In vitro evaluation of antiviral activity in carnivorous plant species.

Sarracenia purpurea is a carnivorous plant historically used to treat smallpox infections. Our previous data found S. purpurea had broad spectrum antiviral activity in vitro. S. purpurea is one of several hundred identified carnivorous species of plants. Carnivorous plants have evolved through convergent evolution in at least ten independent events, usually in response to harsh environments where nutrition from prey is required for growth. These prey are known vectors of plant viruses that might introduce novel biotic stressors and defense pathways in carnivorous plants. This study evaluated the antiviral activity of several non-carnivorous and carnivorous plants from four evolutionarily distinct clades. Results demonstrated that carnivorous plants have evolved antiviral activity, a trait that is not present in related species of non-carnivorous plants. The antiviral trait may be due to the plant-prey relationship with insect vectors and an evolutionary need for carnivorous plants to have more robust antiviral defense systems.

Type I Interferon: Monkeypox/Mpox Viruses Achilles Heel?

Poxviruses are notorious for having acquired/evolved numerous genes to counteract host innate immunity. Chordopoxviruses have acquired/evolved at least three different inhibitors of host necroptotic death: E3, which blocks ZBP1-dependent necroptotic cell death, and vIRD and vMLKL that inhibit necroptosis downstream of initial cell death signaling. While this suggests the importance of the necroptotic cell death pathway in inhibiting chordopoxvirus replication, several chordopoxviruses have lost one or more of these inhibitory functions. Monkeypox/mpox virus (MPXV) has lost a portion of the N-terminus of its E3 homologue. The N-terminus of the vaccinia virus E3 homologue serves to inhibit activation of the interferon-inducible antiviral protein, ZBP1. This likely makes MPXV unique among the orthopoxviruses in being sensitive to interferon (IFN) treatment in many mammals, including humans, which encode a complete necroptotic cell death pathway. Thus, IFN sensitivity may be the Achille's Heel for viruses like MPXV that cannot fully inhibit IFN-inducible, ZBP1-dependent antiviral pathways.

Viral anti-inflammatory serpin reduces immuno-coagulopathic pathology in SARS-CoV-2 mouse models of infection.

SARS-CoV-2 acute respiratory distress syndrome (ARDS) induces uncontrolled lung inflammation and coagulopathy with high mortality. Anti-viral drugs and monoclonal antibodies reduce early COVID-19 severity, but treatments for late-stage immuno-thrombotic syndromes and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases. The myxoma virus-derived Serp-1 protein is a secreted immunomodulatory serpin that targets activated thrombotic, thrombolytic, and complement proteases as a self-defense strategy to combat clearance. Serp-1 is effective in multiple animal models of inflammatory lung disease and vasculitis. Here, we describe systemic treatment with purified PEGylated Serp-1 as a therapy for immuno-coagulopathic complications during ARDS. Treatment with PEGSerp-1 in two mouse-adapted SARS-CoV-2 models in C57Bl/6 and BALB/c mice reduced lung and heart inflammation, with improved outcomes. PEGSerp-1 significantly reduced M1 macrophages in the lung and heart by modifying urokinase-type plasminogen activator receptor (uPAR), thrombotic proteases, and complement membrane attack complex (MAC). Sequential changes in gene expression for uPAR and serpins (complement and plasminogen inhibitors) were observed. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for severe viral ARDS, immuno-coagulopathic responses, and Long COVID.

Multi-modal efficacy of a chimeric vesiculovirus expressing the Morreton glycoprotein in sarcoma.

Vesiculoviruses are attractive oncolytic virus platforms due to their rapid replication, appreciable transgene capacity, broad tropism, limited preexisting immunity, and tumor selectivity through type I interferon response defects in malignant cells. We developed a synthetic chimeric virus (VMG) expressing the glycoprotein (G) from Morreton virus (MorV) and utilizing the remaining structural genes from vesicular stomatitis virus (VSV). VMG exhibited in vitro efficacy by inducing oncolysis in a broad range of sarcoma subtypes across multiple species. Notably, all cell lines tested showed the ability of VMG to yield productive infection with rapid replication kinetics and induction of apoptosis. Furthermore, pilot safety evaluations of VMG in immunocompetent, non-tumor-bearing mice showed an absence of toxicity with intranasal doses as high as 1e10 50% tissue culture infectious dose (TCID50)/kg. Locoregional administration of VMG in vivo resulted in tumor reduction in an immunodeficient Ewing sarcoma xenograft at doses as low as 2e5 TCID50. In a murine syngeneic fibrosarcoma model, while no tumor inhibition was achieved with VMG, there was a robust induction of CD8+ T cells within the tumor. The studies described herein establish the promising potential for VMG to be used as a novel oncolytic virotherapy platform with anticancer effects in sarcoma.

Canonical cellular stress granules are required for arsenite-induced necroptosis mediated by Z-DNA-binding protein 1.

Cellular stress granules arise in cells subjected to stress and promote cell survival. A cellular protein that localizes to stress granules is Z-DNA-binding protein 1 (ZBP1), which plays a major role in necroptosis, a programmed cell death pathway mediated by the kinase RIPK3. Here, we showed that the stress granule inducer arsenite activated RIPK3-dependent necroptosis. This pathway required ZBP1, which localized to arsenite-induced stress granules. RIPK3 localized to stress granules in the presence of ZBP1, leading to the formation of ZBP1-RIPK3 necrosomes, phosphorylation of the RIPK3 effector MLKL, and execution of necroptosis. Cells that did not form stress granules did not induce necroptosis in response to arsenite. Together, these results show that arsenite induces ZBP1-mediated necroptosis in a manner dependent on stress granule formation.

Nuclear export inhibitor Selinexor targeting XPO1 enhances coronavirus replication.

Nucleocytoplasmic transport of proteins using XPO1 (exportin 1) plays a vital role in cell proliferation and survival. Many viruses also exploit this pathway to promote infection and replication. Thus, inhibiting XPO1-mediated nuclear export with selective inhibitors activates multiple antiviral and anti-inflammatory pathways. The XPO1 inhibitor, Selinexor, is an FDA-approved anticancer drug predicted to have antiviral function against many viruses, including SARS-CoV-2. Unexpectedly, we observed that pretreatment of cultured human cells with Selinexor actually enhanced protein expression and replication of coronaviruses, including SARS-CoV-2. Knockdown of cellular XPO1 protein expression significantly enhanced the replication of coronaviruses in human cells. We further demonstrate that Selinexor treatment reduced the formation of unique cytoplasmic antiviral granules that include RNA helicase DHX9 in the virus-infected cells. These results, for the first time, show that the anti-cancer drug Selinexor enhances the replication of coronaviruses in human cells in vitro and thus should be further explored in vivo for the potential impact on the dual use for anticancer and antiviral therapy.

Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers.

BACKGROUND: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

Intranasal Immunization with a Vaccinia Virus Vaccine Vector Expressing Pre-Fusion Stabilized SARS-CoV-2 Spike Fully Protected Mice against Lethal Challenge with the Heavily Mutated Mouse-Adapted SARS2-N501YMA30 Strain of SARS-CoV-2.

The Omicron SARS-CoV-2 variant has been designated as a variant of concern because its spike protein is heavily mutated. In particular, the Omicron spike is mutated at five positions (K417, N440, E484, Q493, and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501YMA30, contains a spike that is also heavily mutated, with mutations at four of the five positions in the Omicron spike associated with neutralizing antibody escape (K417, E484, Q493, and N501). In this manuscript, we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against symptoms and death from SARS2-N501YMA30. Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that the Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus-administered without parenteral injection-can fully protect against the heavily mutated mouse-adapted SARS2-N501YMA30.

Small Hero with Great Powers: Vaccinia Virus E3 Protein and Evasion of the Type I IFN Response.

Poxviridae have developed a plethora of strategies to evade innate and adaptive immunity. In this review, we focused on the vaccinia virus E3 protein, encoded by the E3L gene. E3 is present within the Chordopoxvirinae subfamily (with the exception of the avipoxviruses and molluscum contagiosum virus) and displays pleiotropic effects on the innate immune system. Initial studies identified E3 as a double-stranded RNA (dsRNA)-binding protein (through its C terminus), able to inhibit the activation of protein kinase dependent on RNA (PKR) and the 2'5'-oligoadenylate synthetase (OAS)/RNase L pathway, rendering E3 a protein counteracting the type I interferon (IFN) system. In recent years, N-terminal mutants of E3 unable to bind to Z-form nucleic acids have been shown to induce the cellular death pathway necroptosis. This pathway was dependent on host IFN-inducible Z-DNA-binding protein 1 (ZBP1); full-length E3 is able to inhibit ZBP1-mediated necroptosis. Binding to what was identified as Z-RNA has emerged as a novel mechanism of counteracting the type I IFN system and has broadened our understanding of innate immunity against viral infections. This article gives an overview of the studies leading to our understanding of the vaccinia virus E3 protein function and its involvement in viral pathogenesis. Furthermore, a short summary of other viral systems is provided.

FGFR2-IIIb Expression by Immunohistochemistry Has High Specificity in Cholangiocarcinoma with FGFR2 Genomic Alterations.

BACKGROUND: FGFR2 genomic alterations are observed in 10-20% of cholangiocarcinoma (CCA). Although FGFR2 fusions are an important actionable target, FGFR2 protein expression has not been thoroughly characterized. AIMS: To evaluate FGFR2 protein expression in cholangiocarcinoma harboring FGFR2 genomic alterations. METHODS: FGFR2 protein expression was evaluated in 99 CCA cases with two different antibodies. FGFR2 genomic alterations were confirmed via next-generating sequencing (NGS) or FISH. Primary objective was to determine the specificity and sensitivity of FGFR2 immunohistochemistry staining for detecting FGFR2 genomic alterations. Secondary objectives included overall FGFR2 immunohistochemistry staining in CCA patients, and evaluation of whether FGFR2 expression correlates with clinical outcomes including overall survival (OS), progression-free survival (PFS), and time-to-tumor recurrence (TTR). RESULTS: Immunohistochemistry staining with two antibodies against FGFR2, FPR2-D, and clone 98706 showed high accuracy (78.7% and 91.9%) and specificity (82.9% and 97.7%), and moderate sensitivity (53.9% and 57.1%), respectively, when compared with the standard methods for detecting FGFR2 genomic alterations. In a median follow-up of 72 months, there were no statistically significant differences in OS, PFS, and TTR, for patients with positive or negative FGFR2 staining. CONCLUSION: FGFR2 protein expression by immunohistochemistry has high specificity and therefore could be used to imply the presence of FGFR2 genomic alterations in the context of a positive test. In the case of a negative test, NGS or FISH would be necessary to ascertain cases with FGFR2 genomic alterations.

Intranasal immunization with a vaccinia virus vaccine vector expressing pre-fusion stabilized SARS-CoV-2 spike fully protected mice against lethal challenge with the heavily mutated mouse-adapted SARS2-N501Y MA30 strain of SARS-CoV-2.

The Omicron SARS-CoV-2 variant has been designated a variant of concern because its spike protein is heavily mutated. In particular, Omicron spike is mutated at 5 positions (K417, N440, E484, Q493 and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501Y MA30 , contains a spike that is also heavily mutated, with mutations at 4 of the 5 positions in Omicron spike associated with neutralizing antibody escape (K417, E484, Q493 and N501). In this manuscript we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against disease and death from SARS2-N501Y MA30 . Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus, administered without parenteral injection can fully protect against the heavily mutated mouse-adapted SARS2-N501Y MA30 .

Convergent Loss of the Necroptosis Pathway in Disparate Mammalian Lineages Shapes Viruses Countermeasures.

Programmed cell death is a vital process in the life cycle of organisms. Necroptosis, an evolutionary form of programmed necrosis, contributes to the innate immune response by killing pathogen-infected cells. This virus-host interaction pathway is organized around two components: the receptor-interacting protein kinase 3 (RIPK3), which recruits and phosphorylates the mixed lineage kinase-like protein (MLKL), inducing cellular plasma membrane rupture and cell death. Critically, the presence of necroptotic inhibitors in viral genomes validates necroptosis as an important host defense mechanism. Here, we show, counterintuitively, that in different mammalian lineages, central components of necroptosis, such as RIPK3 and MLKL, are deleted or display inactivating mutations. Frameshifts or premature stop codons are observed in all the studied species of cetaceans and leporids. In carnivores' genomes, the MLKL gene is deleted, while in a small number of species from afrotheria and rodentia premature stop codons are observed in RIPK3 and/or MLKL. Interestingly, we also found a strong correlation between the disruption of necroptosis in leporids and cetaceans and the absence of the N-terminal domain of E3-like homologs (responsible for necroptosis inhibition) in their naturally infecting poxviruses. Overall, our study provides the first comprehensive picture of the molecular evolution of necroptosis in mammals. The loss of necroptosis multiple times during mammalian evolution highlights the importance of gene/pathway loss for species adaptation and suggests that necroptosis is not required for normal mammalian development. Moreover, this study highlights a co-evolutionary relationship between poxviruses and their hosts, emphasizing the role of host adaptation in shaping virus evolution.

Optimization of translation enhancing element use to increase protein expression in a vaccinia virus system.

Since the successful use of vaccinia virus (VACV) in the immunization strategies to eliminate smallpox, research has been focused on the development of recombinant VACV strains expressing proteins from various pathogens. Attempts at decreasing the side effects associated with exposure to recombinant, wild-type viral strains have led to the development of attenuated viruses. Yet while these attenuated VACV's have improved safety profiles compared to unmodified strains, their clinical use has been hindered due to efficacy issues in stimulating a host immune response. This deficiency has largely been attributed to decreased production of the target protein for immunization. Efforts to increase protein production from attenuated VACV strains has largely centered around modulation of viral factors, while manipulation of the translation of viral mRNAs has been largely unexplored. In this study we evaluate the use of translation enhancing element hTEE-658 to increase recombinant protein production in an attenuated VACV system. Optimization of the use of this motif is also attempted by combining it with strategies that have demonstrated effectiveness in previous research. We show that extension of the 5' leader sequence containing hTEE-658 does not improve motif function, nor does the combination with other known translation enhancing elements. However, the sole use of hTEE-658 in an attenuated VACV system is shown to increase protein expression levels beyond those of a standard viral promoter when used with a wild-type virus. Taken together these results highlight the potential for hTEE-658 to improve the effectiveness of attenuated VACV vaccine candidates and give insights into the optimal sequence context for its use in vaccine design.

Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis.

Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.

Subversion of Programed Cell Death by Poxviruses.

Poxviruses have been long regarded as potent inhibitors of apoptotic cell death. More recently, they have been shown to inhibit necroptotic cell death through two distinct strategies. These strategies involve either blocking virus sensing by the host pattern recognition receptor, ZBP1 (also called DAI) or by influencing receptor interacting protein kinase (RIPK)3 signal transduction by inhibition of activation of the executioner of necroptosis, mixed lineage kinase-like protein (MLKL). Vaccinia virus E3 specifically blocks ZBP1 â†’ RIPK3 â†’ MLKL necroptosis, leaving virus-infected cells susceptible to the TNF death-receptor signaling (e.g., TNFR1 â†’ FADD â†’ RIPK1 â†’ RIPK3 â†’ MLKL), and, potentially, TLR3 â†’ TRIF â†’ RIPK3 â†’ MLKL necroptosis. While E3 restriction of necroptosis appears to be common to many poxviruses that infect vertebrate hosts, another modulatory strategy not observed in vaccinia or variola virus manifests through subversion of MLKL activation. Recently described viral mimics of MLKL in other chordopoxviruses inhibit all three modes of necroptotic cell death. As with inhibition of apoptosis, the evolution of potentially redundant viral mechanisms to inhibit programmed necroptotic cell death emphasizes the importance of this pathway in the arms race between pathogens and their hosts.

Detecting Necroptosis in Virus-Infected Cells.

Necroptosis has been implicated as a critical cell death pathway in cancers, Alzheimer's and other neurodegenerative diseases, and virus-infected cells. Necroptosis occurs when mixed-lineage kinase domain-like protein (MLKL) punctures the cytoplasmic membrane allowing a rapid influx of water leading to a loss of cellular integrity. As its role in human disease becomes apparent, methods identifying necroptosis will need to be further developed and optimized. Here we describe identification of necroptosis through quantifying cell death with pathway inhibitors and using western blots to identify end points of MLKL activation and protein-protein interactions leading to it.

Anti-herpes virus activity of the carnivorous botanical, Sarracenia purpurea.

Herpes simplex virus type-1 (HSV-1), one of the most widely spread human viruses in the Herpesviridae family, causes herpes labialis (cold sores) and keratitis (inflammation of the cornea). Conventional treatment for HSV-1 infection includes pharmaceutical drugs, such as acyclovir and docosonal, which are efficacious but maintain the potential for the development of viral drug resistance. Extracts from the carnivorous pitcher plant, Sarracenia purpurea, have previously been shown to inhibit the replication of HSV-1. In this study, we demonstrate that S. purpurea extracts can inhibit the replication of HSV-1 by two distinct mechanisms of action. These extracts directly inhibit extracellular virions or viral attachment to the human host cell as well as inhibiting the expression of viral immediate-early, early and late genes when added at various times post-infection. This botanical has previously been shown to inhibit the replication of poxviruses through the inhibition of early viral gene transcription. These results support a broader anti-viral activity of S. purpurea extracts against both pox and herpes viruses.

The Role of Endophytic/Epiphytic Bacterial Constituents in the Immunostimulatory Activity of the Botanical, Astragalus membranaceus.

Astragalus membranaceus is a staple of Traditional Chinese Medicine being one of the oldest medicinal herbs listed in the material medica of Chinese herbal medicine. Chinese herbalists have used Astragalus to help the human body fight a variety of diseases. Modern herbalists utilize Astragalus primarily as an immunostimulant to prevent common infection and aid in the recovery following infection. Historically, the biological activities associated with Astragalus have been accounted for, at least in part, to several constituents present in the botanical including saponins and polysaccharides. We propose that in addition to these constituents, compounds from endophytic (or epiphytic) bacteria present in (or on) the roots of Astragalus may have an important biological role. Lipopolysaccharides and lipoproteins are major components of Gram-negative bacteria and highly potent activators of the innate immune response. Our data supports a direct correlation between the level of immune gene induction and the level of lipopolysaccharides/lipoproteins present in the Astragalus extract. We demonstrate that extracts from Astragalus specifically activate Toll-like and NOD-like receptors involved in the recognition and response to bacterial constituents and that removal of the lipopolysaccharide/lipoprotein from the Astragalus extract reduced the level of this response. The results support that many immune enhancing botanicals have established a symbiotic relationship with Gram-negative bacteria and that the immune enhancing effect of these botanical extracts on the body may not only be due to endogenous plant compounds, but endophytic (or epiphytic) bacterial components as well.