Targeting ectromelia virus and TNF/NF-κB or STAT3 signaling for effective treatment of viral pneumonia.

Viral causes of pneumonia pose constant threats to global public health, but there are no specific treatments currently available for the condition. Antivirals are ineffective when administered late after the onset of symptoms. Pneumonia is caused by an exaggerated inflammatory cytokine response to infection, but tissue necrosis and damage caused by virus also contribute to lung pathology. We hypothesized that viral pneumonia can be treated effectively if both virus and inflammation are simultaneously targeted. Combined treatment with the antiviral drug cidofovir and etanercept, which targets tumor necrosis factor (TNF), down-regulated nuclear factor kappa B-signaling and effectively reduced morbidity and mortality during respiratory ectromelia virus (ECTV) infection in mice even when treatment was initiated after onset of clinical signs. Treatment with cidofovir alone reduced viral load, but animals died from severe lung pathology. Treatment with etanercept had no effect on viral load but diminished levels of inflammatory cytokines and chemokines including TNF, IL-6, IL-1ß, IL-12p40, TGF-ß, and CCL5 and dampened activation of the STAT3 cytokine-signaling pathway, which transduces signals from multiple cytokines implicated in lung pathology. Consequently, combined treatment with a STAT3 inhibitor and cidofovir was effective in improving clinical disease and lung pathology in ECTV-infected mice. Thus, the simultaneous targeting of virus and a specific inflammatory cytokine or cytokine-signaling pathway is effective in the treatment of pneumonia. This approach might be applicable to pneumonia caused by emerging and re-emerging viruses, like seasonal and pandemic influenza A virus strains and severe acute respiratory syndrome coronavirus 2.

Resistance To Poxvirus Lethality Does Not Require the Necroptosis Proteins RIPK3 or MLKL.

Receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL) are proteins that are critical for necroptosis, a mechanism of programmed cell death that is both activated when apoptosis is inhibited and thought to be antiviral. Here, we investigated the role of RIPK3 and MLKL in controlling the Orthopoxvirus ectromelia virus (ECTV), a natural pathogen of the mouse. We found that C57BL/6 (B6) mice deficient in RIPK3 (Ripk3-/-) or MLKL (Mlkl-/-) were as susceptible as wild-type (WT) B6 mice to ECTV lethality after low-dose intraperitoneal infection and were as resistant as WT B6 mice after ECTV infection through the natural footpad route. Additionally, after footpad infection, Mlkl-/- mice, but not Ripk3-/- mice, endured lower viral titers than WT mice in the draining lymph node (dLN) at three days postinfection and in the spleen or in the liver at seven days postinfection. Despite the improved viral control, Mlkl-/- mice did not differ from WT mice in the expression of interferons or interferon-stimulated genes or in the recruitment of natural killer (NK) cells and inflammatory monocytes (iMOs) to the dLN. Additionally, the CD8 T-cell responses in Mlkl-/- and WT mice were similar, even though in the dLNs of Mlkl-/- mice, professional antigen-presenting cells were more heavily infected. Finally, the histopathology in the livers of Mlkl-/- and WT mice at 7 dpi did not differ. Thus, the mechanism of the increased virus control by Mlkl-/- mice remains to be defined. IMPORTANCE The molecules RIPK3 and MLKL are required for necroptotic cell death, which is widely thought of as an antiviral mechanism. Here we show that C57BL/6 (B6) mice deficient in RIPK3 or MLKL are as susceptible as WT B6 mice to ECTV lethality after a low-dose intraperitoneal infection and are as resistant as WT B6 mice after ECTV infection through the natural footpad route. Mice deficient in MLKL are more efficient than WT mice at controlling virus loads in various organs. This improved viral control is not due to enhanced interferon, natural killer cell, or CD8 T-cell responses. Overall, the data indicate that deficiencies in the molecules that are critical to necroptosis do not necessarily result in worse outcomes following viral infection and may improve virus control.

The establishment of a recombinase polymerase amplification technique for the detection of mouse poxvirus.

BACKGROUND: Ectromelia virus (ECTV) is the causative agent of mousepox in mice. In the past century, ECTV was a serious threat to laboratory mouse colonies worldwide. Recombinase polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method. RESULTS: In this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of the crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed. The lowest detection limit of the ECTV RT- RPA assay was 100 copies of DNA mol-ecules per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 135 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement. CONCLUSIONS: In conclusion, this RPA assay offers a novel alternative for the simple, sensitive, and specific identification of ECTV, especially in low-resource settings.

Ectromelia Virus Affects the Formation and Spatial Organization of Adhesive Structures in Murine Dendritic Cells In Vitro.

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.

The Viral Protein Poly(A) Polymerase Catalytic Subunit Interacts with Guanylate-Binding Proteins 2 to Antagonize the Antiviral Ability of Targeting Ectromelia Virus.

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.

Mechanisms of Antiviral Cytotoxic CD4 T Cell Differentiation.

Cytotoxic CD4 T lymphocytes (CD4-CTL) are important in antiviral immunity. For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection. How viral infections induce CD4-CTL responses remains incompletely understood. We demonstrate here that not only ECTV but also vaccinia virus and lymphocytic choriomeningitis virus induce CD4-CTL, though the response to ECTV is stronger. Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled. We also show that major histocompatibility complex class II molecules on CD11c+ cells are required for CD4-CTL differentiation and for mousepox resistance. Transcriptional analysis indicated that antiviral CD4-CTL and noncytolytic T helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells. Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment, and Runx3, required for CD8 T cell development and effector function. However, at the protein level, CD4-CTL had higher levels of the three transcription factors, suggesting that further posttranscriptional regulation is required for CD4-CTL differentiation. Finally, CRISPR/Cas9-mediated deletion of Runx3 in CD4 T cells inhibited CD4-CTL but not classical Th1 cell differentiation in response to ECTV infection. These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of posttranscriptionally regulated Runx3 in this process. IMPORTANCE While it is well established that cytotoxic CD4 T cells (CD4-CTLs) directly contribute to viral clearance, it remains unclear how CD4-CTL are induced. We now show that CD4-CTLs require sustained antigen presentation and are induced by CD11c-expressing antigen-presenting cells. Moreover, we show that CD4-CTLs are derived from the terminal differentiation of classical T helper 1 (Th1) subset of CD4 cells. Compared to Th1 cells, CD4-CTLs upregulate protein levels of the transcription factors ThPOK, Runx3, and GATA-3 posttranscriptionally. Deletion of Runx3 in differentiated CD4 T cells prevents induction of CD4-CTLs but not classical Th1 cells. These results advance our knowledge of how CD4-CTLs are induced during viral infection.

Loss of Resistance to Mousepox during Chronic Lymphocytic Choriomeningitis Virus Infection Is Associated with Impaired T-Cell Responses and Can Be Rescued by Immunization.

It is well established that chronic viral infections can cause immune suppression, resulting in increased susceptibility to other infectious diseases. However, the effects of chronic viral infection on T-cell responses and vaccination against highly pathogenic viruses are not well understood. We have recently shown that C57BL/6 (B6) mice lose their natural resistance to wild-type (WT) ectromelia virus (ECTV) when chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13). Here we compared the T-cell response to ECTV in previously immunologically naive mice that were chronically infected with CL13 or that were convalescent from acute infection with the Armstrong (Arm) strain of LCMV. Our results show that mice that were chronically infected with CL13 but not those that had recovered from Arm infection have highly defective ECTV-specific CD8+ and CD4+ T-cell responses to WT ECTV. These defects are at least partly due to the chronic infection environment. In contrast to mice infected with WT ECTV, mice chronically infected with CL13 survived without signs of disease when infected with ECTV-Δ036, a mutant ECTV strain that is highly attenuated. Strikingly, mice chronically infected with CL13 mounted a strong CD8+ T-cell response to ECTV-Δ036 and survived without signs of disease after a subsequent challenge with WT ECTV. Our work suggests that enhanced susceptibility to acute viral infections in chronically infected individuals can be partly due to poor T-cell responses but that sufficient T-cell function can be recovered and resistance to acute infection can be restored by immunization with highly attenuated vaccines.IMPORTANCE Chronic viral infections may result in immunosuppression and enhanced susceptibility to infections with other pathogens. For example, we have recently shown that mice chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) are highly susceptible to mousepox, a disease that is caused by ectromelia virus and that is the mouse homolog of human smallpox. Here we show chronic CL13 infection severely disrupts the expansion, proliferation, activation, and cytotoxicity of T cells in response due at least in part to the suppressive effects of the chronic infection milieu. Notably, despite this profound immunodeficiency, mice chronically infected with CL13 could be protected by vaccination with a highly attenuated variant of ECTV. These results demonstrate that protective vaccination of immunosuppressed individuals is possible, provided that proper immunization tools are used.

Chronic Lymphocytic Choriomeningitis Infection Causes Susceptibility to Mousepox and Impairs Natural Killer Cell Maturation and Function.

Chronic viral infections. like those of humans with cytomegalovirus, human immunodeficiency virus (even when under antiretroviral therapy), and hepatitis C virus or those of mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), result in immune dysfunction that predisposes the host to severe infections with unrelated pathogens. It is known that C57BL/6 (B6) mice are resistant to mousepox, a lethal disease caused by the orthopoxvirus ectromelia virus (ECTV), and that this resistance requires natural killer (NK) cells and other immune cells. We show that most B6 mice chronically infected with CL13 succumb to mousepox but that most of those that recovered from acute infection with the LCMV Armstrong (Arm) strain survive. We also show that B6 mice chronically infected with CL13 and those that recovered from Arm infection have a reduced frequency and a reduced number of NK cells. However, at steady state, NK cells in mice that have recovered from Arm infection mature normally and, in response to ECTV, get activated, become more mature, proliferate, and increase their cytotoxicity in vivo Conversely, in mice chronically infected with CL13, NK cells are immature and residually activated, and following ECTV infection, they do not mature, proliferate, or increase their cytotoxicity. Given the well-established importance of NK cells in resistance to mousepox, these data suggest that the NK cell dysfunction caused by CL13 persistence may contribute to the susceptibility of CL13-infected mice to mousepox. Whether chronic infections similarly affect NK cells in humans should be explored.IMPORTANCE Infection of adult mice with the clone 13 (CL13) strain of lymphocytic choriomeningitis virus (LCMV) is extensively used as a model of chronic infection. In this paper, we show that mice chronically infected with CL13 succumb to challenge with ectromelia virus (ECTV; the agent of mousepox) and that natural killer (NK) cells in CL13-infected mice are reduced in numbers and have an immature and partially activated phenotype but do respond to ECTV. These data may provide additional clues why humans chronically infected with certain pathogens are less resistant to viral diseases.

(+)-Camphor and (-)-borneol derivatives as potential anti-orthopoxvirus agents.

Although the World Health Organisation had announced that smallpox was eradicated over 40 years ago, the disease and other related pathogenic poxviruses such as monkeypox remain potential bioterrorist weapons and could also re-emerge as natural infections. We have previously reported (+)-camphor and (-)-borneol derivatives with an antiviral activity against the vaccinia virus. This virus is similar to the variola virus (VARV), the causative agent of smallpox, but can be studied at BSL-2 facilities. In the present study, we evaluated the antiviral activity of the most potent compounds against VARV, cowpox virus, and ectromelia virus (ECTV). Among the compounds tested, 4-bromo-N'-((1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)benzohydrazide 18 is the most effective compound against various orthopoxviruses, including VARV, with an EC50 value of 13.9 µM and a selectivity index of 206. Also, (+)-camphor thiosemicarbazone 9 was found to be active against VARV and ECTV.

Deficiency of Selected Cathepsins Does Not Affect the Inhibitory Action of ECTV on Immune Properties of Dendritic Cells.

Ectromelia virus (ECTV), an orthopoxvirus, undergoes productive replication in conventional dendritic cells (cDCs), resulting in the inhibition of their innate and adaptive immune functions. ECTV replication rate in cDCs is increased due to downregulation of the expression of cathepsins - cystein proteases that orchestrate several steps during DC maturation. Therefore, this study was aimed to determine if downregulation of cathepsins, such as B, L or S, disrupts cDC capacity to induce activating signals in T cells or whether infection of cDCs with ECTV further weakens their functions as antigen-presenting cells. Our results showed that cDCs treated with siRNA against cathepsin B, L and S synthesize similar amounts of pro-inflammatory cytokines and exhibit comparable ability to mature and stimulate alloreactive CD4+ T cells, as untreated wild type (WT) cells. Moreover, ECTV inhibitory effect on cDC innate and adaptive immune functions, observed especially after LPS treatment, was comparable in both cathepsin-silenced and WT cells. Taken together, the absence of cathepsins B, L and S has minimal, if any, impact on the inhibitory effect of ECTV on cDC immune functions. We assume that the virus-mediated inhibition of cathepsin expression in cDCs represents more a survival mechanism than an immune evasion strategy.

ECTV Abolishes the Ability of GM-BM Cells to Stimulate Allogeneic CD4 T Cells in a Mouse Strain-Independent Manner.

Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4+ T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4+ T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4+ T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.

Development of a SYBR Green I real-time PCR for detection and quantitation of orthopoxvirus by using Ectromelia virus.

Ectromelia virus (ECTV) is the causative agent of mousepox, which has devastating effects in laboratory-mouse colonies and causes economic loss in biomedical research. More importantly, ECTV has been extensively used as an excellent model for studies of the pathogenesis and immunobiology of human smallpox. A rapid and sensitive SYBR Green I-based real-time PCR assay was developed and used for the detection and quantitation of orthopoxvirus by using ECTV in this study. Primers targeted to the highly conserved region of major core protein P4b gene of orthopoxvirus were designed and the standard plasmid was constructed. This assay was able to detect a minimum of 10 copies of standard DNA and 5 TCID50 units of ECTV. In addition, no cross-reactions were observed with two DNA viruses, such as herpes simplex virus and swine pseudorabies virus, and one RNA virus, vesicular stomatitis virus. Furthermore, intra- and inter-assay variability data showed that this method had a highly reproducibility and reliability. Moreover, the current assay was faster and had a higher sensitivity for detection of ECTV genomic DNA in cell cultured and clinical test samples. Therefore, the high sensitivity and reproducibility of this SYBR Green real-time PCR approach is a more effective method than the conventional PCR for ECTV diagnosis and quantitation.

Long actin-based cellular protrusions as novel evidence of the cytopathic effect induced in immune cells infected by the ectromelia virus.

The aim of the study was to evaluate the influence of ectromelia virus (ECTV) infection on actin cytoskeleton rearrangement in immune cells, such as macrophages and dendritic cells (DCs). Using scanning electron and fluorescence microscopy analysis we observed the presence of long actin-based cellular extensions, formed by both types of immune cells at later stages of infection with ECTV. Such extensions contained straight tubulin filaments and numerous punctuate mitochondria. Moreover, these long cellular projections extended to a certain length and formed convex structures termed "cytoplasmic packets". These structures contained numerous viral particles and presumably were sites of progeny virions' release via budding. Further, discrete mitochondria and separated tubulin filaments that formed a scaffold for accumulated mitochondria were visible within cytoplasmic packets. ECTV-induced long actin-based protrusions resemble "cytoplasmic corridors" and probably participate in virus dissemination. Our data demonstrate the incredible capacity for adaptation of ECTV to its natural host immune cells, in which it can survive, replicate and induce effective mechanisms for viral spread and dissemination.

Fatal Outbreak in Tonkean Macaques Caused by Possibly Novel Orthopoxvirus, Italy, January 2015 1.

In January 2015, during a 3-week period, 12 captive Tonkean macacques at a sanctuary in Italy died. An orthopoxvirus infection was suspected because of negative-staining electron microscopy results. The diagnosis was confirmed by histology, virus isolation, and molecular analysis performed on different organs from all animals. An epidemiologic investigation was unable to define the infection source in the surrounding area. Trapped rodents were negative by virologic testing, but specific IgG was detected in 27.27% of small rodents and 14.28% of rats. An attenuated live vaccine was administered to the susceptible monkey population, and no adverse reactions were observed; a detectable humoral immune response was induced in most of the vaccinated animals. We performed molecular characterization of the orthopoxvirus isolate by next-generation sequencing. According to the phylogenetic analysis of the 9 conserved genes, the virus could be part of a novel clade, lying between cowpox and ectromelia viruses.

Dendritic cells during mousepox: The role of delayed apoptosis in the pathogenesis of infection.

Dendritic cells (DCs) are effector cells linking the innate immune system with the adaptive immune response. Many viruses eliminate DCs to prevent host response, induce immunosuppression and to maintain chronic infection. In this study, we examined apoptotic response of dendritic cells during in vitro and in vivo infection with ectromelia virus (ECTV), the causative agent of mousepox. ECTV-infected bone marrow dendritic cells (BMDCs) from BALB/c mice underwent apoptosis through mitochondrial pathway at 48 h post infection, up-regulated FasL and decreased expression of anti-apoptotic Bcl-2 and pro-apoptotic Fas. Similar pattern of Bcl-2, Fas and FasL expression was observed for DCs early during in vivo infection of BALB/c mice. Both BMDCs and DCs from BALB/c mice showed no maturation upon ECTV infection. We conclude that ECTV-infected DCs from BALB/c mouse strain help the virus to spread and to maintain infection.

Strategies of NF-κB signaling modulation by ectromelia virus in BALB/3T3 murine fibroblasts.

Nuclear factor κB (NF-κB) is a pleiotropic transcription factor that regulates the expression of immune response genes. NF-κB signaling can be disrupted by pathogens that prevent host immune response. In this work, we examined the influence of ectromelia (mousepox) virus (ECTV) on NF-κB signaling in murine BALB/3T3 fibroblasts. Activation of NF-κB via tumor necrosis factor (TNF) receptor 1 (TNFR1) in these cells induces proinflammatory cytokine secretion. We show that ECTV does not recruit NF-κB to viral factories or induce NF-κB nuclear translocation in BALB/3T3 cells. Additionally, ECTV counteracts TNF-α-induced p65 NF-κB nuclear translocation during the course of infection. Inhibition of TNF-α-induced p65 nuclear translocation was also observed in neighboring cells that underwent fusion with ECTV-infected cells. ECTV inhibits the key step of NF-κB activation, i.e. Ser32 phosphorylation and degradation of inhibitor κBα (IκBα) induced by TNF-α. We also observed that ECTV prevents TNF-α-induced Ser536 of p65 phosphorylation in BALB/3T3 cells. Studying TNFR1 signaling provides information about regulation of inflammatory response and cell survival. Unraveling poxviral immunomodulatory strategies may be helpful in drug target identification as well as in vaccine development.

The roles of CD4+ T cell help, sex, and dose in the induction of protective CD8+ T cells against a lethal poxvirus by mRNA-LNP vaccines.

The role of CD4+ T cells in the induction of protective CD8+ T cells by mRNA lipid nanoparticle (LNP) vaccines is unknown. We used B6 or Tlr9 -/- mice depleted or not of CD4+ T cells and LNP vaccines loaded with mRNAs encoding the ectromelia virus (ECTV) MHC class I H-2 Kb-restricted immunodominant CD8+ T cell epitope TSYKFESV (TSYKFESV mRNA-LNPs) or the ECTV EVM158 protein, which contains TSYKFESV (EVM-158 mRNA-LNPs). Following prime and boost with 10 µg of either vaccine, Kb-TSYKFESV-specific CD8+ T cells fully protected male and female mice from ECTV at 29 (both mRNA-LNPs) or 90 days (EVM158 mRNA-LNPs) post boost (dpb) independently of CD4+ T cells. However, at 29 dpb with 1 µg mRNA-LNPs, males had lower frequencies of Kb-TSYKFESV-specific CD8+ T cells and were much less well protected than females from ECTV, also independently of CD4+ T cells. At 90 dpb with 1 µg EVM158 mRNA-LNPs, the frequencies of Kb-TSYKFESV-specific CD8+ T cells in males and females were similar, and both were similarly partially protected from ECTV, independently of CD4+ T cells. Therefore, at optimal or suboptimal doses of mRNA-LNP vaccines, CD4+ T cell help is unnecessary to induce protective anti-poxvirus CD8+ T cells specific to a dominant epitope. At suboptimal doses, protection of males requires more time to develop.

[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.

Guanylate-Binding Protein 2 Exerts GTPase-Dependent Anti-Ectromelia Virus Effect.

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 GBP2K51A. This study is the first to demonstrate the inhibitory effect of GBP2 on ECTV, and it offers insights into innovative antiviral strategies.

Complete deletion of Ectromelia virus p28 impairs virus genome replication in a mouse strain, cell type, and multiplicity of infection-dependent manner.

p28 is a poxvirus-encoded E3 ubiquitin ligase that possesses an N-terminal KilA-N domain and a C-terminal RING domain. In Ectromelia virus (ECTV), disruption of the p28 RING domain severely attenuated virulence in A strain mice, which normally succumb to ECTV infection. Moreover, this mutant virus exhibited dramatically reduced genome replication and impaired factory formation in A strain mice peritoneal macrophages (PMs) infected at high multiplicity of infection (MOI) These defects were not observed in PMs isolated from C57BL/6 mice which survive ECTV infection, demonstrating that p28 functions in a context-specific manner. To further investigate p28 function, we completely deleted the p28 gene from ECTV (ECTV-Δp28). In contrast to previous findings, we found that the ECTV-Δp28 virus exhibited severely compromised virus production and genome replication in PMs isolated from A strain mice only when infected at low MOI. This defect was minimal in bone marrow-derived macrophages and two cell lines derived from A strain mice. Furthermore, this low MOI defect in virus production was also observed in PMs isolated from the susceptible BALB/c mouse strain, but not PMs isolated from C57BL/6 mice. Taken together, our data demonstrate that the requirement for ECTV p28 to establish a productive infection depends on the MOI, the cell type, as well as the mouse strain.