or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to nonneutralizing anti-L1 MAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and also could bind to virions. By using a variety of techniques, including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light-chain and heavychain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies. [ABSTRACT FROM AUTHOR]

Immunization with modified vaccinia virus Ankara (MVA) can rapidly protect mice against lethal ectromelia virus (ECTV) infection, serving as an experimental model for severe systemic infections. Importantly, this early protective capacity of MVA vaccination completely depends on virus-specific cytotoxic CD8 T cell responses. We used MVA vaccination in the mousepox challenge model using ECTV infection to investigate the previously unknown factors required to elicit rapid protective T cell immunity in normal C57BL/6 mice and in mice lacking the interferon alpha/beta receptor (IFNAR-/-). We found a minimal dose of 105 PFU of MVA vaccine fully sufficient to allow robust protection against lethal mousepox, as assessed by the absence of disease symptoms and failure to detect ECTV in organs from vaccinated animals. Moreover, MVA immunization at low dosage also protected IFNAR-/- mice, indicating efficient activation of cellular immunity even in the absence of type I interferon signaling. When monitoring for virus-specific CD8+ T cell responses in mice vaccinated with the minimal protective dose of MVA, we found significantly enhanced levels of antigen-specific T cells in animals that were MVA vaccinated and ECTV challenged compared to mice that were only vaccinated. The initial priming of naive CD8+ T cells by MVA immunization appears to be highly efficient and, even at low doses, mediates a rapid in vivo burst of pathogen-specific T cells upon challenge. Our findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses. [ABSTRACT FROM AUTHOR]

Modified vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses, including cell migration. Previously, we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) byMVAis necessary for the recruitment of monocytes and T cells, but not neutrophils, to the lung. Here, we identified neutrophil-attracting chemokines produced by MVA-infected primary murine lung fibroblasts and murine bone marrow-derived macrophages. We demonstrate that MVA, but not vaccinia virus (VACV) strain WR, induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA-induced neutrophil chemotaxis in vitro. Finally, intranasal infection of Ccr1-/- mice with MVA, as well as application of the CCR1 antagonist J-113863, revealed a role for CCR1 in leukocyte recruitment, including neutrophils, into the lung. [ABSTRACT FROM AUTHOR]

Superinfection exclusion is a widespread phenomenon that prevents secondary infections by closely related viruses. The vaccinia virus A56 and K2 proteins in the cell membrane can prevent superinfection by interacting with the entry-fusion complex of subsequent viruses. Here, we described another form of exclusion that is established earlier in infection and does not require the A56 or K2 protein. Cells infected with one or more infectious virions excluded hundreds of superinfecting vaccinia virus particles. A related orthopoxvirus, but neither a flavivirus nor a rhabdovirus, was also excluded, indicating selectivity. Although superinfecting vaccinia virus bound to cells, infection was inhibited at the membrane fusion step, thereby preventing core entry into the cytoplasm and early gene expression. In contrast, A56/K2 protein-mediated exclusion occurred subsequent to membrane fusion. Induction of resistance to superinfection depended on viral RNA and protein synthesis by the primary virus but did not require DNA replication. Although superinfection resistance correlated with virus-induced changes in the cytoskeleton, studies with mutant vaccinia viruses indicated that the cytoskeletal changes were not necessary for resistance to superinfection. Interferon-inducible transmembrane proteins, which can inhibit membrane fusion in other viral systems, did not prevent vaccinia virus membrane fusion, suggesting that these interferon-inducible proteins are not involved in superinfection exclusion. While the mechanism remains to be determined, the early establishment of superinfection exclusion may provide a "winnertake- all" reward to the first poxvirus particles that successfully initiate infection and prevent the entry and genome reproduction of defective or less fit particles. [ABSTRACT FROM AUTHOR]

The eradication of smallpox and the cessation of global vaccination led to the increased prevalence of human infections in Central Africa. Serologic and protein-based diagnostic assay for MPXV detection is difficult due to cross-reactive antibodies that do not differentiate between diverse orthopoxvirus (OPXV) species. A previously characterized monoclonal antibody (mAb 69-126-3-7) against MPXV [1] was retested for cross-reactivity with various OPXVs. The 14.5 kDa band protein that reacted with mAb 69-126-3 was identified to be MPXV A29 protein (homolog of vaccinia virus Copenhagen A27). Amino acid sequence analysis of the MPXV A29 with other OPXV homologs identified four amino acid changes. Peptides corresponding to these regions were designed and evaluated for binding to mAb 69-126-3 by ELISA and BioLayer Interferometry (BLI). Further refinement and truncations mapped the specificity of this antibody to a single amino acid difference in a 30-mer peptide compared to other OPXV homologs. This particular residue is proposed to be essential for heparin binding by VACV A27 protein. Despite this substitution, MPXV A29 bound to heparin with similar affinity to that of VACV A27 protein, suggesting flexibility of this motif for heparin binding. Although binding of mAb 69-126-3-7 to MPXV A29 prevented interaction with heparin, it did not have any effect on the infectivity of MPXV. Characterization of 69-126-3-7 mAb antibody allows for the possibility of the generation of a serological based species-specific detection of OPXVs despite high proteomic homology. [ABSTRACT FROM AUTHOR]

Poxviruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCFβ-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1β, Orthopoxvirus-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxvirus infection. Ectromelia virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four ectromelia virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCFβ-TRCP ubiquitin ligase, we investigated the role of the ectromelia virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1β-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, ectromelia virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, ectromelia virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation in vivo. [ABSTRACT FROM AUTHOR]

The process of vaccination introduced by Jenner generated immunity against smallpox and ultimately led to the eradication of the disease. Procedurally, in modern times, the virus is introduced into patients via a process called scarification, performed with a bifurcated needle containing a small amount of virus. What was unappreciated was the role that scarification itself plays in generating protective immunity. In rabbits, protection from lethal disease is induced by intradermal injection of vaccinia virus, whereas a protective response occurs within the first 2 min after scarification with or without virus, suggesting that the scarification process itself is a major contributor to immunoprotection. [ABSTRACT FROM AUTHOR]

CD8 T cells are a key component of immunity to many viral infections. They achieve this through using an array of effector mechanisms, but precisely which component/s are required for protection against a respiratory orthopox virus infection remains unclear. Using a model of respiratory vaccinia virus infection in mice, we could specifically determine the relative contribution of perforin, TRAIL, and IFN-γ-mediated pathways in protection against virus induced morbidity and mortality. Unexpectedly, we observed that protection against death was mediated by IFN-γ without any involvement of the perforin or TRAIL-dependent pathways. IFN-γ mRNA and protein levels in the lung peaked between days 3 and 6 postinfection. This enhanced response coincided with the emergence of virus-specific CD8 T cells in the lung and the cessation of weight loss. Transfer experiments indicated that CD8 T cell-autonomous expression of IFN-γ restricts virus-induced lung pathology and dissemination to visceral tissues and is necessary for clearance of virus. Most significantly, we show that CD8 T cell-derived IFN-γ is sufficient to protect mice in the absence of CD4 and B-lymphocytes. Thus, our findings reveal a previously unappreciated mechanism by which effector CD8 T cells afford protection against a highly virulent respiratory orthopox virus infection. [ABSTRACT FROM AUTHOR]

Recombination plays a critical role in virus evolution. It helps avoid genetic decline and creates novel phenotypes. This promotes survival, and genome sequencing suggests that recombination has facilitated the evolution of human pathogens, including orthopoxviruses such as variola virus. Recombination can also be used to map genes, but although recombinant poxviruses are easily produced in culture, classical attempts to map the vaccinia virus (VACV) genome this way met with little success. We have sequenced recombinants formed when VACV strains TianTan and Dryvax are crossed under different conditions. These were a single round of growth in coinfected cells, five rounds of sequential passage, or recombinants obtained using leporipoxvirus-mediated DNA reactivation. Our studies showed that recombinants contain a patchwork of DNA, with the number of exchanges increasing with passage. Further passage also selected for TianTan DNA and correlated with increased plaque size. The recombinants produced through a single round of coinfection contain a disproportionate number of short conversion tracks (<1 kbp) and exhibited 1 exchange per 12 kbp, close to the ~1 per 8 kbp in the literature. One by-product of this study was that rare mutations were also detected; VACV replication produces ~1 × 10-8 mutation per nucleotide copied per cycle of replication and~1 large (21 kbp) deletion per 70 rounds of passage. Viruses produced using DNA reactivation appeared no different from recombinants produced using ordinary methods. An attractive feature of this approach is that when it is combined with selection for a particular phenotype, it provides a way of mapping and dissecting more complex virus traits. [ABSTRACT FROM AUTHOR]

Background: In case of outbreak of rash illness in remote areas, clinically discriminating monkeypox (MPX) from severe form of chickenpox and from smallpox remains a concern for first responders. Objective: The goal of the study was therefore to use MPX and chickenpox outbreaks in Democratic Republic of Congo (DRC) as a test case for establishing a rapid and specific diagnosis in affected remote areas. Methods: In 2008 and 2009, successive outbreaks of presumed MPX skin rash were reported in Bena Tshiadi, Yangala and Ndesha healthcare districts of the West Kasai province (DRC). Specimens consisting of liquid vesicle dried on filter papers or crusted scabs from healing patients were sampled by first responders. A field analytical facility was deployed nearby in order to carry out a real-time PCR (qPCR) assay using genus consensus primers, consensus orthopoxvirus (OPV) and smallpox-specific probes spanning over the 14 kD fusion protein encoding gene. A PCR-restriction fragment length polymorphism was used on-site as backup method to confirm the presence of monkeypox virus (MPXV) in samples. To complete the differential diagnosis of skin rash, chickenpox was tested in parallel using a commercial qPCR assay. In a post-deployment step, a MPXV-specific pyrosequencing was carried out on all biotinylated amplicons generated on-site in order to confirm the on-site results. Results: Whereas MPXV proved to be the agent causing the rash illness outbreak in the Bena Tshiadi, VZV was the causative agent of the disease in Yangala and Ndesha districts. In addition, each on-site result was later confirmed by MPXV-specific pyrosequencing analysis without any discrepancy. Conclusion: This experience of rapid on-site dual use DNA-based differential diagnosis of rash illnesses demonstrates the potential of combining tests specifically identifying bioterrorism agents and agents causing natural outbreaks. This opens the way to rapid on-site DNA-based identification of a broad spectrum of causative agents in remote areas. [ABSTRACT FROM AUTHOR]

Abstract: Some orthopoxviruses including cowpox virus embed virus particles in dense bodies, comprised of the A-type inclusion (ATI) protein, which may provide long-term environmental protection. This strategy could be beneficial if the host population is sparse or spread is inefficient or indirect. However, the formation of ATI may be neutral or disadvantageous for orthopoxviruses that rely on direct respiratory spread. Disrupted ATI open reading frames in orthopoxviruses such as variola virus, the agent of smallpox, and monkeypox virus suggests that loss of this feature provided positive selection. To test this hypothesis, we constructed cowpox virus mutants with deletion of the ATI gene or another gene required for embedding virions. The ATI deletion mutant caused greater weight loss and higher replication in the respiratory tract than control viruses, supporting our hypothesis. Deletion of the gene for embedding virions had a lesser effect, possibly due to known additional functions of the encoded protein. [Copyright &y& Elsevier]

Highlights: [•] Quantitative real-time PCR assay for enumerating vaccinia virus particles. [•] Applicable to several different species of orthopoxviruses. [•] Yielded similar results to those obtained using OD260 and direct virion counting. [•] Protocol for isolating and enumerating the three forms of progeny orthopoxviruses, IMV, CEV, and EEV. [ABSTRACT FROM AUTHOR]

Abstract: Classical inbred mice are extensively used for virus research. However, we recently found that some wild-derived inbred mouse strains are more susceptible than classical strains to monkeypox virus. Experiments described here indicated that the 50% lethal dose of vaccinia virus (VACV) and cowpox virus (CPXV) were two logs lower in wild-derived inbred CAST/Ei mice than classical inbred BALB/c mice, whereas there was little difference in the susceptibility of the mouse strains to herpes simplex virus. Live bioluminescence imaging was used to follow spread of pathogenic and attenuated VACV strains and CPXV virus from nasal passages to organs in the chest and abdomen of CAST/Ei mice. Luminescence increased first in the head and then simultaneously in the chest and abdomen in a dose-dependent manner. The spreading kinetics was more rapid with VACV than CPXV although the peak photon flux was similar. These data suggest advantages of CAST/Ei mice for orthopoxvirus studies. [Copyright &y& Elsevier]

Human monkeypox is found primarily in forested areas of Central Africa. This article provides a basic review of the clinical, epidemiological, and biological factors that contribute to human disease.Human monkeypox is a zoonotic Orthopoxvirus with a presentation similar to smallpox. Clinical differentiation of the disease from smallpox and varicella is difficult. Laboratory diagnostics are principal components to identification and surveillance of disease, and new tests are needed for a more precise and rapid diagnosis. The majority of human infections occur in Central Africa, where surveillance in rural areas with poor infrastructure is difficult but can be accomplished with evidence-guided tools and educational materials to inform public health workers of important principles. Contemporary epidemiological studies are needed now that populations do not receive routine smallpox vaccination. New therapeutics and vaccines offer hope for the treatment and prevention of monkeypox; however, more research must be done before they are ready to be deployed in an endemic setting. There is a need for more research in the epidemiology, ecology, and biology of the virus in endemic areas to better understand and prevent human infections. [ABSTRACT FROM AUTHOR]

Myxoma virus (MYXV) is one of many animal viruses that exhibit oncolytic properties in transformed human cells. Compared to orthopoxviruses like vaccinia (VACV), MYXV spreads inefficiently, which could compromise its use in treating tumors and their associated metastases. The VACV F11 protein promotes virus exit and rapid spread by inhibiting Rho signalling, which results in a disruption of cortical actin. We have previously shown that although MYXV lacks an F11 homolog, the F11L gene can be introduced into MYXV promoting the spread of this Leporipoxvirus in natural host cells. Here we show that the F11-encoding (F11L+) MYXV strain replicates to higher levels in a number of human cancer cells. We also show that F11L+ MYXV induces better tumor control and prolonged survival of mice bearing MDA-MB-231 cancer cells. Furthermore, we show that this virus also spreads more efficiently from the site of growth in one injected tumor, to a second untreated tumor. While we focused mostly on the use of a modified MYXV we were able to show that the effects of F11 on MYXV growth in cancer cells could be mimicked through the use of pharmacological inhibition or siRNA-mediated silencing of key regulators of cortical actin (RhoA, RhoC, mDia1, or LIMK2). These data suggest that it may be possible to increase the oncolytic efficacy of wild-type MYXV using chemical inhibitors of RhoA/C or their downstream targets. Furthermore, since all viruses must overcome barriers to exit posed by structures like cortical actin, these findings suggest that the oncolytic activity of other viruses may be enhanced through similar strategies. [ABSTRACT FROM AUTHOR]

On May 8, 1980, the World Health Assembly at its 33rd 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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Provides summary table of post-exposure vaccination study data from smallpox eradication. [•] Provides summary table of animal models appropriate for post-exposure vaccination studies. [•] Provides post-exposure efficacy window based on epidemiology and animal studies. [•] Presents a case for optimal animal models for studying post-exposure vaccination efficacy. [Copyright &y& Elsevier]

Several studies have provided evidence that complex relationships between autophagic and apoptotic cell death pathways occur in cancer and virus-infected cells. Previously, we demonstrated that infection of macrophages with Moscow strain of ectromelia virus (ECTV-MOS) induces apoptosis under in vitro and in vivo conditions. Here, we found that autophagy was induced in RAW 264.7 cells during infection with ECTV-MOS. Silencing of beclin 1, an autophagy-related gene, reduced the percentage of late apoptotic cells in virus-infected RAW 264.7 macrophages. Pharmacological modulation of autophagy by wortmannin (inhibitor) or rapamycin (inductor) did not affect or cause increased apoptosis in ECTV-MOS-infected RAW 264.7 cells, respectively. Meantime, blocking apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, increased the formation of autophagosomes in infected macrophages. Taken together, three important points arise from our study. First, autophagy may co-occur with apoptosis in RAW 264.7 cells exposed to ECTV-MOS. Second, at later stages of infection, autophagy may partially participate in the execution of macrophage cell death by enhancing apoptosis. Third, when apoptosis is blocked infected macrophages undergo increased autophagy. Our results provide new information about the relationship between autophagy and apoptosis in ECTV-MOS-infected macrophages. [ABSTRACT FROM AUTHOR]

Background Within the context of an increased epidemiological pressure caused by canine distemper virus ( CDV) in Switzerland together with a potential re-emergence of endemic pathogens such as orthopoxviruses ( OPXV), dual infections are possible among susceptible species. Objective To describe a case of concurrent CDV and OPXV infection in a cat. Animal A 5-year-old, neutered male cat was presented with erythema, crusts and ulcerations around the left eye. High-grade pruritus and a severe conjunctivitis were also present. Methods Formalin-fixed skin biopsy samples were obtained from lesional skin. Histopathology, CDV immunohistochemistry and CDV and OPXV RT- PCR were performed. Results Histopathological examination showed severe epidermal necrosis extending to the follicular walls and a dermal infiltration, predominantly eosinophilic. Intranuclear and intracytoplasmic eosinophilic inclusion bodies were visible in the wall of affected hair follicles, with occasional formation of syncytia. The RT- PCR revealed the contextual presence of both CDV and OPXV. Scattered cells stained positive for CDV by immunohistochemistry. Conclusion and discussion Dual infections with CDV and OPXV, although rare, may occur and represent additional differential diagnoses for ulcerative skin lesions in cats. [ABSTRACT FROM AUTHOR]

Background Despite repeated outbreaks of poxvirus infections unique to the Indian subcontinent region and veterinary research work in this field, much less diagnostic awareness with resultant treatment protocols have been formulated in the human medical field. Aims With this objective in mind, a combined human medical and veterinary study was conducted on a recent outbreak of buffalopox infection in a village in northern India. Methods A team of doctors did the clinical examination and collected swab and serum samples from both cattle and humans, and these were subjected to viral isolation, cell culture, plaque reduction neutralization test, polymerase chain reaction, and partial genome sequencing. Results A clustered foci of 12 human patients aged 11-60 years, 12 buffaloes, and 10 cows were found to be affected with buffalopox infection with some atypical features. Conclusion Awareness, diagnosis, education, early intervention, and formulation of disaster guidelines are needed in view of the potential epidemiologic outbreak, if this happens in the future. [ABSTRACT FROM AUTHOR]

Highlights: [•] Rapid bioinformatics methods for phylogenetic characterization of hundreds of viral genomes are applied to viral pathogens. [•] SNPs are better than PCR to characterize an isolate at all levels of a phylogeny. [•] SNP-based phylogenies match those from whole genome alignments. [•] A highly multiplex SNP assay enables fine resolution genotyping of an unknown in a single hybridization assay. [Copyright &y& Elsevier]

Abstract: Extensive use of Vaccinia virus (VACV) in research has led to associated accidental human exposure in laboratories worldwide. In spite of the social and economic relevance of Bovine Vaccinia outbreaks in Brazil, national data concerning laboratory workers handling these infectious agents are relatively scarce. Therefore, a serological survey was conducted in a Brazilian laboratory to evaluate staff exposure to orthopoxviruses (OPVs). Information concerning direct work with OPVs, vaccination status and laboratory accidents was collected and correlated to serology results. This study presents an opportunity for discussion of routine procedures involving OPVs in laboratories and their intrinsic risks. Aspects of the live attenuated smallpox vaccine are also discussed. [Copyright &y& Elsevier]

Abstract: Since the eradication of Smallpox, researchers have attempted to study Orthopoxvirus pathogenesis and immunity in animal models in order to correlate results human smallpox. A solely human pathogen, Orthopoxvirus variola fails to produce authentic smallpox illness in any other animal species tested to date. In 2003, an outbreak in the USA of Orthopoxvirus monkeypox, revealed the susceptibility of the North American black-tailed prairie dog (Cynomys ludovicianus) to infection and fulminate disease. Prairie dogs infected with Orthopoxvirus monkeypox present with a clinical scenario similar to ordinary smallpox, including prodrome, rash, and high mortality. This study examines if Black-tailed prairie dogs can become infected with O. variola and serve as a surrogate model for the study of human smallpox disease. Substantive evidence of infection is found in immunological seroconversion of animals to either intranasal or intradermal challenges with O. variola, but in the absence of overt illness. [Copyright &y& Elsevier]

A fatal combined infection with canine distemper virus (CDV) and orthopoxvirus (OPXV) in Asian marmots (Marmota caudata) is reported in this article. A total of 7 Asian marmots from a small zoological garden in Switzerland were found dead in hibernation during a routine check in the winter of 2011. The marmots died in February 2011. No clinical signs of disease were observed at any time. The viruses were detected in all individuals for which the tissues were available (n = 3). Detection of the viruses was performed by reverse transcription polymerase chain reaction. The most consistent gross lesion was a neck and thorax edema. A necrotizing pharyngitis and a multifocal necrotizing pneumonia were observed histologically. Numerous large intracytoplasmic eosinophilic inclusions were seen in the epithelial cells of the pharynx, of the airways, and in the skin keratinocytes. Brain lesions were limited to mild multifocal gliosis. Phylogenetic analysis revealed that the marmot CDV strain was closely related to the clusters of CDVs detected in Switzerland in wild carnivores during a local outbreak in 2002 and the 2009–2010 nationwide epidemic, suggesting a spillover of this virus from wildlife. The OPXV was most closely related to a strain of cowpoxvirus, a poxvirus species considered endemic in Europe. This is the first reported instance of CDV infection in a rodent species and of a combined CDV and OPXV infection. [ABSTRACT FROM PUBLISHER]

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox. [ABSTRACT FROM AUTHOR]

Abstract: Vaccinia virus (VACV) strain TianTan was used for much of China's modern history to vaccinate against smallpox, however the only genome sequence contains errors. We have sequenced additional examples of TianTan to obtain a better picture of this important virus. We detected two different subclones. One (TP03) encodes large deletions in the terminal repeats that extend into both VEGF genes and create a small plaque variant. The second clone (TP05) encodes a nearly intact complement of genes in the terminal repeats, except for an insertion of sequences resembling the telomeric 69bp repeats. The TP05 genome spans 196,260bp and encodes 219 genes. The revised sequence documents the integrity of all the genes in the conserved virus core. Phylogenetic methods show that TianTan belongs to a unique clade of VACV, but probably also share a common origin with strains belonging to the Copenhagen/Lister lineage and distinct from the Wyeth/Dryvax lineage. [Copyright &y& Elsevier]

Abstract: The synthesis of 6′-methyl-5′-homoaristeromycin is described from a known 6′-ethyl ester. Antiviral analysis showed the (S)-6′ stereoisomer retained the vaccinia activity of the parent 5′-homoaristeromycin (1) while the (R)-6′ isomer was less active. Both were weaker than 1 towards cowpox. The diastereomers were equally active versus Epstein Barr virus while (S)-6′ was three times more active toward vesicular stomatitis virus than (R)-6′. The diastereomers were inactive towards numerous other viruses. The CC50 for both diastereomers was >300μM. [Copyright &y& Elsevier]

Despite its eradication over 30 years ago, smallpox (as well as other orthopox viruses) remains a pathogen of interest both in terms of biodefense and for its use as a vector for vaccines and immunotherapies. Here we describe the application of mRNA-Seq transcriptome profiling to understanding immune responses in smallpox vaccine recipients. Contrary to other studies examining gene expression in virally infected cell lines, we utilized a mixed population of peripheral blood mononuclear cells in order to capture the essential intercellular interactions that occur in vivo, and would otherwise be lost, using single cell lines or isolated primary cell subsets. In this mixed cell population we were able to detect expression of all annotated vaccinia genes. On the host side, a number of genes encoding cytokines, chemokines, complement factors and intracellular signaling molecules were downregulated upon viral infection, whereas genes encoding histone proteins and the interferon response were upregulated. We also identified a small number of genes that exhibited significantly different expression profiles in subjects with robust humoral immunity compared with those with weaker humoral responses. Our results provide evidence that differential gene regulation patterns may be at work in individuals with robust humoral immunity compared with those with weaker humoral immune responses. [ABSTRACT FROM AUTHOR]

Orthopoxviruses (OPVs), which include the agent of smallpox (variola virus), the zoonotic monkeypox virus, the vaccine and zoonotic species vaccinia virus, and the mouse pathogen ectromelia virus (ECTV), form two types of infectious viral particles: the mature virus (MV), which is cytosolic, and the enveloped virus (EV), which is extracellular. It is believed that MVs are required for viral entry into the host, while EVs are responsible for spread within the host. Following footpad infection of susceptible mice, ECTV spreads lymphohematogenously, entering the liver at 3 to 4 days postinfection (dpi). Afterwards, ECTV spreads intrahepatically, killing the host. We found that antibodies to an MV protein were highly effective at curing mice from ECTV infection when administered after the virus reached the liver. Moreover, a mutant ECTV that does not make EV was able to spread intrahepatically and kill immunodeficient mice. Together, these findings indicate that MVs are sufficient for the spread of ECTV within the fiver and could have implications regarding the pathogenesis of other OPVs, the treatment of emerging OPV infections, as well as strategies for preparedness in case of accidental or intentional release of pathogenic OPVs. [ABSTRACT FROM AUTHOR]

Background: The traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. However, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. Preventive vaccination still remains the most effective control and new vectors have been developed to generate recombinant vaccines against smallpox that induce the same immunogenicity as the traditional one. As protective antibodies are mainly directed against the surface proteins of the two infectious forms of vaccinia, the intracellular mature virions and the extracellular virions, combined proteins from these viral forms can be used to better elicit a complete and protective immunity. Methods: Four novel viral recombinants were constructed based on the fowlpox genetic background, which independently express the vaccinia virus L1 and A27 proteins present on the mature virions, and the A33 and B5 proteins present on the extracellular virions. The correct expression of the transgenes was determined by RT-PCR, Western blotting, and immunofluorescence. Results and conclusions: Using immunoprecipitation and Western blotting, the ability of the proteins expressed by the four novel FPL1R, FPA27L, FPA33R and FPB5R recombinants to be recognized by VV-specific hyperimmune mouse sera was demonstrated. By neutralisation assays, recombinant virus particles released by infected chick embryo fibroblasts were shown not be recognised by hyperimmune sera. This thus demonstrates that the L1R, A27L, A33R and B5R gene products are not inserted into the new viral progeny. Fowlpox virus replicates only in avian species, but it is permissive for entry and transgene expression in mammalian cells, while being immunologically non--crossreactive with vaccinia virus. These recombinants might therefore represent safer and more promising immunogens that can circumvent neutralisation by vector-generated immunity in smallpox-vaccine-experienced humans. [ABSTRACT FROM AUTHOR]

Abstract: Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections. [Copyright &y& Elsevier]

Plasma pharmacokinetics of ST-246, smallpox therapeutic, was evaluated in mice, rabbits, monkeys and dogs following repeat oral administrations by gavage. The dog showed the lowest Tmax of 0.83 h and the monkey, the highest value of 3.25 h. A 2- to 4-fold greater dose-normalized Cmax was observed for the dog compared to the other species. The mouse showed the highest dose-normalized AUC, which was 2-fold greater than that for the rabbit and monkey both of which by approximation, recorded the lowest value. The Cl/F increased across species from 0.05 L/h for mouse to 42.52 L/h for dog. The mouse showed the lowest VD/F of 0.41 L and the monkey, the highest VD/F of 392.95 L. The calculated extraction ratios were 0.104, 0.363, 0.231 and 0.591 for mouse, rabbit, monkey and dog, respectively. The dog showed the lowest terminal half-life of 3.10 h and the monkey, the highest value of 9.94 h. The simple allometric human VD/F and MLP-corrected Cl/F were 2311.51 L and 51.35 L/h, respectively, with calculated human extraction ratio of 0.153 and terminal half-life of 31.20 h. Overall, a species-specific difference was observed for Cl/F with this parameter increasing across species from mouse to dog. The human MLP-corrected Cl/F, terminal half-life, extraction ratios were in close proximity to the observed estimates. In addition, the first-in-humans (FIH) dose of 485 mg, determined from the MLP-corrected allometry Cl/F, was well within the dose range of 400 mg and 600 mg administered in healthy adult human volunteers. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare no conflict of interest.

Monkeypox virus (MPXV), which belongs to the orthopoxvirus genus, causes zoonotic viral disease. This review discusses the biology, epidemiology, and evolution of MPXV infection, particularly cellular, human, and viral factors, virus transmission dynamics, infection, and persistence in nature. This review also describes the role of recombination, gene loss, and gene gain in MPXV evol-vement and the role of signal transduction in MPXV infection and provides an overview of the current access to therapeutic options for the treatment and prevention of MPXV. Finally, this review highlighted gaps in knowledge and proposed future research endeavors to address the unresolved questions., Competing Interests: Conflict-of-interest statement: The authors declare that they have no competing interests., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

The aim of the work was an experimental evaluation of the characteristics of the kit for the rapid immunochemical detection of orthopoxviruses (OPV). The kit is based on the method of one-stage dot-immunoassay on flat protein arrays using gold conjugates and a silver developer. Rabbit polyclonal antibodies against the vaccinia virus were used as capture and detection reagents. The sensitivity of detection of OPV and the specificity of the analysis were assessed using culture crude preparations (monkeypox virus, vaccinia virus, rabbitpox virus, cowpox virus, and ectromelia virus), a suspension from a crust from a human vaccination site as well as blood and tissue suspensions of infected rabbits. It has been shown that the assay using the kit makes it possible to detect OPV within 36 min at a temperature of 18-40 °C in unpurified culture samples of the virus and clinical samples in the range of 10 3 -10 4 PFU/mL. Tests of the kit did not reveal cross-reactivity with uninfected cell cultures and viral pathogens of exanthematous infections (measles, rubella and chicken pox). The kit can be used to detect or exclude the presence of a virus threat in samples and can be useful in various aspects of biosecurity. The simplicity of analysis, the possibility of visual accounting the and interpretation of the results make it possible to use the test in laboratories with a high level of biological protection and in out-of-laboratory conditions.

Before the international spread of monkeypox in May 2022, PCR kits for the detection of orthopoxviruses, and specifically monkeypox virus, were rarely available. Here we describe the evaluation of 11 recently developed commercially available PCR kits for the detection of monkeypox virus DNA. All tested kits are currently intended for research use only and clinical performance still needs to be assessed in more detail, but all were suitable for diagnostics of monkeypox virus, with variations in specificity rather than sensitivity.

Monkeypox is caused by a sylvatic, double-stranded DNA zoonotic virus. Since 1 January 2022, monkeypox cases have been reported to WHO from 106 Member States across six WHO regions, and as of 2 October 2022, a total of 68,900 confirmed cases, including 25 deaths, occurred. Here, by using a whole genome approach, we perform a genetic and phylodynamic survey of the monkeypox virus Clade IIb B.1, which is the lineage causing the current multi-country outbreak. Results suggest that outbreaks seem to be isolated and localized in several epidemic clusters with geographic consistency. Currently, monkeypox appears to be a virus with a flattened genetic variability in terms of evolutionary path, with a very slow rate of growth in the population size. This scenario confirms that the monkeypox virus lacks the evolutionary advantage, given by the high level of mutation rate, which is very strong in RNA viruses. Of course, constant genome-based monitoring must be performed over time in order to detect the change in its genetic composition, if any.

The monkeypox virus (MPXV) is the cause of a zoonotic infection similar to smallpox. Although it is endemic to Africa, it has recently begun to circulate in other parts of the world. In July 2022, the World Health Organization declared monkeypox an international public health emergency. This review aims to provide an overview of this neglected zoonotic pathogen. MPXV circulates as two distinct clades, the Central African and West African, with case fatality rates of 10.6% and 3.6%, respectively. The risk of infection is greater for those who work with animals or infected individuals. The virus' entry into the human body provokes both natural and acquired immunity. Although natural killer cells, CD4 + T cells, and CD8 + T cells play an essential role in eradicating MPXV, there is still a gap in the understanding of the host immune response to the virus. Currently, there are no specific therapeutic guidelines for treating monkeypox; however, some antiviral drugs such as tecovirimat and cidofovir may help to abate the severity of the disease. The use of nonpharmaceutical interventions and immunization can reduce the risk of infection. Increased surveillance and identification of monkeypox cases are crucial to understand the constantly shifting epidemiology of this resurging and intimidating disease. The present review provides a detailed perspective on the emergence and circulation of MPXV in human populations, infection risks, human immune response, disease diagnosis and prevention strategies, and future implications, and highlights the importance of the research community engaging more with this disease for an effective global response., (Copyright © 2022. Published by Elsevier Ltd.)

The spread of the monkeypox virus infection among humans in many countries outside of Africa, which started in 2022, is now drawing the attention of the medical and scientific communities to the fact that immunization against this infection is sorely needed. According to current guidelines, immunization of people with the first-generation smallpox vaccine based on the vaccinia virus (VACV) LIVP strain, which is licensed in Russia, should be performed via transepidermal inoculation (skin scarification, s.s.). However, the long past experience of using this vaccination technique suggests that it does not ensure virus inoculation into patients' skin with enough reliability. The procedure of intradermal (i.d.) injection of a vaccine can be an alternative to s.s. inoculation. The effectiveness of i.d. vaccination can depend on the virus injection site on the body. Therefore, the aim of this study was to compare the development of the humoral and cellular immune responses in BALB/c mice immunized with the LIVP VACV strain, which was administered either by s.s. inoculation or i.d. injection into the same tail region of the animal. A virus dose of 105 pfu was used in both cases. ELISA of serum samples revealed no significant difference in the dynamics and level of production of VACV-specific IgM and IgG after i.d. or s.s. vaccination. A ELISpot analysis of splenocytes from the vaccinated mice showed that i.d. administration of VACV LIVP to mice induces a significantly greater T-cell immune response compared to s.s. inoculation. In order to assess the protective potency, on day 45 post immunization, mice were intranasally infected with lethal doses of either the cowpox virus (CPXV) or the ectromelia virus (ECTV), which is evolutionarily distant from the VACV and CPXV. Both vaccination techniques ensured complete protection of mice against infection with the CPXV. However, when mice were infected with a highly virulent strain of ECTV, 50% survived in the i.d. immunized group, whereas only 17% survived in the s.s. immunized group. It appears, therefore, that i.d. injection of the VACV can elicit a more potent protective immunity against orthopoxviruses compared to the conventional s.s. technique., (Copyright ® 2022 National Research University Higher School of Economics.)

Abstract: The development of an effective prophylactic vaccine is still necessary to improve the safety of the conventional although-discontinued smallpox vaccine, and to protect from the threat of deliberate release of variola virus. This need also arises from the number of new cases of animal orthopoxvirus infections each year, and to reduce the risk to animal handlers. Fowlpox (FP) recombinants only replicate in avian species and have been developed against human infectious diseases, as they can elicit an effective immune response, are not cross-reactive immunologically with vaccinia, and represent safer and more promising immunogens for immunocompromised individuals. The aim of this study was the characterisation of two new fowlpox recombinants expressing the A33R vaccinia virus gene either alone (FPA33R) or with the green fluorescent protein (FPA33R-GFP) to verify whether GFP can affect the expression of the transgene. The results show that both FPA33R and FPA33R-GFP can express A33R correctly, but A33R mRNA and protein synthesis are higher by FPA33R than by FPA33R-GFP. Therefore, GFP co-expression does not prevent, but can reduce the level of a vaccine protein, and may affect the protective efficacy of the immune response. [Copyright &y& Elsevier]

Abstract: The commercially available Orthopox BioThreat® Alert assay for orthopoxvirus (OPV) detection is piloted. This antibody-based lateral-flow assay labels and captures OPV viral agents to detect their presence. Serial dilutions of cultured Vaccinia virus (VACV) and Monkeypox virus (MPXV) were used to evaluate the sensitivity of the Tetracore assay by visual and quantitative determinations; specificity was assessed using a small but diverse set of diagnostically relevant blinded samples from viral lesions submitted for routine OPV diagnostic testing. The BioThreat® Alert assay reproducibly detected samples at concentrations of 107 pfu/ml for VACV and MPXV and positively identified samples containing 106 pfu/ml in 4 of 7 independent experiments. The assay correctly identified 9 of 11 OPV clinical samples and had only one false positive when testing 11 non-OPV samples. Results suggest applicability for use of the BioThreat® Alert assay as a rapid screening assay and point of care diagnosis for suspect human monkeypox cases. [Copyright &y& Elsevier]