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.

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.

Comparative Pathogenesis, Genomics and Phylogeography of Mousepox.

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.

Mechanism of action of the viral chemokine-binding protein E163 from ectromelia virus.

Chemokines interact with glycosaminoglycans (GAGs) at the cellular surface and to specific cell-surface receptors to activate signaling pathways. The GAG interaction allows the formation of a chemotactic gradient of chemokine required for cell haptotaxis and chemokine oligomerization. Poxviruses encode secreted chemokine-binding proteins with no sequence similarity to their cellular counterparts to modulate the host immune system. The E163 protein from ectromelia virus, the causative agent of mousepox, binds chemokines through their GAG-binding domain. In addition, E163 interacts with GAGs to be anchored at the cell surface, but its ability to interfere with chemokine-GAG interactions has not been demonstrated. We report the identification of the GAG-binding regions in E163 and the generation of mutant forms deficient of GAG binding. Chemokine binding assays show that some of the E163 GAG-binding sites are also involved in the interaction with chemokines. By using recombinant GAG-binding mutant forms we demonstrate that E163 prevents the interaction of chemokines with cell-surface GAGs, providing mechanisms for the immunomodulatory activity of the viral chemokine-binding protein E163.

The Virology of Taterapox Virus In Vitro.

Taterapox virus (TATV) is phylogenetically the closest related virus to variola-the etiological agent of smallpox. Despite the similarity, few studies have evaluated the virus. In vivo, TATV can infect several animals but produces an inapparent infection in wild-type mice; however, TATV does cause morbidity and mortality in some immunocompromised strains. We employed in vitro techniques to compare TATV to ectromelia (ECTV) and vaccinia (VACV) viruses. Both ECTV and TATV replicate efficiently in primate cell lines but TATV replicates poorly in murine cells lines. Furthermore, TATV induces cytopathic effects, but to a lesser extent than ECTV, and changes cytoskeletal networks differently than both ECTV and VACV. Bioinformatic studies revealed differences in several immunomodulator open reading frames that could contribute to the reduced virulence of TATV, which were supported by in vitro cytokine assays.

Ectromelia virus lacking the E3L ortholog is replication-defective and nonpathogenic but does induce protective immunity in a mouse strain susceptible to lethal mousepox.

All known orthopoxviruses, including ectromelia virus (ECTV), contain a gene in the E3L family. The protein product of this gene, E3, is a double-stranded RNA-binding protein. It can impact host range and is used by orthopoxviruses to combat cellular defense pathways, such as PKR and RNase L. In this work, we constructed an ECTV mutant with a targeted disruption of the E3L open reading frame (ECTVΔE3L). Infection with this virus resulted in an abortive replication cycle in all cell lines tested. We detected limited transcription of late genes but no significant translation of these mRNAs. Notably, the replication defects of ECTVΔE3L were rescued in human and mouse cells lacking PKR. ECTVΔE3L was nonpathogenic in BALB/c mice, a strain susceptible to lethal mousepox disease. However, infection with ECTVΔE3L induced protective immunity upon subsequent challenge with wild-type virus. In summary, E3L is an essential gene for ECTV.

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies.

Evaluation of Taterapox Virus in Small Animals.

Taterapox virus (TATV), which was isolated from an African gerbil (Tatera kempi) in 1975, is the most closely related virus to variola; however, only the original report has examined its virology. We have evaluated the tropism of TATV in vivo in small animals. We found that TATV does not infect Graphiurus kelleni, a species of African dormouse, but does induce seroconversion in the Mongolian gerbil (Meriones unguiculatus) and in mice; however, in wild-type mice and gerbils, the virus produces an unapparent infection. Following intranasal and footpad inoculations with 1 × 106 plaque forming units (PFU) of TATV, immunocompromised stat1-/- mice showed signs of disease but did not die; however, SCID mice were susceptible to intranasal and footpad infections with 100% mortality observed by Day 35 and Day 54, respectively. We show that death is unlikely to be a result of the virus mutating to have increased virulence and that SCID mice are capable of transmitting TATV to C57BL/6 and C57BL/6 stat1-/- animals; however, transmission did not occur from TATV inoculated wild-type or stat1-/- mice. Comparisons with ectromelia (the etiological agent of mousepox) suggest that TATV behaves differently both at the site of inoculation and in the immune response that it triggers.

Infection with diverse immune-modulating poxviruses elicits different compositional shifts in the mouse gut microbiome.

It is often not possible to demonstrate causality within the context of gut microbiota dysbiosis-linked diseases. Thus, we need a better understanding of the mechanisms whereby an altered host immunophysiology shapes its resident microbiota. In this regard, immune-modulating poxvirus strains and mutants could differentially alter gut mucosal immunity in the context of a natural immune response, providing a controlled natural in vivo setting to deepen our understanding of the immune determinants of microbiome composition. This study represents a proof-of-concept that the use of an existing collection of different immune-modulating poxviruses may represent an innovative tool in gut microbiome research. To this end, 16S rRNA amplicon sequencing and RNAseq transcriptome profiling were employed as proxies for microbiota composition and gut immunophysiological status in the analysis of caecal samples from control mice and mice infected with various poxvirus types. Our results show that different poxvirus species and mutants elicit different shifts in the mice mucosa-associated microbiota and, in some instances, significant concomitant shifts in gut transcriptome profiles, thus providing an initial validation to the proposed model.

A homolog of the variola virus B22 membrane protein contributes to ectromelia virus pathogenicity in the mouse footpad model.

Most poxviruses encode a homolog of a ~200,000-kDa membrane protein originally identified in variola virus. We investigated the importance of the ectromelia virus (ECTV) homolog C15 in a natural infection model. In cultured mouse cells, the replication of a mutant virus with stop codons near the N-terminus (ECTV-C15Stop) was indistinguishable from a control virus (ECTV-C15Rev). However, for a range of doses injected into the footpads of BALB/c mice there was less mortality with the mutant. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less ECTV-C15Stop in popliteal and inguinal lymph nodes, spleen and liver indicating decreased virus spread and replication. The latter results were supported by immunohistochemical analyses. Decreased spread was evidently due to immune modulatory activity of C15, rather than to an intrinsic viral function, as the survival of infected mice depended on CD4+ and CD8+ T cells.

Quantum dot-fluorescence in situ hybridisation for Ectromelia virus detection based on biotin-streptavidin interactions.

Ectromelia virus (ECTV) is an pathogen that can lead to a lethal, acute toxic disease known as mousepox in mice. Prevention and control of ECTV infection requires the establishment of a rapid and sensitive diagnostic system for detecting the virus. In the present study, we developed a method of quantum-dot-fluorescence based in situ hybridisation for detecting ECTV genome DNA. Using biotin-dUTP to replace dTTP, biotin was incorporated into a DNA probe during polymerase chain reaction. High sensitivity and specificity of ECTV DNA detection were displayed by fluorescent quantum dots based on biotin-streptavidin interactions. ECTV DNA was then detected by streptavidin-conjugated quantum dots that bound the biotin-labelled probe. Results indicated that the established method can visualise ECTV genomic DNA in both infected cells and mouse tissues. To our knowledge, this is the first study reporting quantum-dot-fluorescence based in situ hybridisation for the detection of viral nucleic acids, providing a reference for the identification and detection of other viruses.

Deficiency in Th2 cytokine responses exacerbate orthopoxvirus infection.

Ectromelia virus (ECTV) causes mousepox in mice, a disease very similar to smallpox in humans. ECTV and variola virus (VARV), the agent of smallpox, are closely related orthopoxviruses. Mousepox is an excellent small animal model to study the genetic and immunologic basis for resistance and susceptibility of humans to smallpox. Resistance to mousepox is dependent on a strong polarized type 1 immune response, associated with robust natural killer (NK) cell, cytotoxic T lymphocyte (CTL) and gamma interferon (IFN-γ) responses. In contrast, ECTV-susceptible mice generate a type 2 response, associated with weak NK cell, CTL and IFN-γ responses but robust IL-4 responses. Nonetheless, susceptible strains infected with mutant ECTV lacking virus-encoded IFN-γ binding protein (vIFN-γbp) (ECTV-IFN-γbpΔ) control virus replication through generation of type 1 response. Since the IL-4/IL-13/STAT-6 signaling pathways polarize type 2/T helper 2 (Th2) responses with a corresponding suppression of IFN-γ production, we investigated whether the combined absence of vIFN-γbp, and one or more host genes involved in Th2 response development, influence generation of protective immunity. Most mutant mouse strains infected with wild-type (WT) virus succumbed to disease more rapidly than WT animals. Conversely, the disease outcome was significantly improved in WT mice infected with ECTV-IFN-γbpΔ but absence of IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage. Deficiency in IL-13 or STAT-6 resulted in defective CTL responses, higher mortality rates and accelerated deaths. Deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Factors contributing to susceptibility or resistance to mousepox are far more complex than a balance between Th1 and Th2 responses.

Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV.

Ectromelia virus encodes an anti-apoptotic protein that regulates cell death.

Apoptosis serves as a powerful defense against damaged or pathogen-infected cells. Since apoptosis is an effective defense against viral infection, many viruses including poxviruses, encode proteins to prevent or delay apoptosis. Here we show that ectromelia virus, the causative agent of mousepox encodes an anti-apoptotic protein EVM025. Here we demonstrate that expression of functional EVM025 is crucial to prevent apoptosis triggered by virus infection and staurosporine. We demonstrate that the expression of EVM025 prevents the conformational activation of the pro-apoptotic proteins Bak and Bax, allowing the maintenance of mitochondrial membrane integrity upon infection with ECTV. Additionally, EVM025 interacted with intracellular Bak. We were able to demonstrate that EVM025 ability to inhibit Bax activation is a function of its ability to inhibit the activity of an upstream BH3 only protein Bim. Collectively, our data indicates that EVM025 inhibits apoptosis by sequestering Bak and inhibiting the activity of Bak and Bax.

Ectromelia virus encodes a family of Ankyrin/F-box proteins that regulate NFκB.

A notable feature of poxviruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein ßTrCP. Here, we show that ectromelia virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and this inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which ectromelia virus inhibits NFκB.

The genome sequence of ectromelia virus Naval and Cornell isolates from outbreaks in North America.

Ectromelia virus (ECTV) is the causative agent of mousepox, a disease of laboratory mouse colonies and an excellent model for human smallpox. We report the genome sequence of two isolates from outbreaks in laboratory mouse colonies in the USA in 1995 and 1999: ECTV-Naval and ECTV-Cornell, respectively. The genome of ECTV-Naval and ECTV-Cornell was sequenced by the 454-Roche technology. The ECTV-Naval genome was also sequenced by the Sanger and Illumina technologies in order to evaluate these technologies for poxvirus genome sequencing. Genomic comparisons revealed that ECTV-Naval and ECTV-Cornell correspond to the same virus isolated from independent outbreaks. Both ECTV-Naval and ECTV-Cornell are extremely virulent in susceptible BALB/c mice, similar to ECTV-Moscow. This is consistent with the ECTV-Naval genome sharing 98.2% DNA sequence identity with that of ECTV-Moscow, and indicates that the genetic differences with ECTV-Moscow do not affect the virulence of ECTV-Naval in the mousepox model of footpad infection.

Initial characterization of vaccinia virus B4 suggests a role in virus spread.

Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus.

Changes in the mitochondrial network during ectromelia virus infection of permissive L929 cells.

Mitochondria are extremely important organelles in the life of a cell. Recent studies indicate that mitochondria also play a fundamental role in the cellular innate immune mechanisms against viral infections. Moreover, mitochondria are able to alter their shape continuously through fusion and fission. These tightly regulated processes are activated or inhibited under physiological or pathological (e.g. viral infection) conditions to help restore homeostasis. However, many types of viruses, such as orthopoxviruses, have developed various strategies to evade the mitochondrial-mediated antiviral innate immune responses. Moreover, orthopoxviruses exploit the mitochondria for their survival. Such viral activity has been reported during vaccinia virus (VACV) infection. Our study shows that the Moscow strain of ectromelia virus (ECTV-MOS), an orthopoxvirus, alters the mitochondrial network in permissive L929 cells. Upon infection, the branching structure of the mitochondrial network collapses and becomes disorganized followed by destruction of mitochondrial tubules during the late stage of infection. Small, discrete mitochondria co-localize with progeny virions, close to the cell membrane. Furthermore, clustering of mitochondria is observed around viral factories, particularly between the nucleus and viroplasm. Our findings suggest that ECTV-MOS modulates mitochondrial cellular distribution during later stages of the replication cycle, probably enabling viral replication and/or assembly as well as transport of progeny virions inside the cell. However, this requires further investigation.

Expression of a non-coding RNA in ectromelia virus is required for normal plaque formation.

Poxviruses are dsDNA viruses with large genomes. Many genes in the genome remain uncharacterized, and recent studies have demonstrated that the poxvirus transcriptome includes numerous so-called anomalous transcripts not associated with open reading frames. Here, we characterize the expression and role of an apparently non-coding RNA in orthopoxviruses, which we call viral hairpin RNA (vhRNA). Using a bioinformatics approach, we predicted expression of a transcript not associated with an open reading frame that is likely to form a stem-loop structure due to the presence of a 21 nt palindromic sequence. Expression of the transcript as early as 2 h post-infection was confirmed by northern blot and analysis of publicly available vaccinia virus infected cell transcriptomes. The transcription start site was determined by RACE PCE and transcriptome analysis, and early and late promoter sequences were identified. Finally, to test the function of the transcript we generated an ectromelia virus knockout, which failed to form plaques in cell culture. The important role of the transcript in viral replication was further demonstrated using siRNA. Although the function of the transcript remains unknown, our work contributes to evidence of an increasingly complex poxvirus transcriptome, suggesting that transcripts such as vhRNA not associated with an annotated open reading frame can play an important role in viral replication.

Characterization of ectromelia virus deficient in EVM036, the homolog of vaccinia virus F13L, and its application for rapid generation of recombinant viruses.

The orthopoxvirus (OPV) vaccinia virus (VACV) requires an intact F13L gene to produce enveloped virions (EV) and to form plaques in cell monolayers. Simultaneous introduction of an exogenous gene and F13L into F13L-deficient VACV results in expression of the foreign gene and restoration of plaque size. This is used as a method to rapidly generate VACV recombinants without the need for drug selection. However, whether other OPVs require the orthologs of F13L to generate EV and form plaques, whether F13L orthologs and EV are important for OPV pathogenesis in natural hosts, and whether a system based on F13L ortholog deficiency can be used to generate recombinant OPVs other than VACV have not been reported. The F13L ortholog in ectromelia virus (ECTV), the agent of mousepox, is EVM036. We show that ECTV lacking EVM036 formed small plaques and was highly attenuated in vivo but still induced strong antibody responses. Reintroduction of EVM036 in tandem with the DsRed gene resulted in a virus that expressed DsRed in infected cells but was indistinguishable from wild-type ECTV in terms of plaque size and in vivo virulence. Thus, our data show that, like F13L in VACV, EVM036 is required for ECTV plaque formation and that EVM036 and EV are important for ECTV virulence. Our experiments also suggest that OPVs deficient in F13L orthologs could serve as safer anti-OPV vaccines. Further, our results demonstrate that ECTV deficient in EVM036 can be exploited for the rapid generation of fully virulent ECTV expressing foreign genes of interest.