Search Results (27)

The use of experimental animals with unified quality standards is an important condition for ensuring the effectiveness of scientific research. Ectromelia virus (ECTV), murine hepatitis virus (MHV), reovirus type 3 (Reo-3), and murine parvoviruses (MUV) are the four pathogens that need to be eliminated from SPF (Specific Pathogen-Free) level mice. These four pathogens present fast transmission and high pathogenicity, making it difficult to control. The previously described detection methods present substantial limitations in efficiency and accuracy. Thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and diagnosis efforts. In this study, we developed a one-step multiplex real-time PCR (mrt-PCR) detection method that can simultaneously detect four key viral pathogens causing diseases in laboratory mice without cross-reactivity with other mouse susceptible pathogens. We tested 128 suspected diseased mouse tissue samples collected from Beijing, and the results showed that this new method has higher sensitivity and specificity than ordinary PCR. The detection limit for ECTV, MHV, and MUV was determined to be 1.08 × 10¹ copies/μL, 1.14 × 10¹ copies/μL, 2.38 ×10¹ copies/μL, and 1.08 × 10¹ copies/μL, respectively. In addition, the assay showed excellent reproducibility, with a coefficient of variation below 1.5%, strong linear correlation (R² > 0.996), and amplification efficiency between 90% and 100%. In summary, the mrt-PCR serves not only as a rapid and accurate detection and early prevention method for laboratory mice but also constitutes a robust tool for microbial quality control in laboratory mice. Full article

Since smallpox vaccination was discontinued in 1980, there has been a resurgence of poxvirus infections, particularly the monkeypox virus. Without a global recommendation to use the smallpox vaccine, the population is not immune, posing a severe threat to public health. Given these circumstances, it is crucial to understand the relationship between poxviruses and their hosts. Therefore, this study focuses on the ectromelia virus, the causative agent of mousepox, which serves as an excellent model for studying poxvirus pathogenesis. Additionally, we investigated the role of mitochondria in innate antiviral immunity during ECTV infection, focusing specifically on mitochondrial antiviral signaling protein. The study used a Moscow strain of ECTV and L929 mouse fibroblasts. Cells were treated with ECTV and chemical modulators of mitochondrial network: Mdivi-1 and CCCP. Our investigation revealed that an elongated mitochondrial network attenuates the suppression of MAVS-dependent immunity by ECTV and reduces ECTV replication in L929 fibroblasts compared to cells with an unaltered mitochondrial network. Conversely, a fragmented mitochondrial network reduces the number of progeny virions while increasing the inhibition of the virus-induced immune response during infection. In conclusion, our study showed that modifications of mitochondrial network morphology alter MAVS-dependent immunity in ECTV-infected mouse L929 fibroblasts. Full article

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs’ immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs’ organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation. Full article

Conventional dendritic cells (cDCs) are innate immune cells that play a pivotal role in inducing antiviral adaptive immune responses due to their extraordinary ability to prime and polarize naïve T cells into different effector T helper (Th) subsets. The two major subpopulations of cDCs, cDC1 (CD8α⁺ in mice and CD141⁺ in human) and cDC2 (CD11b⁺ in mice and CD1c⁺ in human), can preferentially polarize T cells toward a Th1 and Th2 phenotype, respectively. During infection with ectromelia virus (ECTV), an orthopoxvirus from the Poxviridae family, the timing and activation of an appropriate Th immune response contributes to the resistance (Th1) or susceptibility (Th2) of inbred mouse strains to the lethal form of mousepox. Due to the high plasticity and diverse properties of cDC subpopulations in regulating the quality of a specific immune response, in the present study we compared the ability of splenic cDC1 and cDC2 originating from different ECTV-infected mouse strains to mature, activate, and polarize the Th immune response during mousepox. Our results demonstrated that during early stages of mousepox, both cDC subsets from resistant C57BL/6 and susceptible BALB/c mice were activated upon in vivo ECTV infection. These cells exhibited elevated levels of surface MHC class I and II, and co-stimulatory molecules and showed enhanced potential to produce cytokines. However, both cDC subsets from BALB/c mice displayed a higher maturation status than that of their counterparts from C57BL/6 mice. Despite their higher activation status, cDC1 and cDC2 from susceptible mice produced low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, and the ability of these cells to stimulate the proliferation and Th1 polarization of allogeneic CD4⁺ T cells was severely compromised. In contrast, both cDC subsets from resistant mice produced significant amounts of Th1-polarizing cytokines and demonstrated greater capability in differentiating allogeneic T cells into Th1 cells compared to cDCs from BALB/c mice. Collectively, our results indicate that in the early stages of mousepox, splenic cDC subpopulations from the resistant mouse strain can better elicit a Th1 cell-mediated response than the susceptible strain can, probably contributing to the induction of the protective immune responses necessary for the control of virus dissemination and for survival from ECTV challenge. Full article

The recent spread of the monkeypox virus among humans has heightened concerns regarding orthopoxvirus infections. Consequently, conducting a comprehensive study on the immunobiology of the monkeypox virus is imperative for the development of effective therapeutics. Ectromelia virus (ECTV) closely resembles the genetic and disease characteristics of monkeypox virus, making it a valuable research tool for studying orthopoxvirus–host interactions. Guanylate-binding proteins (GBPs), highly expressed interferon-stimulated genes (ISGs), have antagonistic effects against various intracellular pathogenic microorganisms. Our previous research has shown that GBP2 has a mild but statistically significant inhibitory effect on ECTV infection. The presence of a significant number of molecules in the poxvirus genome that encode the host immune response raises questions about whether it also includes proteins that counteract the antiviral activity of GBP2. Using IP/MS and co-IP technology, we discovered that the poly(A) polymerase catalytic subunit (PAPL) protein of ECTV is a viral regulatory molecule that interacts with GBP2. Further studies have shown that PAPL antagonizes the antiviral activity of GBP2 by reducing its protein levels. Knocking out the PAPL gene of ECTV with the CRISPR/Cas9 system significantly diminishes the replication ability of the virus, indicating the indispensable role of PAPL in the replication process of ECTV. In conclusion, our study presents preliminary evidence supporting the significance of PAPL as a virulence factor that can interact with GBP2. Full article

Guanylate-binding proteins (GBPs) are highly expressed interferon-stimulated genes (ISGs) that play significant roles in protecting against invading pathogens. Although their functions in response to RNA viruses have been extensively investigated, there is limited information available regarding their role in DNA viruses, particularly poxviruses. Ectromelia virus (ECTV), a member of the orthopoxvirus genus, is a large double-stranded DNA virus closely related to the monkeypox virus and variola virus. It has been intensively studied as a highly effective model virus. According to the study, GBP2 overexpression suppresses ECTV replication in a dose-dependent manner, while GBP2 knockdown promotes ECTV infection. Additionally, it was discovered that GBP2 primarily functions through its N-terminal GTPase activity, and the inhibitory effect of GBP2 was disrupted in the GTP-binding-impaired mutant GBP2^K51A. This study is the first to demonstrate the inhibitory effect of GBP2 on ECTV, and it offers insights into innovative antiviral strategies. Full article

The phenomenon of pathogen co-infection detected in a half-fed Ixodes persulcatus tick taken from a human in the south of the Far East was studied. Research was carried out on PEK, Vero, and Vero-E6 cell lines, outbred mice, and chicken embryos using ELISA, PCR, IMFA, plaque formation, and electron microscopy. The tick contained an antigen and a genetic marker of the tick-borne encephalitis virus (TBEV). The patient had post-vaccination antibodies in a titer of 1:200, as a result of which, obviously, an antibody-dependent elimination of TBEV occurred. The tick-borne co-isolate also contained an unknown pathogen (Kiparis-144 virus), which, in our opinion, was a trigger for the activation of chronic infection in suckling white mice. In the laboratory co-isolate, ectromelia virus was present, as evidenced by paw edema during the intradermal infection of mice, characteristic rashes on the chorioallantoic envelope of chicken embryos, and typical plaques on Vero-E6. The Kiparis-144 virus was not pathogenic for white mice and chicken embryos, but it successfully multiplied in the PEK, Vero, and Vero-E6 lines. Viral co-infection was confirmed by electron microscopy. Passaging on mice contributed to an increase in the virulence of the co-isolate, whose titer increased by 10,000 times by the fifth passage, which poses an epidemiological danger. Full article

In west and central Africa, monkeypox occurs mainly in older children, adolescents and young adults. In two large epidemiology studies of monkeypox outbreaks, the investigators observed a sizable number of coinfections of chickenpox (varicella) and monkeypox. Based on a review of the literature, we propose that chickenpox (human herpesvirus-3 infection) is a risk factor for acquisition of monkeypox infection. Our hypothesis states that the chickenpox skin lesion provides an entry site for the monkeypox virus, which is harbored on a fomite in the environment of the patient. The fact that monkeypox can enter via a scratch or abrasion is a known mechanism of spread for three other poxviruses, including mousepox (ectromelia), orf and molluscum contagiosum. There are many similarities in pathogenesis between certain poxviruses and chickenpox, including a viremia with a cellular stress response leading to high levels of the IL-6 cytokine. One very revealing observation in the two epidemiology studies was that the number of pox as well as the severity of disease in children with chickenpox and monkeypox coinfection was not greater than found in children with monkeypox alone. Based on the above observations, we conclude that, when chickenpox precedes monkeypox, priming of the immune system by the earlier chickenpox infection moderates the severity of the secondary infection with monkeypox. This conclusion also has important public health implications about chickenpox surveillance. Full article

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 10³–10⁴ 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. Full article

Tumour necrosis factor (TNF) is an inflammatory cytokine produced in response to viral infections that promotes the recruitment and activation of leukocytes to sites of infection. This TNF-based host response is essential to limit virus spreading, thus poxviruses have evolutionarily adopted diverse molecular mechanisms to counteract TNF antiviral action. These include the expression of poxvirus-encoded soluble receptors or proteins able to bind and neutralize TNF and other members of the TNF ligand superfamily, acting as decoy receptors. This article reviews in detail the various TNF decoy receptors identified to date in the genomes from different poxvirus species, with a special focus on their impact on poxvirus pathogenesis and their potential use as therapeutic molecules. Full article

The ubiquitin system has emerged as a master regulator of many, if not all, cellular functions. With its large repertoire of conjugating and ligating enzymes, the ubiquitin system holds a unique mechanism to provide selectivity and specificity in manipulating protein function. As intracellular parasites viruses have evolved to modulate the cellular environment to facilitate replication and subvert antiviral responses. Poxviruses are a large family of dsDNA viruses with large coding capacity that is used to synthetise proteins and enzymes needed for replication and morphogenesis as well as suppression of host responses. This review summarises our current knowledge on how poxvirus functions rely on the cellular ubiquitin system, and how poxviruses exploit this system to their own advantage, either facilitating uncoating and genome release and replication or rewiring ubiquitin ligases to downregulate critical antiviral factors. Whilst much remains to be known about the intricate interactions established between poxviruses and the host ubiquitin system, our knowledge has revealed crucial viral processes and important restriction factors that open novel avenues for antiviral treatment and provide fundamental insights on the biology of poxviruses and other virus families. Full article

Ectromelia virus (ECTV), the causative agent of mousepox, has threatened laboratory mouse colonies worldwide for almost a century. Mousepox has been valuable for the understanding of poxvirus pathogenesis and immune evasion. Here, we have monitored in parallel the pathogenesis of nine ECTVs in BALB/cJ mice and report the full-length genome sequence of eight novel ECTV isolates or strains, including the first ECTV isolated from a field mouse, ECTV-MouKre. This approach allowed us to identify several genes, absent in strains attenuated through serial passages in culture, that may play a role in virulence and a set of putative genes that may be involved in enhancing viral growth in vitro. We identified a putative strong inhibitor of the host inflammatory response in ECTV-MouKre, an isolate that did not cause local foot swelling and developed a moderate virulence. Most of the ECTVs, except ECTV-Hampstead, encode a truncated version of the P4c protein that impairs the recruitment of virions into the A-type inclusion bodies, and our data suggest that P4c may play a role in viral dissemination and transmission. This is the first comprehensive report that sheds light into the phylogenetic and geographic relationship of the worldwide outbreak dynamics for the ECTV species. Full article

Dendritic cells (DCs) and macrophages are the first line of antiviral immunity. Viral pathogens exploit these cell populations for their efficient replication and dissemination via the modulation of intracellular signaling pathways. Disruption of the noncanonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling has frequently been observed in lymphoid cells upon infection with oncogenic viruses. However, several nononcogenic viruses have been shown to manipulate the noncanonical NF-κB signaling in different cell types. This study demonstrates the modulating effect of ectromelia virus (ECTV) on the components of the noncanonical NF-κB signaling pathway in established murine cell lines: JAWS II DCs and RAW 264.7 macrophages. ECTV affected the activation of TRAF2, cIAP1, RelB, and p100 upon cell treatment with both canonical and noncanonical NF-κB stimuli and thus impeded DNA binding by RelB and p52. ECTV also inhibited the expression of numerous genes related to the noncanonical NF-κB pathway and RelB-dependent gene expression in the cells treated with canonical and noncanonical NF-κB activators. Thus, our data strongly suggest that ECTV influenced the noncanonical NF-κB signaling components in the in vitro models. These findings provide new insights into the noncanonical NF-κB signaling components and their manipulation by poxviruses in vitro. Full article

The inhibition of tumor necrosis factor (TNF) through the use of either antibodies or soluble receptors is a highly effective strategy for the clinical control of chronic inflammatory conditions such as rheumatoid arthritis. Different viruses have similarly exploited this concept by expressing a set of specifically tailored secreted TNF decoy receptors to block host inflammatory responses. Poxviruses have been shown to encode at least two distinct molecules, termed Cytokine response modifier D (CrmD) and CrmB, in which a TNF inhibitor is combined with a chemokine inhibitor on the same molecule. The ectromelia virus CrmD protein was found to be a critical determinant of virulence in vivo, being able to control local inflammation to allow further viral spread and the establishment of a lethal infection. Strikingly, both the TNF and the chemokine inhibitory domains are required for the full activity of CrmD, suggesting a model in which inhibition of TNF is supported by the concomitant blockade of a reduced set of chemokines. Inspired by this model, we reasoned that a similar strategy could be applied to modify the clinically used human TNF receptor (etanercept), producing a generation of novel, more effective therapeutic agents. Here we show the analysis of a set of fusion proteins derived from etanercept by addition of a viral chemokine-binding protein. A bifunctional inhibitor capable of binding to and blocking the activity of TNF as well as a set of chemokines is generated that is active in the prevention of arthritis in a murine disease model. Full article

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that infects a wide range of mammals. CPXV-specific DNA and antibodies were detected in different vole species, such as common voles (Microtus arvalis) and bank voles (Myodes glareolus). Therefore, voles are the putative main reservoir host of CPXV. However, CPXV was up to now only isolated from common voles. Here we report the detection and isolation of a bank vole-derived CPXV strain (GerMygEK 938/17) resulting from a large-scale screening of bank voles collected in Thuringia, Germany, during 2017 and 2018. Phylogenetic analysis using the complete viral genome sequence indicated a high similarity of the novel strain to CPXV clade 3 and to OPV “Abatino” but also to Ectromelia virus (ECTV) strains. Phenotypic characterization of CPXV GerMygEK 938/17 using inoculation of embryonated chicken eggs displayed hemorrhagic pock lesions on the chorioallantoic membrane that are typical for CPXV but not for ECTV. CPXV GerMygEK 938/17 replicated in vole-derived kidney cell lines but at lower level than on Vero76 cell line. In conclusion, the first bank vole-derived CPXV isolate provides new insights into the genetic variability of CPXV in the putative reservoir host and is a valuable tool for further studies about CPXV-host interaction and molecular evolution of OPV. Full article